WO2011015040A1 - 用于宮颈上皮內瘤样病变和宮致癌的诊断和预后的方法与組合物 - Google Patents

用于宮颈上皮內瘤样病变和宮致癌的诊断和预后的方法与組合物 Download PDF

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WO2011015040A1
WO2011015040A1 PCT/CN2010/001202 CN2010001202W WO2011015040A1 WO 2011015040 A1 WO2011015040 A1 WO 2011015040A1 CN 2010001202 W CN2010001202 W CN 2010001202W WO 2011015040 A1 WO2011015040 A1 WO 2011015040A1
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mirna
nucleic acid
cervical
sequence
level
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PCT/CN2010/001202
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English (en)
French (fr)
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秦文彦
董鹏
邓沱
马彩玲
麦凯斯
温浩
程京
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博奥生物有限公司
清华大学
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Priority to EP10805944A priority Critical patent/EP2463381A4/en
Priority to IN1979DEN2012 priority patent/IN2012DN01979A/en
Priority to US13/389,428 priority patent/US8957039B2/en
Priority to CN201080035889.7A priority patent/CN102510905B/zh
Publication of WO2011015040A1 publication Critical patent/WO2011015040A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • This application relates to the diagnosis, prognosis, clinical management and improvement of survival rate of diseases such as cervical intraepithelial neoplasa (CIN) and cervical cancer (cervical cancer) based on the microRNA level of the patient.
  • CIN cervical intraepithelial neoplasa
  • cervical cancer cervical cancer
  • Cervical cancer is the second largest female cancer in the world, with nearly 500,000 new cases each year (Parkin et al. 2005;). In 2002, cervical cancer caused approximately 274,000 deaths, a major cause of death among young women due to cancer (Zur Hausen, 2002). Cervical cancer usually originates from cell transformation caused by persistent infection with high-risk human papilloma virus (HPV) (Scheffner et al, 1990). Almost all squamous cell carcinomas and most stratified epithelial adenocarcinomas are HPV positive. Although HPV can cause cancer by destroying many signaling pathways that inhibit tumorigenesis, this alone is not enough to cause cancer (Burk, 1999). Other host factors are needed to develop a malignant phenotype.
  • HPV human papilloma virus
  • Cervical precancerous lesions are also known as cervical intraepithelial neoplasia.
  • SIL squamous intraepithelial lesion
  • CIN cervical intraepithelial neoplasia
  • cervical lesions are classified into AGUS or AGCUS (atypical glandular cells of undetermined significance), LSIL (low grade squamous intraepithelial lesion) and HSIL (high grade squamous intraepithelial lesion).
  • the classification of cervical lesions depends on single cells.
  • the extent of the lesion also depends on the extent to which the lesion develops within the cervical epithelium.
  • cervical lesions are classified as CIN1 (corresponding to mild lesions or LSIL) CIN2 (corresponding to moderate lesions or HSIL) and CIN3 (corresponding to severe lesions or HSIL). Most CIN1 will return to normal after a period of time, but about 11% develop into CIN3.
  • CIN1 carcinoma in situ
  • MicroRNAs are a class of small, non-coding, single-stranded regulatory RNAs that interact with the 3'-untranslated region (3'-UTR) of a target mRNA molecule by partial pairing (Yekta et al., 2004), they act as control elements in the regulatory network of gene expression (Fatica et al., 2006). Bioinformatics analysis predicts that a miRNA can regulate hundreds of target genes and comprehensively and finely regulate a large number of cellular signaling pathways (Hwang and Mendell, 2006; Lewis et al., 2005).
  • MiR-133 expression abnormalities have also been reported in many other diseases, including colorectal cancer (Bandres et al, 2006), tongue squamous cell carcinoma (Wong et al., 2008), esophageal squamous cell carcinoma (Guo et Al., 2008) and pancreatic ductal carcinoma (Szafranska et al., 2007).
  • the present invention provides a system for detecting the expression level of a microRNA (miRNA) or a precursor thereof, comprising a plurality of probes, wherein at least 50% can detect any one of the miRNAs having the nucleic acid sequences listed in SEQ ID NOs: 1-20 or The homologue.
  • the invention also provides methods for diagnosing cancer and prognosis based on the level of miRNA expression or the genetic status of the corresponding miRNA gene, particularly for the diagnosis and prognosis of cervical intraepithelial neoplasia and cervical cancer.
  • Cervical intraepithelial neoplasia And drugs and methods of treatment for cervical cancer comprising a component which alters the expression level of at least one miRNA having the nucleic acid sequence of SEQ ID NOs: 1 to 20 or a homolog thereof, and a pharmaceutically acceptable carrier.
  • the invention provides a system for detecting the level of miRNA expression, comprising a plurality of probes, wherein at least 50% of the miRNAs having the nucleic acid sequences listed in SEQ ID NOs: 1-20 or their homologs can be detected. body.
  • at least 50% of the probes can detect at least 5 miRNAs with the nucleic acid sequences listed in SEQ ID Nos. 1-20 or their homologs.
  • at least 50% of the probes can detect at least 10 miRNAs having the nucleic acid sequences listed in SEQ ID NOs: 1-20 or their homologs.
  • At least 50% of the probes can detect a miRNA having the nucleic acid sequence listed in SEQ ID NOs: 1-13 or a homolog thereof and a miRNA having the nucleic acid sequence of SEQ ID NO: 14-20 or Their homologs. In still another case, at least 50% of all miRNAs having the nucleic acid sequences listed in SEQ ID Nos. 1-20 or their homologs can be detected.
  • expression of at least one (including, for example, at least 2, 3, 5, 10, 13) miRNAs having the nucleic acid sequences listed in SEQ ID NOs: 1-13 or their homologs can be detected.
  • expression levels of at least one (including, for example, at least 2, 3, 5, 7 of one) miRNAs having the nucleic acid sequences listed under SEQ ID Nos. 14-20 or their homologs can be detected.
  • expression levels of at least one (including, for example, at least 2, 3, 5, 10, 13) miRNAs having the nucleic acid sequences listed in SEQ ID NOs: 1-13 or their homologs can be detected and at least The expression level of one (including, for example, at least 2, 3, 5, 7) miRNAs having the nucleic acid sequences listed in SEQ ID Nos. 14-20 or their homologs.
  • the system comprises at least one (including, for example, at least 2, 5, 10, 15, 20, 25, 30, 35 and 40) probes capable of detecting a nucleic acid sequence having the sequence number 1-20 miRNAs or their homologs.
  • At least one miRNA is has-miR-133b or a homolog thereof.
  • the miRNA comprises hsa-miR-133a, hsa-miR-133b, hsa-miR-140-3p, hsa-miR-143*, hsa-miR-145, hsa-miR-223, hsa-miR -99b, hsa-miR-221, hsa-miR-320a, hsa-miR-100, hsa-miR-199a-5p, hsa-miR-127-3p, hsa-miR-214 or their homologs.
  • the miRNA comprises hsa-miR-203, hsa-miR-190, hsa-miR-200b, hsa-miR-200c, hsa-miR-200a, hsa-miR-31, hsa-miR-141 or Their same Source body.
  • the probe may be 20, and the nucleic acid sequence of the 20 probes may be as follows: a) or b) : a) the complete complement of the 20 nucleic acids represented by the sequence 1 to the sequence 20 in the sequence listing. b) Connect 10-30 T at the 5' end of the 20 nucleic acids described in a), specifically 19 1 ⁇
  • the invention provides a method of detecting a cervical cancer or a cervical intraepithelial neoplasia sample, comprising: a) detecting a level of miRNA expression in a sample using a system comprising a plurality of probes, at least 50 of the probes % can detect a miRNA having the nucleic acid sequence listed in SEQ ID NO: 1-20 or a homolog thereof; b) align the miRNA expression level with a reference level; c) if the miRNA expression level of the sample exhibits a characteristic change , the classification of whether the sample is cancer or intraepithelial neoplasia.
  • a characteristic change in the level of miRNA expression comprises a significant increase in the expression level of at least one miRNA having the nucleic acid sequence set forth in SEQ ID NOs: 1-13 or their homologs.
  • a characteristic change in the level of miRNA expression comprises a significant increase in the expression level of has-miR-133b or its homolog.
  • a characteristic change in the level of miRNA expression comprises a significant decrease in the expression level of at least one miRNA having the nucleic acid sequence set forth in SEQ ID NOs: 14-20 or their homologs.
  • the characteristic change of the miRNA expression level comprises at least one miRNA having the nucleic acid sequence of SEQ ID NOs: 1 to 13 or a homologous expression level thereof, and at least one having a sequence The expression levels of miRNAs or their homologs of the nucleic acid sequences listed in No. 14-20 are actually decreased.
  • a characteristic change in the level of miRNA expression comprises at least three miRNAs having the nucleic acid sequences listed in SEQ ID NOs: 1-13 or their homologous expression levels that actually rise, and at least three have SEQ ID NO: 14-20 The expression levels of miRNAs or their homologs of the nucleic acid sequences listed are indeed degraded.
  • a characteristic change in the level of miRNA expression comprises at least five miRNAs having the nucleic acid sequences listed in SEQ ID NOs: 1-13 or their homologous expression levels are truly elevated, and at least five have a SEQ ID NO: 14-20 The expression levels of miRNAs or their homologs of the nucleic acid sequences listed are indeed degraded.
  • a characteristic change in the level of miRNA expression encompasses a true increase in the expression levels of all miRNAs or their homologs with the nucleic acid sequences listed in SEQ ID NOs: 1-13, and all have the SEQ ID NO: 14-20 The expression levels of miRNAs or their homologs of nucleic acid sequences are actually declining.
  • a method comprising detecting a genetic state of at least one miRNA in a sample using a system comprising a plurality of probes, at least 50% of which can detect a miRNA having the nucleic acid sequence listed in SEQ ID NOs: 1-20 or the same
  • the source body in which a characteristic change in the genetic state of the miRNA, suggests that the sample is a cancerous or intraepithelial neoplasia.
  • a characteristic change in the genetic state of a miRNA includes amplification of at least one miRNA having the nucleic acid sequence set forth in SEQ ID NOs: 1-13 or a homolog thereof.
  • a characteristic change in the genetic state of the miRNA includes amplification of has-miR-133b or its homolog.
  • a characteristic change in the genetic state of the miRNA includes deletion of at least one miRNA having the nucleic acid sequence set forth in SEQ ID NO: 14-20 or a homolog thereof.
  • the characteristic change of the genetic state of the miRNA includes amplification of at least one miRNA having the nucleic acid sequence of SEQ ID NO: 1-13 or a homolog thereof, and at least one having the serial number Deletions of miRNAs or their homologs of the nucleic acid sequences listed in Nos. 14-20.
  • a characteristic change in the genetic state of the miRNA includes amplification of at least three miRNAs having the nucleic acid sequences listed in SEQ ID NOs: 1-13 or their homologs, and at least three having the sequence number 14-20 Deletions of miRNAs or their homologs of the nucleic acid sequences listed.
  • the characteristic change in the genetic state of the miRNA comprises amplification of at least five miRNAs having the nucleic acid sequences listed in SEQ ID NOs: 1-13 or their homologs, and at least five having the sequence number 14-20 Deletion of miRNAs or their homologs of the nucleic acid sequences listed.
  • the invention provides a method of diagnosing cervical cancer or cervical intraepithelial neoplasia, the method comprising detecting whether a miRNA expression level of an individual sample has a characteristic change, comprising: a) using a plurality of probes Systematic detection of miRNA expression levels in samples, at least 50% of these probes can detect a miRNA having the nucleic acid sequence listed in SEQ ID NOs: 1-20 or their homologs; b) compare miRNA expression levels to reference levels (c) If the miRNA expression level of the sample exhibits a characteristic change, the sample is judged to be cancer or intraepithelial neoplasia.
  • a method of diagnosing cervical cancer or cervical intraepithelial neoplasia comprising detecting the genetic status of a miRNA of an individual sample.
  • the method comprises detecting a gene status of at least one miRNA in a sample using a system comprising a plurality of probes, at least 50% of which can detect a miRNA having the nucleic acid sequence listed in SEQ ID NOs: 1-20 or a homolog thereof, A characteristic change in the state of the miRNA gene suggests that the sample is a cancerous or intraepithelial neoplasia.
  • the invention provides a method of prognosis for a patient with cervical cancer or cervical intraepithelial neoplasia, the method comprising: a) detecting a miRNA in a sample using a system comprising a plurality of probes Expression level, at least 50% of these probes can detect a miRNA with the nucleic acid sequence listed in SEQ ID NOs: 1-20 or their homologs; b) Align miRNA expression levels with reference levels, miRNA expression levels The characteristic changes suggest an individual's high or low survival rate. In some cases, the method also includes appropriate treatments for the individual.
  • Also provided herein is a method for prognosis of a patient with cervical cancer or cervical intraepithelial neoplasia comprising detecting a gene status of at least one miRNA in the sample using a system comprising a plurality of probes, at least 50 of the probes % can detect a miRNA having the nucleic acid sequence listed in SEQ ID NO: 1-20 or a homolog thereof, and a characteristic change in the state of the miRNA gene implies a high or low survival rate of the individual.
  • the method also includes appropriate treatments for the individual.
  • Also provided herein is a method for classifying patients with cervical intraepithelial neoplasia and/or cervical cancer based on, for example, detecting the expression level of a miRNA or a homolog thereof using a system comprising a plurality of probes, these probes At least 50% of them can detect a miRNA having the nucleic acid sequence listed in SEQ ID NOs: 1-20 or a homolog thereof.
  • determining the level of cervical intraepithelial neoplasia and/or cervical cancer differentiation in an individual comprising detecting the expression level of a miRNA or a homolog thereof using a system comprising a plurality of probes, at least 50 of these probes.
  • % can detect a miRNA having the nucleic acid sequence listed in SEQ ID NO: 1-20 or a homolog thereof, where the expression level of the miRNA is used as a cervical intraepithelial neoplasia and/or cervical cancer to determine the individual. The basis of the level of differentiation.
  • the present invention provides a method for the diagnosis of colorectal cancer, tongue squamous cell carcinoma, esophageal squamous cell carcinoma, and pancreatic ductal carcinoma.
  • the method comprises detecting whether there is a characteristic change in the expression level of miR-133b in an individual sample, the detection method comprises: a) detecting the level of miR-133b expression in the sample; b) comparing the expression level of the sample miR-133b with a reference level; c) If the miRNA level in the sample shows a characteristic change, then the sample is determined to be cancer.
  • the method comprises detecting the genetic status of miR-133b in an individual sample, and if the genetic status of the miRNA in the sample exhibits a characteristic change, the sample is implicated as a cancer.
  • the invention provides a therapeutic agent for an individual with cervical cancer or cervical intraepithelial neoplasia, comprising at least one miRNA having the nucleic acid sequence of SEQ ID NOs: 1-13 or a homolog thereof A component of the expression level of the body, and a pharmaceutically acceptable carrier.
  • the drug also includes miRNAs that increase at least one of the nucleic acid sequences set forth in SEQ ID NOs: 14-20 Or components of the expression level of their homologs.
  • a therapeutic agent for an individual with cervical cancer or cervical intraepithelial neoplasia including an expression level that can increase the expression level of at least one miRNA having the nucleic acid sequence listed in SEQ ID NO: 14-20 or a homolog thereof Ingredients, as well as pharmaceutically acceptable carriers.
  • treatment regimens for individuals with cervical cancer or cervical intraepithelial neoplasia using expression levels comprising miRNAs or their homologs that can reduce at least one of the nucleic acid sequences set forth in SEQ ID NOs: 1-13.
  • Ingredients, as well as pharmaceutically acceptable carriers are also provided herein.
  • treatment regimens for individuals with cervical cancer or cervical intraepithelial neoplasia using expression levels comprising miRNAs or homologs thereof that increase at least one of the nucleic acid sequences set forth in SEQ ID NOs: 14-20 Ingredients, as well as pharmaceutically acceptable carriers.
  • the invention provides an oligonucleotide primer for amplifying an RNA sequence comprising a nucleotide sequence having the following characteristics: a) under high stringency conditions, with a nucleic acid sequence or Its complementary sequence hybridizes, as listed in the sequence listing; b) has at least 90% similarity to a nucleic acid sequence or its complement, such as those listed in the sequence listing.
  • the primer comprises a nucleic acid sequence shown in the sequence listing or a complement thereof.
  • the primers include DNA,
  • RNA RNA, PNA or a derivative thereof.
  • the primers can be labeled.
  • the markers belong to the following categories: chemical, enzymatic, immunogenic, radioactive, fluorescent, chemiluminescent, and FRET (fluorescence resonance energy transfer) labels.
  • the invention also provides kits for the methods described herein. Chart description
  • FIG. 1 provides a differentially expressed miRNA clustering map analyzed by a micro-array significance analysis of Microarrays (SAMs).
  • SAMs Microarrays
  • M 20 bp DNA Ladder Marker (product of Bao Bioengineering (Dalian) Co., Ltd.); N: Negative control, using plasmid without hsa-miR-133b gene as template for amplification reaction; P: positive control, containing hsa- The plasmid of miR-133b gene was used as a template for amplification reaction; 1-5: 5 cases of cervical cancer tissue cDNA were used as template for amplification reaction.
  • Figure 3 provides the quantitative expression of hsa-miR-133b at different stages of cervical carcinogenesis and development using quantitative RT-PCR; each point in the figure represents the expression of hsa-miR-133b in a cervical tissue sample.
  • CIN 2 Cervical intraepithelial neoplasia stage II
  • CIN 3 Cervical intraepithelial neoplasia III.
  • Figure 4 shows the expression of hsa-miR-133b at different stages of cervical carcinogenesis and development by in situ hybridization.
  • H&E Hematoxylin and eosin staining
  • Ki-67 Ki-67 antibody immunohistochemical staining
  • Hsa-miR-133b probe digoxigenin-labeled hsa-miR-133b detection probe; Scramble-miR probe: digoxigenin-labeled random sequence probe (negative control).
  • the digoxigenin-labeled hsa-miR-133b detection probe and the digoxigenin-labeled random sequence probe were purchased from Exiqon, Denmark (www.exiqon.com).
  • Figure 5 shows CaSki cells stably expressing hsa-miR-133b (CaSki-miR-133b) and negative control CaSki cells (CaSki-NC) subcutaneously forming tumors in severely immunodeficient mice (SCID mice). Growth curve.
  • Figure 6 shows the results of the number of metastases formed on the surface of the lungs after 60 days of injection of SiHa cells (SiHa-miR-133b) stably expressing hsa-miR-133b and negative control SiHai cells (SiHa-NC) into SCID mice.
  • SiHa-miR-133b SiHa cells
  • SiHa-NC negative control SiHai cells
  • the invention is based, in part, on the study of miRNA expression profiles, which include 6 normal cervical tissues, 11 CIN2 cervical tissues, 9 cervical cancer tissues, and 11 invasive cancer tissues. . Comparing the miRNA expression profiles of cervical cancer tissue samples with normal cervical tissue samples, 20 miRNAs were up-regulated or down-regulated in normal tissue tissues compared to normal tissues. Real-time fluorescence PCR and in situ hybridization were used to confirm the expression level of has-miR-133b in normal cervical tissues, cervical intraepithelial neoplasia and cervical cancer tissues. Cytological and zoological experiments have shown that has-miR-133b promotes the formation and metastasis of cervical cancer.
  • the invention provides a system for detecting the level of expression of a miRNA or the genetic status of its gene. Oligonucleotide primers that amplify miRNAs are also provided.
  • the invention provides a method for classifying and prognosing a cancer patient, particularly a cervical intraepithelial neoplasia and/or a cervical cancer patient, based on the level of miRNA expression or the genetic status of the gene.
  • the invention provides a medicament and method of treatment comprising a composition that alters the level of miRNA expression.
  • An "individual” herein refers to a vertebrate, mammal or human. Mammals include, but are not limited to, livestock animals, sport animals, pets, primates, mice, and rats. In some cases, an individual is a human. In some cases, an individual is an animal model for studying cervical cancer. It will be understood that when the individual does not refer to a human, the miRNA refers to the corresponding homolog or ortholog of the human miRNA.
  • tissue sample herein refers to a tissue sample derived from the cervix.
  • tissue samples are fresh.
  • tissue samples are frozen.
  • tissue samples are saved.
  • tissue samples are preserved in formalin.
  • tissue samples are paraffin embedded. As described below, depending on the method used, the tissue can be used entirely, or the tissue can be separated into small pieces, cell clusters or individual cells using various methods known in the art.
  • Cervical cancer includes, but is not limited to, cervical squamous cell carcinoma or cervical adenocarcinoma.
  • homolog refers to a nucleic acid that differs from a natural nucleic acid (such as a "prototype” or “wild-type” nucleic acid) by minor modification of the native nucleic acid, but it remains The same basic nucleotide structure of the natural nucleic acid. These changes include, but are not limited to, one or more nucleotide changes, including deletions, insertions, and/or substitutions.
  • a homologue may have enhanced, attenuated or substantially similar characteristics as compared to a native nucleic acid.
  • the homologous nucleic acid can be complementary or paired with the native nucleic acid. Homologues can be produced by techniques known in the art for the production of nucleic acids, including but not limited to: DNA recombination techniques, chemical synthesis, and the like.
  • complementary or paired refers to a sequence in which two nucleic acid sequences have at least 50% identity. More suitably, the two nucleic acid sequences have at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical sequences. “Complementary or paired” also means that two nucleic acid sequences are low, Hybridization can be carried out under neutral and/or high stringency conditions.
  • nucleic acid sequences have at least 90% identity. More suitably, the two nucleic acid sequences have at least 95%, 96%, 97%, 98%, 99% or 100% identical sequences.
  • highly complementary or highly matched also means that two nucleic acid sequences can hybridize under high stringency conditions.
  • hybridization reaction In general, the effects of hybridization stability on ion concentration and temperature.
  • the hybridization reaction is carried out under conditions of low stringency and then washed under different but more stringent conditions.
  • a medium stringency hybridization reaction refers to a condition that allows a nucleic acid molecule, such as a probe, to bind to a complementary nucleic acid molecule.
  • Hybridized nucleic acid molecules typically have at least 60% similarity, including at least 70%, 75%, 80%, 85%, 90%, or 95% similarity.
  • Medium stringency hybridization conditions were equivalent to 50% formamide, 5 x Denhardt's solution, 5 SSPE, 0.2% SDS, 42 °C reaction, followed by 0.2x SSPE, 0.2% SDS, 42 °C.
  • High stringency hybridization conditions were equivalent to 50% formamide, 5 ⁇ Denhardt's solution, 5 ⁇ SSPE, 0.2% SDS, 42 ° C reaction, and then washed in O. l x SSPE, 0.1% SDS, 65 °C.
  • the low stringency hybridization conditions were equivalent to 10% formamide, 5 X Denhardt's solution, 6 SSPE, 0.2% SDS, 22 °C reaction, and then washed at l x SSPE, 0.2% SDS, 37 °C.
  • Denhardt's solution contains 1% polysucrose,
  • SSPE bovine serum albumin
  • 20x SSPE contains 3M sodium chloride, 0.2M sodium phosphate and 0.025M ethylenediaminetetraacetic acid.
  • Other suitable medium rigor and high stringency hybridization fluids and conditions are well known to those of ordinary skill in the art and are described elsewhere, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 nd ed., Cold Spring Harbor Press, Plainview, NY (1989);. Wo P Ausubel et al, Short Protocols in Molecular Biology, 4 th ed "John Wiley & Sons (1999).
  • the "gene state” herein refers to the structure, copy number and position on the chromosome of the miRNA gene.
  • a “characteristic change” in the state of a miRNA gene refers to, for example, deletion or amplification, changes in copy number, or changes in position on a chromosome.
  • a "characteristic change" in the level of miRNA expression herein can be simply understood as a significant increase or decrease in the expression level of a miRNA in a sample compared to the reference level. Characteristic changes can also refer to a significant increase or decrease in the expression levels of multiple miRNAs. It can also mean that the expression levels of some miRNAs are significantly increased while the expression levels of other miRNAs are significantly decreased.
  • reference level refers to a level at which a particular miRNA is considered “normal.” In some cases, the reference level is based on non-cancerous cervical intraepithelial or cervical tissue in the same individual. The level of expression of this miRNA. In some cases, the reference level is based on the level of expression of this miRNA in an individual who does not have cervical intraepithelial neoplasia or cervical cancer. In some cases, the reference level is based on the average expression level of this miRNA in a population of individuals not suffering from cervical intraepithelial neoplasia or cervical cancer. In some cases, the reference level is derived from a sample library, including the sample being tested. The reference level can be determined in advance or simultaneously with the sample to be tested.
  • the reference level can be the level of another miRNA, the level of another RNA, such as U6, or the level of another nucleic acid, such as DNA.
  • the level of expression of the miRNA can be compared to other nucleic acid expression levels of the same sample or reference sample.
  • Reference samples may be derived from the same tissue or different tissues, or may be derived from the same individual or different individuals.
  • the "reference value” herein may be an absolute value, a relative value, a value having an upper limit and a lower limit, a series of values, an average value, a median value, an intermediate value, or a value compared with a specific control or reference value.
  • a “significant” change herein refers to a change that can be detected by the methods described herein, or a statistically significant change.
  • “significant rise” refers to a 5% increase in miRNA levels, including, for example, at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%. Or more.
  • a “significant decline” refers to a 5% decrease in miRNA levels, including, for example, at least a 6% decrease.
  • Probes herein refer to, for example, DNA, RNA, PNA, LNA, combinations thereof, and/or modifications thereof. They may also include modified oligonucleotide backbones. In some cases, these probes comprise at least 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive oligonucleosides that are identical or complementary to the miRNA. acid. A probe sequence can comprise two or more such complementary sequences. In some cases, the 5' or 3' end of the probe may be attached with a reactive group (such as an amino group) so that the probe is attached to the substrate. For example, the probe provided in Case 1 is 20, 20 probes.
  • the nucleic acid sequence is a complete complement of 20 nucleic acids shown in SEQ ID NO: 1 to SEQ ID NO: 20, and 19 T is ligated to the 5' end of each probe sequence, and the 5' end of the probe is amino-modified.
  • the probe can be used as, for example, an oligonucleotide for in situ hybridization or as a primer for a PCR amplification reaction (such as the primers provided in Cases 2 and 3).
  • System for detecting miRNA expression levels and gene status provides a number of systems for detecting characteristic changes in miRNA expression levels in cervical intraepithelial neoplasia or cervical cancer patients.
  • a system for detecting the status of a miRNA gene is also provided. These systems can be used for a variety of purposes including, for example, cervical intraepithelial neoplasia or cervical cancer diagnosis, cervical intraepithelial neoplasia or cervical cancer patient classification, and survival prognosis for cervical intraepithelial neoplasia or cervical cancer patients.
  • the miRNAs described herein can also be used in one or more of the following aspects: based on one or more miRNA expression levels or gene status of an individual cervical epithelium or cervical tissue sample, for a cervical intraepithelial neoplasia or cervical cancer patient Classification, predicting the risk of developing cervical intraepithelial neoplasia or cervical cancer, monitoring the development of tumors in patients with cervical intraepithelial neoplasia or cervical cancer, and treating patients with cervical intraepithelial neoplasia or cervical cancer monitor.
  • the systems described herein include probes that detect the status of a miRNA or its gene.
  • the following discussion will focus on systems that can detect miRNA expression levels, and it will be readily understood by those of ordinary skill in the art that aspects of the description are also applicable to the inclusion of detection gene deletions, amplifications, and/or miRNA gene copies.
  • a system of probes that change the number (collectively referred to as the gene state of the miRNA).
  • a system comprising a plurality of probes that can detect different miRNAs in a sample, at least 15% (including, for example, at least 20%, 30%, 40%, 50 The probes of %, 60%, 70%, 80%, 90% or 95%) can detect one of the miRNAs in Table 1 or their homologs.
  • the system comprises (including must contain or can contain) at least 2, 5, 10, 20, 30, 40 or 50 probes, each of which can detect one of the miRNAs in Table 1 or Their homologs.
  • the systems described herein can comprise two or more probes that detect the same miRNA.
  • the probe is multiple (e.g., 2, 3, 4, 5, 6, 7 or more) copies on the microarray.
  • the system contains different probes that detect the same miRNA.
  • these probes may bind to different regions of the miRNA (overlapping or non-overlapping).
  • any probe that can detect miRNA levels can be used.
  • the probe can be an oligonucleotide. It is understood that in order to detect miRNA, partial sequence variation is acceptable. Thus, an oligonucleotide sequence (or its complement) can be slightly different from the miRNA sequences described herein. It is readily understood by those of ordinary skill in the art that such sequence changes do not significantly affect the ability of the oligonucleotide to detect miRNA levels. For example, homologs or variants of these oligonucleotide molecules possess a high degree of sequence similarity when aligned by standard methods.
  • the invention comprises an oligonucleotide sequence having at least 40% of the miRNAs described herein, including, for example, at least 50%, 60%, 70%, 80%, 90%, 95% or greater sequence homology.
  • the oligonucleotide comprises a portion that detects the miRNA and another part. Another part can be used, for example, to attach an oligonucleotide to a substrate.
  • another portion contains a non-specific sequence (eg, polyT) that increases the distance of the complementary sequence from the surface of the substrate.
  • Oligonucleotides as described herein include, for example, DNA, RNA, PNA, LNA, combinations thereof, and/or modifications thereof. They may also include modified oligonucleotide backbones. In some cases, these oligonucleotides comprise at least 9, 10, 12, 13, 14, 15, 16, 17, 18, 20, 20 or more consecutive oligos that are identical or complementary to the miRNA, either completely or partially. Nucleotide. An oligonucleotide sequence may comprise two or more such complementary sequences. In some cases, the 5' or 3' end of the oligonucleotide may be linked to a reactive group (e.g., an amino group) such that the oligonucleotide is attached to the substrate.
  • a reactive group e.g., an amino group
  • the system is a microarray containing probes. "microarray” and used here
  • Arrays can be substituted for each other, with an array of surfaces, preferably an ordered array, with predicted sites (e.g., hybridization) that bind to biochemical samples (targets) of unknown characteristics.
  • a microarray refers to a unique collection of oligonucleotide probes immobilized at a specific location on a substrate.
  • a microarray containing a plurality of probes wherein each probe can detect a different miRNA in the sample, at least 15% (including, for example, at least 20% :
  • the probes of 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%) can detect one of the miRNAs listed in Table 1 or their homologs.
  • a microarray for detecting the genetic status of a gene corresponding to a miRNA referred to herein is provided herein.
  • Microarrays that detect the genetic state of a gene are known in the art.
  • the system can comprise an inverted probe molecule that detects a sequence tag of the genetic state.
  • the array can be formed on a substrate made of paper, glass, plastic (eg, polypropylene, nylon, polystyrene), polyacrylamide, nitrocellulose, silicon, fiber, or other suitable solid or semi-solid support. It is also in the form of a plane (such as a slide, a silicon wafer) or a three-dimensional (such as a needle tip, fiber, beads, particles, micropores, capillaries).
  • plastic eg, polypropylene, nylon, polystyrene
  • polyacrylamide nitrocellulose
  • silicon fiber
  • fiber or other suitable solid or semi-solid support. It is also in the form of a plane (such as a slide, a silicon wafer) or a three-dimensional (such as a needle tip, fiber, beads, particles, micropores, capillaries).
  • the probe is an oligonucleotide.
  • Oligonucleotides can be attached to a substrate to form an array by: (but not limited to): (i) in situ synthesis using photolithography techniques (eg, high density oligonucleotide arrays); (ii) Low density sites in the medium are sampled on glass, nylon or nitrocellulose; (iii) mask; and (iv) latticed to nylon or nitrocellulose hybrid membranes.
  • Oligonucleotides can also be immobilized non-covalently on a substrate, such as anchor hybridization, and contained in micropores or capillaries in the form of magnetic beads or mobile phases.
  • nucleic acids there are several well-known techniques in the art for attaching nucleic acids to solid substrates such as glass. Surgery.
  • One method is to embed modified bases or analogs on the amplified nucleic acids comprising groups that can be attached to a solid substrate, such as amine-monoamine derivatives or other positively charged groups.
  • the amplified product is then attached to a solid substrate such as a slide.
  • the slide may be coated with an aldehyde group or other reactive group that can form a covalent linkage with the reactive group on the amplified product, such that the amplification product is interspersed with the slide.
  • a covalent bond is formed.
  • Microarrays containing amplification products can be produced by Biodot spotting (BioDot, Inc.
  • the amplified product can be spotted onto an aldehyde-coated slide and then processed according to published procedures by CSchena et al., Proc. Natl. Acad. Sci. USA (1995), 93: 10614-10619).
  • the array can also be spotted by a robot to glass, nylon (Ramsay, G Nature Biotechnol. (1998), 16:40-44), polypropylene (Matson, et al, Anal Biochem. (1995), 224(1): 110 -6) and silicon wafers (Marshall and Hodgson, Nature Biotechnol.
  • One of the methods of making a microarray is to form a high density nucleic acid array.
  • the technique used has the technique of rapidly depositing polynucleotides (Blanchard, et al., Biosensors & Bioelectronics, 11: 687-690).
  • Other methods of making microarrays can also be used, such as masks CMaskos and Southern, Nucleic. Acids Res. (1992), 20: 1679-1684).
  • any of the above arrays can be used, such as a lattice on a nylon hybrid membrane.
  • This invention found 20 miRNAs whose expression levels were associated with cervical intraepithelial neoplasia or cervical cancer. These miRNAs are listed in Table 1. Table 1 provides the miRNA name, sequence, and chromosomal location. Information on miRNAs can be found at http://miRNA.sanger.ac.uk/ (Griffths- Jones, et al., Nucleic Acids Research, 2006, Vol. 34, Database issue). The method of diagnosing cervical intraepithelial neoplasia or cervical cancer can be based on the expression level or gene status of any one of the miRNAs shown in Table 1.
  • the system described herein can be used to detect expression levels of one or more miRNAs in Table 1, and to cervical intraepithelial neoplasia based on the expression levels of one or more miRNAs in Table 1. Diagnosis of lesions or cervical cancer.
  • the effectiveness of the method can generally be facilitated by the use of at least two miRNAs. In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20 miRNAs in Table 1 were utilized.
  • the expression level or gene status of at least 2 (eg, at least 2, 3, 5, 10 or more) miRNAs of SEQ ID NOs: 1-13 are detected.
  • the expression level or gene status of at least 2 (e.g., at least 2, 5, 7 or more) miRNAs of SEQ ID NOs: 14-20 are detected.
  • detecting the expression level or gene status of at least 2 (eg, at least 2, 3, 5, 10 or more) miRNAs of SEQ ID NOs: 1-13, and at least 2 (eg, at least 2, 5, 7 or more) Multiple) expression levels or gene status of miRNAs of SEQ ID NOs: 14-20 are examined.
  • the levels of the corresponding homologs of the miRNAs described herein are detected.
  • the MiRNA "corresponding homolog” refers to at least 50% of the sequences (including, for example, at least 60%, 70%, 80%, 90%, 95%, 98%, or 99%) of the same miRNA as the miRNA herein.
  • a homolog of miRNA 1 has at least 50% (including, for example, at least 60%, 70%, 80%, 90%, 95%, 98%, or 99%) of the same sequence.
  • a miRNA sequence having at least 95% identical to a reference sequence (e.g., number 1) is considered to be identical to the reference sequence. If the miRNA sequence contains 5 nucleotides different from the reference sequence per 100 nucleotides. These five different points can be missing, replaced, inserted, can occur anywhere in the sequence, can be scattered independently in the reference sequence, or can form one or more consecutive segments.
  • One aspect of the invention provides a method of diagnosing cervical intraepithelial neoplasia and cervical cancer for an individual comprising: a) detecting at least one miRNA in a tissue sample suspected of having a cervical intraepithelial neoplasia and cervical cancer (eg, at least one The expression levels of the miRNAs in Table 1 or their homologs; b) When the expression level of the miRNA in the test tissue is characteristically changed compared with the reference level, it is predicted that there may be cervical intraepithelial neoplasia or cervical cancer. .
  • the method further includes from the individual Sampling cervical tissue.
  • the method further comprises extracting the miRNA from the tissue sample.
  • methods for providing information for the diagnosis of cervical intraepithelial neoplasia and cervical cancer including: a) for tissue samples suspected of having cervical intraepithelial neoplasia or cervical cancer, a test table Expression level of at least one miRNA or a homolog thereof in the tissue; b) providing expression levels of miRNAs for diagnosing cervical intraepithelial neoplasia and cervical cancer.
  • the expression level of MiRNA is the basis for the diagnosis of cervical intraepithelial neoplasia and cervical cancer. At least one characteristic change of miRNA indicates cervical intraepithelial neoplasia or cervical cancer.
  • the level of at least one (including at least 2, 3, 5, 10, 13) miRNAs of SEQ ID NO: 1-13 is detected.
  • the level of at least one of the detected miRNAs rises significantly, it is predicted to have a cervical intraepithelial neoplasia or cervical cancer.
  • Cervical intraepithelial neoplasia or cervical cancer is predicted when at least one miRNA of SEQ ID NO: 1-13 rises significantly and when at least one miRNA of SEQ ID NO: 14-20 is significantly decreased. In some cases, when at least two miRNAs of SEQ ID NO: 1-13 rise significantly and when at least two miRNAs of SEQ ID NO: 14-20 are significantly decreased, it is predicted to have cervical intraepithelial neoplasia or cervical cancer. . In some cases, when the level of SEQ ID NO: 1-13 is significantly increased and when the level of SEQ ID NO: 14-20 miRNA is significantly decreased, it is predicted to have cervical intraepithelial neoplasia or cervical cancer.
  • the level of miRNA expression in a tissue sample can also reflect changes in the state of the miRNA gene. For example, changes in the deletion, amplification or copy number of the miRNA gene can be reflected.
  • a diagnostic method for cervical intraepithelial neoplasia and/or cervical cancer including analysis of at least one miRNA gene in a cervical epithelial or cervical tissue sample of an individual suspected of having cervical cancer (for example, at least one genetic state corresponding to the gene of the miRNA in Table 1.
  • Significant changes in the genetic status of miRNA genes relative to control samples indicate cervical intraepithelial neoplasia or cervical cancer.
  • the change in genetic status is due to the miRNA gene Missing or amplifying.
  • the change in genetic status is due to a change in the copy number of the miRNA gene.
  • a diagnostic method for cervical intraepithelial neoplasia and/or cervical cancer comprises analyzing at least one miRNA gene corresponding to Table 1 in a cervical epithelial or cervical tissue sample of an individual suspected of having cancer. Missing or amplifying condition.
  • the miRNA gene is deleted or amplified relative to the control sample, it is predicted to have cervical intraepithelial neoplasia or cervical cancer.
  • this method involves the analysis of amplification of at least one miRNA gene of SEQ ID NO: 1-13, and amplification compared to a control sample indicates cervical intraepithelial neoplasia or cervical cancer.
  • this method involves the analysis of at least one miRNA gene deletion with sequence number 14-20, and a deletion compared to the control sample indicates cervical intraepithelial neoplasia or cervical cancer.
  • the method further includes sampling cervical epithelium or cervical tissue from an individual suspected of having cancer.
  • the method further includes extracting DNA from the cervical epithelium or cervical tissue.
  • a diagnostic method for cervical intraepithelial neoplasia and/or cervical cancer comprising detecting at least one miRNA listed in Table 1 or a corresponding miRNA of a cervical epithelial or cervical tissue sample of an individual suspected of having cancer.
  • the copy number of the homologous gene When the number of copies of miRNA genes located on autosomes or sex chromosomes is not two, it is predicted to have cervical intraepithelial neoplasia or cervical cancer.
  • the method comprises detecting a copy number of a gene corresponding to at least one miRNA of SEQ ID NO: 1-13 in an individual sample, when more than two copies of the miRNA gene are located on an autosomal or sex chromosome Changes indicate cervical intraepithelial neoplasia or cervical cancer.
  • the method comprises detecting a copy number of a gene corresponding to at least one miRNA of SEQ ID NO: 14-20 in an individual sample, and when less than two copies of the miRNA gene located on the autosome or sex chromosome are altered It is indicated that there is cervical intraepithelial neoplasia or cervical cancer.
  • the method further comprises sampling cervical epithelial or cervical tissue from an individual suspected of having cancer.
  • the method further comprises extracting DNA from the cervical epithelium or cervical tissue.
  • the diagnosis method of cervical intraepithelial neoplasia and cervical cancer is based on detecting the expression level of miRNA.
  • the invention provides a method for the diagnosis of colorectal cancer, tongue squamous cell carcinoma, esophageal squamous cell carcinoma, and pancreatic ductal carcinoma, comprising: a) detecting miR-133b in a tissue sample suspected of having cancerous The level of expression; b) the miR-133b expression level of the sample is compared with the reference level. When the miR-133b level in the sample shows a characteristic change, it indicates colorectal cancer, tongue squamous cell carcinoma, esophageal squamous cell carcinoma or Pancreatic ductal carcinoma.
  • the miR-133b gene provides a method for the diagnosis of colorectal cancer, tongue squamous cell carcinoma, esophageal squamous cell carcinoma, and pancreatic ductal carcinoma, including analysis of the genetic status of the miR-133b gene in an individual tissue suspected of having cancer.
  • the miR-133b gene is characteristically altered relative to the control sample, it is indicative of colorectal cancer, tongue squamous cell carcinoma, esophageal squamous cell carcinoma, or pancreatic ductal carcinoma.
  • the change in the genetic state of the test gene is based on the deletion or amplification of the miR-133b gene.
  • changes in the genetic status of the test gene are based on changes in the copy number of the miR-133b gene.
  • the "expression level” and “level” herein are interchangeable and each denotes the amount or ratio of accumulation of a miRNA molecule or a precursor thereof. This concept can be used to indicate the absolute amount of miRNA in a sample (e.g., the intensity of the hybridization signal), or the ratio relative to a control sample (e.g., the ratio of hybridization signals to control samples in the sample).
  • the control may be another miRNA in the same sample that does not change in the expression level of cervical intraepithelial neoplasia or cervical cancer tissue, or may be derived from different samples (eg, a non-cancerous tissue sample of the same individual or another one) The same miRNA of a tissue sample of an individual with cervical intraepithelial neoplasia or cervical cancer.
  • a "precursor" or "miRNA precursor" of a MiRNA molecule refers to a miRNA gene transcript that has not been fully processed, typically an approximately 70 base RNA transcript. MiRNA precursors are typically digested with RNase (eg Dicer, Argonaut, or RNAase III) to yield an active miRNA molecule approximately 19-25 bases in length.
  • RNase eg Dicer, Argonaut, or RNAase III
  • Levels of miRNA in cervical epithelial or cervical tissue samples refers to the level of miRNA in a tissue sample.
  • miRNA levels in cervical epithelial or cervical tissue samples are obtained by direct detection of miRNA levels in cervical epithelial or cervical tissue samples, whereas miRNA levels in cervical epithelial or cervical tissue samples can also be passed through lymph node samples (eg most miRNA levels in close lymph nodes or lymphoids, serum, blood or other closest biological flow samples such as saliva are reflected.
  • lymph node samples eg most miRNA levels in close lymph nodes or lymphoids, serum, blood or other closest biological flow samples such as saliva are reflected.
  • detection of miRNA levels is based on miRNA levels in lymphoid samples (such as lymph node fragments or needle-sucking samples).
  • the detection of miRNA levels is based on miRNA levels in blood or serum.
  • the level of miRNA detection is based on miRNA levels of cervical epithelium or cervical tissue scrapers.
  • the determination of miRNA levels is the level of miRNA in a sample obtained by an endoscopic sampling procedure (eg, by RT-PCR analysis). Detection of miRNA levels in samples other than cervical intraepithelial neoplasia or cervical cancer tissue can be used alone or in combination.
  • the level of miRNA can be detected first from serum and then analyzed for operational regionality. The level in the lymph nodes. This multi-step analysis can provide more information and increase the credibility of the diagnosis.
  • miRNA levels can be detected at different stages, for example, before surgery, during surgery, after surgery, before, during, or after treatment of a tumor.
  • Methods for detecting miRNA levels are also known in the art. For example, Northern blot, in situ hybridization, RT-PCR, and microarrays can be used (Einat, Methods Mol. Biol. (2006),
  • total RNA can be reprecipitated by homogenizing the cells in a nucleic acid extract, followed by centrifugation.
  • the DNA is removed by DNase digestion and then precipitated.
  • RNA molecules are separated by agarose gel electrophoresis according to standard techniques and then transferred to a nitrocellulose filter by techniques such as Northern blotting.
  • the RNA is immobilized on the filter by heating.
  • Specific RNA is detected and quantified by appropriately labeled DNA or RNA probes complementary to the RNA to be tested. Detection of a probe that hybridizes to a miRNA using autoradiography can be achieved by exposing the hybridized membrane to a film. Concentration scanning of the exposed film provides accurate RNA transcription levels.
  • RNA transcription levels can also be calculated from the hybridization points by photo processing software.
  • levels of miRNA transcripts can be obtained by in situ hybridization techniques. This technique involves placing the entire cell or tissue on a coverslip and then detecting the nucleic acid in the cell or tissue in a solution containing a radioactive or labeled probe (e. g., a cRNA probe).
  • a radioactive or labeled probe e. g., a cRNA probe
  • the level of miRNA can also be detected by RT-PCR.
  • the level of miRNA can be compared by comparison with standard internal controls, such as the mRNA level of the "housekeeping gene" in the same sample. Suitable "housekeeping genes” for use as internal controls include myosin, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) or human U6.
  • G3PDH glyceraldehyde-3-phosphate dehydrogenase
  • Quantitative RT-PCR or variants thereof are well known to those of ordinary skill in the art. In some specific cases, real-time quantitative PCR (qRT-PCR) may be more sensitive than detection of miRNA levels in early cancer by classical tissue section staining.
  • the qRT-PCR method for detecting miRNA levels may provide a sensitive and specific tool for the diagnosis and prognosis of cervical intraepithelial neoplasia or cervical cancer.
  • This invention provides a method for detecting miRNA levels in a sample of an individual (e.g., a diseased individual, such as a cancer patient) by RT-PCR. Specifically, the level of miRNA was detected by qRT-PCR.
  • the level of miRNA is detected by a microarray, such as the microarray described herein.
  • Probes for use in one or more of the methods described above can be obtained by methods known in the art, such as recombinant DNA or chemical synthesis.
  • hybridization probes can be labeled with different labels, such as radioisotopes, fluorescent probes, reporter enzymes, biotin or other ligands. This detectable label can be spectrophotometrically detected by coupling to a chromatographic or spectroscopic indicator. Methods of labeling and detecting such probes are known in the art.
  • the level of miRNA may be derived from different time points of the individual.
  • This "sequence" sample is most suitable for the monitoring of cervical intraepithelial neoplasia or cervical cancer development in cervical intraepithelial neoplasia or cervical cancer in the present invention. Sequence sampling is performed in any time series, for example every six months, every year, every two years or longer. The comparison between the detection level and the reference level can be performed after each sampling, or the data can be saved to a certain amount for analysis.
  • the miRNA compared to the reference level can be any 1, 2, 3, 4, 5, 6, ⁇ , 8, 9, 10, 15, 18, 19 or 20 miRNAs or homologs thereof in Table 1. body.
  • the process of comparing miRNA levels to reference levels can take any form suitable for the value of the detected miRNA.
  • the intensity of the hybridization signal can be qualitatively compared by visual comparison.
  • it can be done by comparing observed data or representative data (such as observation plot representations such as histograms or lines).
  • the process of comparison can be manual (such as visual observation by the practitioner of the method) or automated.
  • a comparison between the levels of the detected miRNA and the reference level is compared (e.g., by comparing the "fold” or percentage difference between the level of the detected miRNA and the reference level).
  • the "fold difference” herein refers to a numerical representation of the difference in amplitude between the level at which the miRNA is detected and the reference value.
  • Table 1 provides the altered miRNAs detected in all of the exemplary methods. Characteristic changes in MiRNA levels are used as a basis for the diagnosis of cervical intraepithelial neoplasia and cervical cancer. For example, in some cases, when at least one miRNA level of SEQ ID NO: 1-13 is detected, a true rise in at least one of the detected miRNA levels is indicative of a cervical intraepithelial neoplasia or cervical cancer. In some cases, at least one detected when at least one miRNA level of SEQ ID NO: 14-20 is detected A true decline in miRNA levels is indicative of cervical intraepithelial neoplasia or cervical cancer.
  • At least one miRNA of SEQ ID NO: 1-13 and at least one miRNA level of SEQ ID NO: 14-20 are detected, at least one of the detected miRNA levels of No. 1-13 is actually increased and at least one is detected.
  • a true decline in miRNA levels 14-20 predicts cervical intraepithelial neoplasia or cervical cancer.
  • the levels of all miRNAs shown in Table 1 were detected, at least one true rise in miRNA levels 1-13 and a true decrease in at least one miRNA level 14-20 predicted a cervical epithelium Tumor-like lesions or cervical cancer.
  • a true rise in at least two levels of miRNAs 1-13 and a true decrease in at least two levels of 14-20 miRNAs indicate cervical intraepithelial neoplasia or cervical cancer.
  • the "majority" result is considered. For example, when 5 miRNAs are used, 3 of which predict cervical intraepithelial neoplasia or cervical cancer, the results are predicted to indicate that the individual is a cervical intraepithelial neoplasia or cervical cancer. However, in some cases, the diagnosis of cervical intraepithelial neoplasia or cervical cancer requires characteristic changes in at least one or more specific miRNAs.
  • hsa-miR-133b one of them is hsa-miR-133b, and in some cases, a true rise in hsa-miR-133b levels may be a prerequisite for the diagnosis of cervical intraepithelial neoplasia or cervical cancer. Diagnostic method based on miRNA genetic state
  • Also provided herein is a method of diagnosing cervical intraepithelial neoplasia or cervical cancer based on the genetic status of at least one of the miRNAs or their homologs shown in Table 1 in an individual sample.
  • the genetic status is assessed by analyzing the deletion or amplification of at least one miRNA gene in the sample, and if the miRNA gene is deleted or amplified relative to the control sample, the individual is predicted to have a cervical intraepithelial neoplasia. Lesion or cervical cancer.
  • Deletion or expansion of the MiRNA gene can be achieved by detecting the genetic structure or sequence in a cervical epithelial or cervical tissue sample cell of an individual suspected of having an intraepithelial neoplasia or cervical cancer, and in a control sample The gene structure or sequence is compared. Any technique suitable for detecting changes in gene structure or sequence can be used to implement current methods. For example, detection of deletion or amplification of a miRNA gene can be detected by hybridization of a subject's genomic DNA by Southern blot using a specific nucleic acid probe for the miRNA. Sequence analysis and single-strand conformation polymorphism can also be used for analysis.
  • Deletion or amplification of miRNA genes can also be performed by PCR to extend fragment detection of these genes, and then sequence or length analysis of fragments amplified from individual DNA samples by sequencing or electrophoresis is identical to the control DNA sample.
  • Deletion of the MiRNA gene can also be achieved by detecting the absence of a chromosomal marker near the miRNA gene.
  • the status of a miRNA gene in an individual cell can also be assessed by detecting the copy number of at least one gene in the sample.
  • the copy number of the miRNA gene on the autosome and sex chromosome is not two, it indicates that the individual has cervical intraepithelial neoplasia or cervical cancer.
  • Any method that can detect gene copy number can be used to implement current methods, including Southern blot and PCR amplification techniques.
  • Another method for detecting miRNA gene copy numbers in cervical epithelial or cervical tissue samples relies on the fact that many miRNAs or gene clusters are closely linked to chromosomal markers or other genes. In an individual, if the marker or gene adjacent to the miRNA gene is heterozygous, the loss of one copy number of the miRNA gene can be reflected by the loss of heterozygosity of the chromosomal marker or gene. Methods for detecting loss of heterozygosity of a chromosomal marker are known in the art.
  • control sample may be a tissue sample derived from a body that does not have a cervical intraepithelial neoplasia or a cervical cancer, or a tissue sample collected from a population.
  • the genetic status of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18 or 20 miRNAs or their homologs in Table 1 can be used for detection.
  • the "majority" result will prevail.
  • the results are considered to indicate that the individual is a cervical intraepithelial neoplasia or cervical cancer.
  • the diagnosis of cervical intraepithelial neoplasia or cervical cancer requires characteristic changes in at least one or more specific miRNA genes.
  • a variety of techniques are available for detecting the genetic status of a miRNA gene. These include, for example, allele-specific primer amplification on microarrays, PCR/LDR universal arrays, microsphere-based single-strand amplification, sequential labeling molecule inversion probes, and combinatorial hybridization sequencing.
  • the genetic status is evaluated by analyzing the deletion or amplification of the miR-133b gene in the sample.
  • the miR-133b gene is deleted or amplified relative to the control sample, it indicates that the individual is colorectal cancer, tongue scale Cell carcinoma, esophageal squamous cell carcinoma or pancreatic ductal carcinoma.
  • Another aspect of the invention provides a prognostic approach for patients with cervical intraepithelial neoplasia or cervical cancer, including survival prognosis for individuals with cervical intraepithelial neoplasia or cervical cancer.
  • the prognostic method of the present invention is useful for determining an appropriate treatment for cervical intraepithelial neoplasia or cervical cancer.
  • survival prognosis can help decide whether to adopt a more conservative or more aggressive treatment, or a combination of treatments.
  • this prognosis can help determine if it is necessary and/or effective to use a drug that helps survive (such as the drug mentioned here).
  • a cervical intraepithelial neoplasia or cervical cancer including: (a) detecting at least one of an individual's cervical intraepithelial neoplasia or cervical cancer tissue sample. The level of a miRNA; (b) comparing the miRNA level of the sample to a threshold, wherein the miRNA level relative threshold is positively or negatively correlated with the individual survival rate.
  • “positive correlation” refers to a relative threshold miRNA with low levels suggesting a low survival rate in individuals with cervical intraepithelial neoplasia or cervical cancer, and vice versa.
  • negative correlation refers to a relative threshold miRNA high level suggesting a low survival rate in individuals with cervical intraepithelial neoplasia or cervical cancer, and vice versa.
  • the miRNA gene is located on any of chromosomes 6, 18 and 20. Specifically, at least one miRNA is hsa-miR-133b.
  • herein are methods for prognosis of survival for a cervical intraepithelial neoplasia or cervical cancer, including: (a) detecting at least one of an individual's cervical intraepithelial neoplasia or cervical cancer tissue sample. The level of a miRNA; (b) comparing the miRNA level of the sample to a threshold, wherein the relative threshold of the miRNA level is inversely related to the individual survival rate, and at least one of the miRNAs is hsa-miR-133b or its corresponding homologue .
  • the miRNA levels described herein can also reflect changes in the genetic status of miRNAs (such as the miRNAs described herein). Specifically, here is a method for prognosis of survival for a cervical intraepithelial neoplasia or cervical cancer, including analysis of the genetic status of at least one miRNA gene (eg, a miRNA gene of hsa-miR-133b or its homolog), A change in genetic status relative to a control sample implies a high or low survival rate.
  • miRNAs such as the miRNAs described herein.
  • here is a method for prognosis of survival for individuals with cervical intraepithelial neoplasia or cervical cancer, including analysis of hsa-miR-133b or amplification of at least one miRNA gene corresponding to its homolog. Increasing the situation, when the miRNA gene is amplified relative to the control sample, the individual survival rate is low.
  • cervical intraepithelial neoplasia A variant of cervical cancer provides a means of survival prognosis, including analysis of hsa-miR-133b or copy number of at least one miRNA gene corresponding to its homolog, and low survival rate when the miRNA gene is more than two copies . Use probes to detect miRNA levels
  • a method of using one or more probes (or systems comprising one or more probes) for prognosis of cervical intraepithelial neoplasia or cervical cancer is provided herein, wherein the probe can be detected in the sample miRNA, and the level of miRNA is positively or negatively correlated with individual survival rate relative to threshold.
  • a method of using one or more probes for prognosis of cervical intraepithelial neoplasia or cervical cancer wherein the relative threshold of miRNA levels is inversely related to individual survival, and at least one of the miRNAs Is hsa-miR-133b or its homolog.
  • a method of producing a reagent (or system) for prognosis of a cervical intraepithelial neoplasia or cervical cancer using one or more probes wherein the probe can detect miRNA in the sample, And the level of miRNA is positively or negatively correlated with the individual survival rate relative to the threshold.
  • the survival discussed herein can be disease free survival or overall survival.
  • Disease-free survival refers to a patient who is diagnosed without tumor recurrence and/or survival under conditions of proliferation, such as a surviving patient without a recurrent tumor.
  • Total survival refers to the overall survival of a confirmed patient regardless of tumor recurrence. Threshold
  • Some of the methods and uses herein involve survival prognosis based on miRNA levels, while miRNA levels are relative threshold levels.
  • the threshold can be determined by a number of methods. It is assumed that the threshold obtained can accurately provide a miRNA level, and the survival rate of a group of patients above this level is different from the survival rate of another group of patients below this level.
  • Threshold values can be determined, for example, by non-cancerous cervical intraepithelial neoplasia or cervical cancer tissue samples. Set. Thresholds can also be determined by analyzing miRNA levels in a population of cervical intraepithelial neoplasia or cervical cancer patients. This can be done, for example, by a histogram analysis that includes all individuals of the population being tested, with one axis representing the miRNA level and the other axis representing the individual survival rate. A population is divided into two or more independent groups based on different miRNA levels. It is then determined that the miRNA level thresholds for these populations are best distinguished.
  • the threshold may be based on an average of miRNA levels with a high survival population and an average of miRNA levels with a low survival population.
  • the threshold can also represent two or more miRNA levels.
  • the levels of two or more miRNAs can be expressed by the ratio of each miRNA level.
  • the threshold may be a value suitable for each individual intraepithelial neoplasia or cervical cancer, or may be set to a different value depending on the particular population. For example, the threshold for older women can be different from that for young women. Further, a threshold can also be set for each individual. For example, the threshold can be the ratio of a miRNA level in a cervical intraepithelial neoplasia or cervical cancer tissue to that of a non-cancerous tissue in the same individual.
  • the threshold level can be verified using univariate or multivariate analysis. These methods can determine the correlation between one or more variables and the results. In certain cases, the method can determine the association between miRNA levels and disease-free survival or overall survival in cancer patients. Any of these analytical methods are known to those skilled in the art and can be analyzed using these methods.
  • An example of a univariate analysis is the Kaplan-Meir analysis or the Cox proportional hazard regression model.
  • a population sample of sufficient size can be utilized to determine a threshold, such as by a histogram analysis to divide a population into two or more groups of patients with different miRNA levels. This group contains at least
  • confirmation of the threshold obtained may also include at least 25 patients, including, for example, at least 50, 75, 100, 125, 150 or 200 patients.
  • a threshold can separate the two groups of patients. Further, multiple thresholds can differentiate patients into multiple groups. For example, two thresholds can divide patients into three groups of high, medium, and low levels of miRNA. Data with different thresholds can be used to plot a curve, such as a continuous curve, to describe the patient's disease-free or overall survival rate based on the patient's miRNA level. Based on the "continuous" miRNA levels established by this curve, the likelihood of a patient's disease-free or overall survival rate is proportional to the patient's miRNA level. This curve can represent the level of two or more miRNAs.
  • the miRNAs used in this invention for prognosis of cancer patients can be used in combination.
  • a combination of two or more miRNAs can increase the significance or credibility of the prognosis.
  • the level of MiRNA can also be used in conjunction with another indicator.
  • This indicator is statistically significant in predicting disease-free or overall survival in patients with cervical intraepithelial neoplasia or cervical cancer.
  • Such indicators include, for example, pathological indicators (e.g., age, tumor size, tumor histology, clinical stage, family history, etc.).
  • the clinical stage of cancer is a statistically significant indicator of disease-free or overall survival. Therefore, the threshold of miRNAs can be different when used as an indicator of no disease or overall survival in another cervical intraepithelial neoplasia or cervical cancer.
  • the method comprises: (a) detecting at least one miRNA level in an individual cervical intraepithelial neoplasia or cervical cancer tissue; (b) classifying a cervical intraepithelial neoplasia or cervical cancer patient based on miRNA levels .
  • Patients in the high miRNA level group had a lower survival rate than the low miRNA level group, and at least one of the miRNAs was hsa-miR-133b.
  • the patients are assigned to a cohort.
  • Individual disease-free survival or overall survival is estimated by assessing the disease-free survival or overall survival of the cohort.
  • one sample may be detected as a low level of miRNA.
  • This patient will be assigned to a low-level group of miRNAs. Because patients with low levels of miRNA are known to have a high disease-free or overall survival rate, the patient has a high disease-free or overall survival rate.
  • the methods described herein may further comprise the step of determining a suitable treatment for an individual. It is generally believed that the survival rate of cancer patients at an early stage is different from the survival rate of cancer patients at an advanced stage. For example, the prognosis of stage I may indicate continued growth and/or metastasis of the cancer, while the prognosis of stage IV may indicate the effectiveness of the cancer treatment. These factors will be considered in determining the appropriate treatment. Individual treatment drugs and treatment methods for cervical cancer or cervical intraepithelial neoplasia
  • a therapeutic agent comprising a component that reduces miRNA levels and a pharmaceutically acceptable carrier, wherein at least one miRNA is hsa-miR-133b, hsa-miR-104- 3p, hsa-miR-143*.
  • at least one miRNA is hsa-miR-133b.
  • at least one miRNA is hsa-miR-104-3p.
  • at least one miRNA is hsa-miR-143*.
  • the component is a double stranded RNA (eg, a short or small interfering RNA, or "siRNA”), an antisense nucleic acid, or an enzymatically active RNA molecule such as a ribozyme.
  • siRNA short or small interfering RNA
  • drugs to reduce specific miRNA levels such as hsa-miR-133b, hsa-miR-100, hsa-miR-104-3p, hsa-miR-143*, ⁇ plus cervical intraepithelial neoplasia Or survival rate of patients with cervical cancer.
  • RNA molecules that reduces miRNA levels can be used in the methods of the invention.
  • suitable components for inhibiting miRNA gene expression include, but are not limited to, double-stranded RNA (eg, short or small interfering RNA, or "siRNA"), antisense nucleic acids, enzymatically active RNA molecules such as ribozymes, small molecules Complex, as well as protein. These components may be used singly or in combination with other components (other components as described herein). These components can reduce miRNA levels either directly (e.g., by inhibiting miRNA expression or its function) or indirectly (e.g., acting on the genetic state of the miRNA gene).
  • a particular miRNA gene can inhibit its expression by inducing RNA interference.
  • the method comprises a double strand having at least 70%, including, for example, at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence homology to a portion of the miRNA gene product RNA
  • dsRNA molecules are carried out. Specifically, the dsRNA molecule is a short or small interfering RNA (“siRNA”).
  • siRNAs that can be effectively used in these current methods can be short double stranded RNAs of 10-30 nucleotides in length (including, for example, about 12-28, 14-26, 16-24, or 18-22 nucleosides). acid).
  • the siRNA can contain a sense RNA strand and a complementary pair of antisense RNA strands, which are double-stranded by annealing according to the Watson-Crick base complementary pairing principle.
  • the sense strand contains a nucleic acid sequence that is substantially identical to the target miRNA.
  • the sense strand and the antisense strand of the siRNA may be composed of two single-stranded RNAs that are complementary paired, or may be composed of two portions of a complementary pair of one molecule joined together by a single-stranded "hairpin" structure.
  • siRNA may differ from naturally occurring RNA by insertion, deletion, substitution and/or transformation of one or more nucleotides. These changes include the addition of non-nucleotide material (e.g., added to the end or inside of the siRNA), resulting in modification of the siRNA against ribozyme digestion, or replacement of one or more nucleotides in the siRNA with deoxynucleotides. In some cases, one or both strands of the siRNA may contain a 3' overhang.
  • the siRNA can be obtained by chemical or biological methods, or expressed from a recombinant plasmid or viral vector, which will be explained below.
  • antisense nucleotide refers to a method capable of binding a target RNA by means of RNA-RNA or RNA-DNA interaction, Thereby changing the nucleic acid molecule of the target RNA activity.
  • Antisense nucleotides suitable for use in current methods can be single-stranded nucleic acids (eg, RNA, DNA, RNA-DNA chimeras, PNA and LNA) that are complementary to sequences adjacent to the miRNA.
  • the antisense nucleic acid comprises at least 70% (eg, at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) complementary to the miRNA contiguous sequence Paired nucleic acid sequences.
  • the antisense nucleic acid is about 10-30 nucleotides in length (including, for example, about 12-28, -26.
  • Antisense nucleic acids can also include modifications to the nucleic acid backbone or sugars and bases (or their equivalents) to enhance target specificity, resistance to nuclease degradation, transport or other efficacy related features. These modifications include cholesterol family molecules, double helix inserts such as acridine, or one or more components with nuclease resistance.
  • Antisense nucleic acids can be obtained by chemical or biological methods, or expressed from recombinant plasmids or viral vectors, as will be explained below.
  • nucleic acid having enzymatic activity refers to a nucleic acid containing a substrate binding region which is complementary to a contiguous sequence of a miRNA and which specifically cleaves the miRNA.
  • the enzymatically active nucleic acid binding region is 50-100% complementary to the miRNA contiguous sequence (including, for example, 75-95% complementarity or 95-100% complementarity).
  • An enzymatically active nucleic acid can also be modified on base, sugar, and phosphate components.
  • a typical enzymatically active nucleic acid that can be used in current methods is the ribozyme.
  • Nucleic acids having enzymatic activity can be obtained by chemical or biological methods, or expressed from recombinant plasmids or viral vectors, as will be explained below.
  • nucleic acid molecules into cells, including cancer cells. These methods include microinjection, electroporation, lipofection, calcium phosphate-mediated transfection, DEAE dextran-mediated transfection, microparticle bombardment, transport by colloidal dispersion (eg, macromolecular complexes) , gel particles, water-oil emulsifiers, colloidal ions, mixed colloidal ions and liposomes), as well as with antibodies, gramicidin, artificial virus coats or other intracellular vectors such as TAT.
  • colloidal dispersion eg, macromolecular complexes
  • gel particles e.g, water-oil emulsifiers, colloidal ions, mixed colloidal ions and liposomes
  • antibodies gramicidin, artificial virus coats or other intracellular vectors such as TAT.
  • Nucleic acid agents can also be introduced into mammalian cells in vitro or in vivo by vectors known in the literature. Suitable vectors are viral vectors and non-viral vectors, such as plasmid vectors. These vectors are useful in providing therapeutically effective doses of antisense RNA or siRNA.
  • a virus-based system has the advantage of being able to introduce heterologous nucleic acids into a wide variety of cells with relative efficiency.
  • Suitable viral vectors for introducing nucleic acids are herpes simplex virus vectors, vaccinia virus Vector, cytomegalovirus vector, murine Moloney leukemia vector, adenoviral vector, adeno-associated virus vector, retroviral vector and lentivirus.
  • the tropism of viral vectors can be controlled by the use of envelope proteins or surface antigens of other viruses.
  • an adeno-associated viral vector can achieve the tropism of these viruses by surface proteins of oral vesicular virus, rabies virus, Ebola virus, Mokola and the like.
  • the nucleic acid or vector of the present invention may contain any inducible promoter or enhancer so that expression of antisense RNA or siRNA can be induced by stimulation or addition of a molecule.
  • induction systems include, for example, the tetracycline-inducing system, the heavy metal-induced metallothionein promoter, the ecdysone or related steroids such as the ketone-responsive insect steroid hormone, steroids such as glucocorticoids and estrogen-induced mouse mammary tumor virus (MMTV). ), as well as a heat shock promoter induced by temperature changes.
  • the dose of a drug is sufficient to alter the level (e.g., decrease) of its target miRNA, then this dose can be said to be an effective dose of the drug.
  • a drug can reduce the level of target miRNA to at least 10%, 20%, 30%, 40%, or 50% of the difference from the threshold.
  • Typical doses of the drugs (such as nucleic acid drugs) provided here include 0.1-3000 mg/kg body weight, 10-2000 mg/kg body weight, 50-1000 mg/kg body weight, and 100-500 mg/kg body weight, but not Limited to these ranges.
  • the dose of a drug is 100-500 mg/g tumor weight.
  • Typical dosing frequencies include, but are not limited to, at least once every three weeks, once every two weeks, once a week, twice a week, three times a week, four times a week, every Friday, every Saturday, Or once a day.
  • the interval between two administrations may be less than one week, such as less than every six, five, four, three, two, or one day.
  • the time interval between administrations is fixed. For example, it can be administered every day, every two days, every three days, every four days, every five days, every six days or every week. In some cases, it can be administered twice, three times or more per day.
  • the administration time of the drug may be long-term, such as from about one month to three years.
  • one administration can be as long as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30 or 36 months.
  • the drug cannot be discontinued during administration.
  • the interval between each dose may not be longer than one week.
  • the medicaments described herein can be administered to an individual by any route within the art, including, but not limited to, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intraalveolar, intramuscular, respiratory. Tube, subcutaneous, cerebrospinal, trans-cutaneous, trans-pleural, topical, inhalation (eg spray), transmucosal (eg through the nasal mucosa), through the gastrointestinal, intra-articular, intraurethral, intraventricular, rectal (eg by suppository), intravaginal (eg by vaginal suppository), intracranial, intrahepatic, and intratumoral. In some cases, it can be administered systemically. In some cases, it can be administered topically.
  • the drug contains a component that lowers the level of a miRNA that is one of hsa-miR-133b or its homolog. In some cases, at least one miRNA is hsa-miR-133b. In some cases, the component is an siRNA. In some cases, the component is an antisense RNA. In some cases, the component is a ribozyme.
  • the drug is sterile.
  • the drug is heat-free.
  • Suitable pharmaceutically acceptable carriers are water, aqueous solutions, standard salt solutions, 0.4% salt solutions, 0.3% glycine and hyaluronic acid.
  • the drug may also contain conventional pharmaceutical excipients and/or be added.
  • Suitable pharmaceutical excipients include stabilizers, antioxidants, osmo-regulators, buffers, pH adjusters.
  • Suitable additives include, for example, physiologically non-rejecting buffers, chelating agents (such as DTPA and DTPA bisamide), and calcium chelating complexes (such as calcium DTPA and CaNaDTPA bisamide), calcium or sodium salts (such as calcium chloride). , calcium ascorbate, calcium gluconate and calcium lactate).
  • the drug in this invention may be a liquid package or a lyophilized product.
  • a pharmaceutically acceptable conventional non-toxic solid carrier can be used.
  • Pharmaceutically acceptable solid carriers include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • This invention also provides a method of increasing the survival rate of cervical intraepithelial neoplasia or cervical cancer patients. Specifically, there is provided a method for improving the survival rate of cervical intraepithelial neoplasia or cervical cancer, including administering to the individual an effective dose of a drug that reduces miRNA levels, which is negative relative to the threshold and individual survival rate. Correlation. Specifically, drug manufacturers are provided with drugs that reduce miRNA levels, improve cervical intraepithelial neoplasia, or survival rate in patients with cervical cancer. This miRNA level is inversely related to patient survival.
  • a cervical intraepithelial neoplasia or cervical cancer comprising administering to the individual an effective amount of a drug that reduces miRNA levels, the miRNA being selected from the group consisting of hsa miRNA group of -miR-133b, hsa-miR-140-3p and their homologs.
  • the drug manufacturer is provided with a drug that increases the survival rate of cervical intraepithelial neoplasia or cervical cancer. This drug can reduce miRNA levels. miRNA group of hsa-miR-133b, hsa-miR-140-3p and their homologs.
  • the methods described herein can further comprise the step of prognosing (e.g., by the methods described herein) for individual survival rates prior to use of the drug.
  • the level of more than one miRNA is decreased.
  • ingredients that reduce the levels of two or more miRNAs can be used.
  • two or more components can be used to reduce two or more miRNA levels.
  • methods for increasing the survival rate of cervical intraepithelial neoplasia or cervical cancer are provided herein, including administering to the individual one or more effective doses that reduce the level of at least two miRNAs, These miRNAs are selected from the group of miRNAs comprising hsa-miR-133b, hsa-miR-140-3p, hsa-miR-143* and their homologs.
  • the drug manufacturer is provided with one or more drugs that increase the survival rate of cervical intraepithelial neoplasia or cervical cancer, which can reduce at least two miRNA levels selected from hsa miRNA group of -miR-133b, hsa-miR-143*, hsa-miR-140-3p and their homologs.
  • methods for increasing the survival rate of cervical intraepithelial neoplasia or cervical cancer are provided herein, including administering one or more to the individual to reduce hsa-miR-133b, hsa-miR-143*, hsa-miR-140-3p and their homologous levels of the drug.
  • drug manufacturers are provided with one or more drugs that increase the survival rate of cervical intraepithelial neoplasia or cervical cancer. These drugs can reduce hsa-miR-133b, hsa-miR-143*, The level of hsa-miR-140-3p and their homologs. Oligonucleotide primers for amplification of miRNA
  • the invention provides an oligonucleotide primer for amplifying an RNA sequence comprising a nucleotide sequence having the following characteristics: a) under high stringency conditions, with a nucleic acid sequence or Its complementary sequence hybridizes, as indicated by the sequences listed in the sequence listing; b) has at least 90% similarity to a nucleic acid sequence or its complement, such as those listed in the sequence listing.
  • Current primers may comprise any suitable nucleic acid, such as DNA, RNA, PNA or derivatives thereof. Primers containing a nucleic acid sequence shown in the sequence listing or a complementary sequence thereof are preferred. Labeled primers are also preferred, such as chemical, enzymatic, immunological, radioactive, fluorescent, luminescent, and FRET labels.
  • Oligonucleotide primers can be produced by any suitable method.
  • primers can be chemically synthesized (see Ausubel (Ed.) Current Protocols in Molecular Biology, 2.11. Synthesis and purification of oligonucleotides, John Wiley & Sons, Inc. (2000)), isolated from natural sources, recombinantly produced or combined with these methods.
  • Synthetic oligonucleotides can also be referred to the method of Matteucci et al., J. Am. Chem. Soc, 3: 3185-3191 (1981).
  • automatic synthesis may be better, for example, synthesis on a DNA synthesizer using cyanoethyl phosphoramidite chemistry. The method of chemically synthesizing primers is preferred.
  • the bases used to synthesize the oligonucleotide primers can select natural bases such as adenine, cytosine, guanine, uracil, and thymine.
  • Natural or “synthetic" bases may also be selected, such as 8-oxoguanine, 6-mercaptoguanine, 4-acetylcytosine, 5-(carboxyl-hydroxyethyl)uridine, 2' -0-methylcytidine, 5-carboxymethylamino-methyl-2-thymidine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2'-0-methyl pseudouridine, ⁇ -D-galactosylcyclopentene, 2'-methoxyguanosine, hypoxanthine, N6-isopentenyladenosine, 1-methyladenosine, 1-methyl pseudouridine, 1 -methylguanosine, 1-methylinosine, 2,2-bismethylguanosine, 2-methyladen
  • oligonucleotide analogs eg, phosphodiester-modified oligonucleotides such as methyl phosphate, phosphotriester, phosphorothioate, dithiophosphate, Or phosphoramidate.
  • Primers can be protected from degradation by "3' end capping", by which ribozyme inhibition is linked to the phosphodiester at the 3' end of the replacement oligonucleotide (Shaw et al., Nucleic Acids Res., 19:747 (1991) )).
  • the phosphoramidate, phosphorothioate and methyl phosphate have the same function.
  • a broader modification of the phosphodiester backbone can increase the stability of the oligonucleotide, promote the affinity of the oligonucleotide and the cell permeability of the oligonucleotide (Milligan et al., J. Med. Chem. , 36: 1923 (1993)).
  • Skeletal analogs include methyl phosphate, phosphotriester, phosphorothioate, dithiophosphate, phosphoramidate, boron phosphate.
  • Phosphorothioate and methyl phosphate modified oligonucleotides can be obtained by automated oligonucleotide synthesis and are therefore more popular.
  • the oligonucleotide may be a "peptide nucleic acid" as described in the following literature (Milligan et al., J. Med. Chem, 36:. 1923 0 only requirement is that a portion of oligonucleotide primer must contain a sequence, at least a portion of the RNA sequence capable of binding to a target sequence (1993)). Kit
  • This invention also provides kits for use in the various methods described herein.
  • kits comprising a system (e.g., a microarray) for detecting miRNA levels as described herein.
  • the kit may additionally include reagents for detection.
  • the kit also includes instructions for describing in detail the methods of operation mentioned herein, and/or providing a web site with such instructions.
  • kits comprising a system (e.g., a microarray) for use in the diagnosis of cervical intraepithelial neoplasia or cervical cancer as described herein. It may also include one or more control samples to determine the reference level, and/or how to obtain information on the reference level. In some cases, the kit may also include instructions for using the kit for the diagnosis of cervical intraepithelial neoplasia or cervical cancer.
  • a system e.g., a microarray
  • the kit may also include instructions for using the kit for the diagnosis of cervical intraepithelial neoplasia or cervical cancer.
  • kits comprising a classification system (e.g., a microarray) for a cervical intraepithelial neoplasia or cervical cancer patient described herein.
  • a classification system e.g., a microarray
  • one or more control samples may be included to determine individual classification, and/or information about the control sample, and in some cases, kit usage instructions for the individual classification.
  • kits comprising a system for the diagnosis of colorectal cancer, tongue squamous cell carcinoma, esophageal squamous cell carcinoma, and pancreatic ductal carcinoma described herein (eg, a microarray) ). It may also include one or more control samples to determine the reference level, and/or how to obtain information on the reference level. In some cases, the kit may also include instructions for using the kit for the diagnosis of colorectal cancer, tongue squamous cell carcinoma, esophageal squamous cell carcinoma, and pancreatic ductal carcinoma.
  • kits comprising a classification system (eg, a microarray) of a colorectal cancer, a tongue squamous cell carcinoma, an esophageal squamous cell carcinoma, and a pancreatic ductal carcinoma patient described herein.
  • a classification system eg, a microarray
  • one or more control samples may be included to determine individual classification, and/or information about the control sample, and in some cases, kit usage instructions for the individual classification.
  • kits are provided herein for prognosis of survival rates for patients with cervical intraepithelial neoplasia or cervical cancer.
  • kits include, for example, probes that detect miRNAs.
  • the kit can contain a control sample that determines the threshold, and/or information on how to obtain the threshold.
  • the instructions for using the kit for prognosis of patients are also included.
  • the kit may include an agent that reduces the level of miRNA, or a drug that contains such an agent, to help improve survival.
  • kits referred to herein may also include reagents including, but not limited to, substrates, labels, reagents, reagents for labeling miRNA, reagents for isolating miRNA, hybridization and detection of negative or positive controls, Tubes and/or other accessories, reagents for collecting tissue samples, buffers, hybrid cassettes, coverslips, etc., and possibly software packages (eg analysis of miRNA levels and/or miRNA level characteristics using the statistical methods mentioned here) sexual changes), and any password and/or username used to obtain database information.
  • reagents including, but not limited to, substrates, labels, reagents, reagents for labeling miRNA, reagents for isolating miRNA, hybridization and detection of negative or positive controls, Tubes and/or other accessories, reagents for collecting tissue samples, buffers, hybrid cassettes, coverslips, etc., and possibly software packages (eg analysis of miRNA levels and/or miRNA level characteristics using the statistical methods mentioned here) sexual changes), and any password and
  • kits can include a drug that reduces a level of a miRNA, and instructions for using it to improve the survival rate of a cervical intraepithelial neoplasia or cervical cancer patient.
  • the kit may also include one or more carriers or other agents for delivering the drug complex.
  • the kit also includes instructions for use of the drug complex.
  • the MiRNA microarray includes 509 mature miRNA sequences, including 435 human mature miRNAs (including the reported 122 predicted miRNA sequences (Xie et al) (http:// ⁇ microma.sanger.ac.uk). , 2005)), 196 rat mature miRNAs, 261 mouse mature miRNAs.
  • each probe sequence was designed to be approximately 40 nt oligonucleotides (the 3' end is a miRNA sequence and the 5' end is 19 poly polyT, 5' terminal C6 amino modification). Oligonucleotide probes were synthesized at MWG Biotech and dissolved in EasyArrayTM spotting solution (Capital Bio Corp.) at a concentration of 40 ⁇ M. Using SmartArrayTM microarrayer (CapitalBio Corp.) point-to-point chips, each probe has three repeat points.
  • RNA ligase 2 units of T4 RNA ligase (New England Biolabs, Beijing, China) used 4 g of small molecular weight RNA with 500 ng of 5'-phosphate-cytosine-uracil-cy3-3, (Dharmacon; Lafayette, CO ) Mark.
  • the labeling reaction was carried out at 16 ° C for 4 hours.
  • the labeled RNA was precipitated with 0.3 M sodium acetate and 2.5 volumes of ethanol, washed with ethanol, and air-dried, and the labeled RNA was resuspended in 15 ⁇ of a hybridization solution containing 3 x SSC, 0.2% SDS and 15% formaldehyde.
  • RNA force B was applied to the miRNA chip and covered with a LifterSlipTM (Erie; Portsmouth, H) coverslip.
  • Hybridization was carried out in a hybridization cassette placed in a three-stage tilting mixer BioMixeTM (Capital Bio Corp.) to distribute the hybridization solution evenly across the entire surface of the slide to avoid edge effects. The efficiency of this procedure is demonstrated on our mRNA expression profile platform.
  • Hybridization overnight at 50 °C.
  • the microarray chip was then washed twice in succession: the first time in a 0.2% SDS, 2xSSC cleaning solution for 5 minutes at 50 °C, and the second time in a 0.2% SSC cleaning solution for 5 minutes at room temperature.
  • the array was then scanned with the confocal scanner LuxScanTM and the resulting image was soft with L U xS Can TM 3.0 TM Analysis (all from CapitalBio Corp.).
  • the average of each miRNA repeat point was background corrected, corrected, and further analyzed.
  • the method of calibration is to use the median value of each chip for correction.
  • the chip data is then filtered to remove genes with signals below 500 in all samples, and then through microarray significance analysis software.
  • RT-PCR distinguishes hsa-miR-133a and hsa-miR-133b expression in cervical cancer
  • hsa-miR-133a and hsa-miR-133b are two miRNAs whose expression levels are significantly increased in cervical cancer tissues.
  • the mature hsa-miR-133a and hsa-miR-133b sequences differ only by one nucleotide at the 3'-end, as shown in Table 1. This difference is indistinguishable when using microarray chips for miRNA expression profiling.
  • the hsa-miR-133a precursor differs greatly from the hsa-miR-133b precursor (pre-hsa-miR-133b) sequence, based on the sequence of the two precursors.
  • Design-specific RT-PCR primers differentiate the expression of h S a-miR-133a and hsa-miR-133b in cells or tissues. Sequence information on pre-hsa-miR-133a and pre-hsa-miR-133b can be found at http: ⁇ miRNA. sanger. ac.uk/ (Griffths- Jones, et al, Nucleic Acids Research, 2006, Vol. 34, Database issue).
  • RNA extraction from 6 normal cervix and 6 cervical cancer FFPE tissues was as described in Case 1.
  • Reverse transcription reaction containing 10 ng/ ⁇ total RNA, 25 nM reverse transcription primer, 1 x reverse transcription buffer, 0.25 mM dNTP, 7.5 U ThermoScriptTM reverse transcriptase and 0.25 U/ml RNase inhibitor (Invitrogen, Carlsbad, CA ).
  • the 20 ⁇ reaction was incubated in a MJ Research PTC-225 Thermocycler for 30 min at 60 °C, 5 min at 85 °C, and then stopped at 4 °C.
  • the 50 ⁇ MiRNA precursor amplification reaction system includes 200 nM dNTP, 1 PCR buffer, 15 nM forward primer and 15 nM reverse primer, 2 ⁇ reverse transcription product, 1.25 U HotStar® Taq DNA polymerase (Qiagen). The reaction conditions were incubated at 95 ° C for 10 min, followed by 40 cycles of incubation at 95 ° C for 15 s and incubation at 70 ° C for 20 s. The amplified products after the reaction were each subjected to 5 ⁇ for 1.5% agarose electrophoresis, and the results are shown in Fig. 2 .
  • RNA extraction from FFPE tissue is as described in Case 1.
  • the reverse transcription reaction kit was purchased from Exiqon.
  • the 10 ⁇ reaction system contained 10 ng total RNA, 2 ⁇ reverse transcription primer, 1 RT buffer, 0.2 mM dNTP, 0.5 ⁇ reverse transcriptase and 0.5 ⁇ RNase inhibitor.
  • the reaction system was incubated at 50 °C for 30 min in MJ Research PTC-225 Thermocycler, incubated at 85 °C for 5 min, and then stopped at 4 °C. All reverse transcription reactions, including no template control, were repeated twice.
  • Real-time fluorescent PCR was performed using the miRCURY LNATM microRNA PCR System kit (Exiqon, Vedbaek, Denmark) and the LightCycler instrument (Roche Diagnostics, Mannheim, Germany).
  • the 20 ⁇ PCR system consisted of 4 ⁇ l of 10-fold diluted reverse transcription product, 10 ⁇ SYBR® Green master mix, 1 ⁇ LNTM PCR primer and 1 ⁇ Universal PCR primer. The reaction conditions were incubated at 95 ° C for 10 min, followed by 60 cycles of incubation at 95 ° C for 10 s and incubation at 60 ° C for 20 s.
  • the hybrid reaction solution contained 50 nM miRCURY LNATM miR-133b probe or Scramble-miR probe (Exiqon) and reacted overnight at 45 °C.
  • the experimental results are shown in Figure 4. H&E staining and Ki-67 immunohistochemical results clearly showed normal epithelium, intraepithelial neoplasia stage III and invasive carcinoma tissues in cervical tissues.
  • SCID Severe combined immunodeficiency
  • the miR-133b gene in ex vivo human genomic DNA was amplified using primer 5' CTGACAGGATCCGTAAGAGGACATTCTGGACAAGGCAAGC 3' and 5' CGCACGAATTCATTCCTGGGAGCATAAGAATATGGTGAAA 3 '.
  • the amplified product was digested with BamHI and EcoRI and cloned into pcDNA 3.1 -neomycin vector (Invitrogen, Carlsbad, CA). Recombinant plasmid tested Pre-verification.
  • the recombinant plasmid containing the miR-133b gene and the empty vector plasmid (negative control) were transfected with CaSki (purchased from the Basic Medical Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), and the stable cell strain was screened with 800 ⁇ ⁇ / ⁇ 1 G418. Overexpression of the miR-133b gene in stable cell lines was verified by real-time fluorescent PCR.
  • a miR-133b expression plasmid containing an anti- puromycin gene was constructed.
  • the construction process was as follows: The first step was to digest the plasmid pcDNA3.1-neomycin and the plasmid containing the miR-133b gene in Case 5 using Bgl II and Pvu II enzymes. The digested product was subjected to 1% agarose electrophoresis, and the small fragment DNA was purified by gelatinization. In the second step, the pSIREN-RetroQ vector (Clontech, Mountain View, CA) was digested with EcoR I, and the digested product was treated with T4 DNA polymerase to make it blunted.
  • the vector was then digested with Bgl II, and the digested product was subjected to 1% agarose electrophoresis, and the large fragment was purified by gelatinization.
  • the third step uses the T4 DNA linker to link the small fragment DNA of the first step with the large fragment DNA of the second step.
  • the recombinant plasmid was screened after transformation into E. coli and verified by sequencing.
  • Recombinant plasmid containing miR-133b gene and empty vector plasmid (negative control) were transfected into SiHa cells (purchased from the Cell Resource Center of Shanghai Institute of Life Sciences, Chinese Academy of Sciences), and then screened for stable cell lines with 5 ⁇ ⁇ / ⁇ 1 puromycin. .
  • Overexpression of the miR-133b gene in stable cell lines was verified by real-time fluorescent PCR.
  • SiHa-miR-133b SiHa cells stably expressing hsa-miR-133b or negative control SiHa cells (SiHa-NC) were injected into SCID mice through the tail vein. Seven cells were inoculated into each cell. The inoculated SCID mice were housed in a constant temperature (25 °C ⁇ 2 °C), constant humidity (45% ⁇ 50%), sterile purification barrier system. During the experiment, SiHa-miR-133b inoculated mice died two. After 60 days, all rats were sacrificed, the lungs were removed, and the number of metastases on the lung surface was counted after fixation with Bouin's solution. The test results are shown in Figure 6.
  • SiHa cells with high expression of hsa-miR-133b formed more metastatic tumors in the lungs of SCID mice than negative control SiHa cells, indicating that hsa-miR-133b has a role in promoting metastasis formation.
  • Fatica A., Rosa, A., Fazi, R, Ballarino, M., Morlando, M., De Angelis,

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Description

说 明 书 用于宮颈上皮內瘤样病变和宮致癌的诊断和预后的方法与組合物
技术领域
[0001] 本项申请涉及根据病人的 microRNA水平来进行疾病 （例如宫颈上 皮内瘤样病变 ( cervical intraepithelial neoplasa, CIN)和 颈癌 ( cervical cancer) ) 诊断、 预后、 指导临床处理和改善生存率的系统与方法。
[0002] 在世界范围内， 宫颈癌是女性第二大肿瘤， 每年有近 50万新发病 例 （Parkin et al. 2005；)。 2002年， 宫颈癌导致了大约 27.4万人死亡， 是年轻妇 女因患癌症而死亡的主要因素 （zur Hausen, 2002)。 宫颈癌通常起源于持续感染 高危型人乳头瘤病毒 （human papilloma virus , HPV ) 而导致的细胞转化 (Scheffner et al, 1990)。 几乎所有的鳞状细胞癌和大部分复层上皮腺癌都是 HPV 阳性。 虽然 HPV可以通过破坏肿瘤抑制的众多信号通路导致癌症， 但是单独这 一个因素并不足以导致癌变 （Burk, 1999)。要发展到恶性表型还需要其他的宿主 因素。
[0003] 宫颈癌前病变也称为宫颈上皮内瘤样病变。 宫颈上皮内瘤样病变有 两个不同的分类系统： SIL ( squamous intraepithelial lesion)系统和 CIN (cervical intraepithelial neoplasia)系统。 虽然这两个系统描述的内容相同， 但是它们之间 也存在重要的不同。 SIL系统通常是通过巴氏涂片法关注单个细胞的病变， 然后 将这些细胞的病变程度进行分类。根据 SIL系统，宫颈病变分为 AGUS或 AGCUS ( atypical glandular cells of undetermined significance )、 LSIL (low grade squamous intraepithelial lesion) 禾口 HSIL (high grade squamous intraepithelial lesion) CIN 系统对宫颈病变的分类既依赖于单个细胞病变的程度也依赖于病变在宫颈上皮 层内发展的程度。根据 CIN系统，宫颈病变分为 CIN1 (对应于轻度病变或 LSIL) CIN2 (对应于中度病变或 HSIL) 和 CIN3 (对应于重度病变或 HSIL)。 大部分 CIN1经过一段时间后会回复到正常状态， 但是大约有 11%发展成 CIN3。 只有 非常小的一部分 CIN1 发展成宫颈癌。 CIN2大约有 43%能够回复正常， 20%发 展成 CIN3。 虽然部分 CIN3能够自然恢复， 但是这种程度的病变通常都做临床 处理，因为其下一个阶段就是癌症。 CIN3有时候也称为原位癌（carcinoma in situ, CIS)。
[0004] 广泛用于筛查宫颈癌和宫颈上皮内瘤样病变的方法有三个： 细胞学 筛查、应用醋酸肉眼观察和 HPV检测。 目前还没有办法区分发展中的 CIN和正 在回复的 CIN。 在筛查阳性的妇女中， 过度治疗很常见。
[0005] MicroRNA (miRNA)是一类小的非编码的单链调控型 RNA， 它们 通过部分配对与靶标 mRNA分子的 3'非翻译区（3'-UTR)发生相互作用 （Yekta et al., 2004), 它们作为控制元件参与基因表达的调控网络 (Fatica et al., 2006)。 生 物信息学分析预测一个 miRNA可以调节几百个靶基因，全面并且精细地调控大 量的细胞信号通路 （Hwang and Mendell, 2006; Lewis et al., 2005)。
[0006] miRNA表达水平的改变与各种癌症发生和发展相关， 它们既可以 是癌基因也可以是抑癌基因 （Chen, 2005)。 已有的研究表明大约有 50%的 miRNA基因位于癌症关联的基因组区域， 此类区域被称为 "脆性位点" （Calin et al., 2004)。此外， 大量的功能研究表明不同的 miRNA在癌症发展的不同阶段 ——从肿瘤发生 (He et al., 2007; Voorhoeve et al., 2006)到侵润与迁移 (； Budhu et al.，
2008; Ma et al, 2007)——有截然不同的作用。 MiRNA在宫颈癌中的表达异常已 经有报道 (Lee et al., 2008; Wang et al., 2008), 但是这些表达异常的 miRNA在宫 颈癌中形成中的作用还不清楚。
[0007] MiR-133 表达异常在很多其他疾病中也有报道， 包括结肠直肠癌 (Bandres et al, 2006), 舌头鳞状细胞癌 (Wong et al., 2008)， 食管鳞状细胞癌 (Guo et al., 2008)和胰腺导管癌 (Szafranska et al., 2007)。
[0008] 在此涉及到的所有公开的出版物、 专利、 专利申请和公开的专利申 请都以参考文献的方式列出。 发明摘要
[0009] 此发明提供了一个检测 microRNA (miRNA) 或其前体表达水平的 系统， 包括大量探针， 其中至少 50%可以检测任意一个具有序列号 1-20号所列 核酸序列的 miRNA或它们的同源体。 此发明还提供了基于 miRNA表达水平或 相应的 miRNA基因的遗传状态诊断癌症和预后的方法，特别是对宫颈上皮内瘤 样病变和宫颈癌的诊断和预后。 此发明进一步提供了针对宫颈上皮内瘤样病变 和宫颈癌的药物和治疗方法， 包含改变至少一个具有序列号 1-20号所列核酸序 列的 miRNA或它们的同源体的表达水平的成分， 以及药学上可以接受的载体。
[0010] 因此， 一方面， 此发明提供了一个检测 miRNA表达水平的系统， 包括大量探针， 其中至少 50%可以检测一个具有序列号 1-20号所列核酸序列的 miRNA或它们的同源体。 某一情况下， 至少 50%的探针可以检测至少 5个具 有序列号 1-20号所列核酸序列的 miRNA或它们的同源体。 另一情况下， 至少 50%的探针可以检测至少 10个具有序列号 1-20号所列核酸序列的 miRNA或它 们的同源体。 再另一情况下， 至少 50%的探针可以检测一个具有序列号 1-13号 所列核酸序列的 miRNA或它们的同源体和一个具有序列号 14-20号所列核酸序 列的 miRNA或它们的同源体。还有一种情况， 至少 50%可以检测所有具有序列 号 1-20号所列核酸序列的 miRNA或它们的同源体。
[0011] 在某些情况下， 可以检测至少一个 （包括例如至少 2， 3， 5， 10， 13其中一个） 具有序列号 1-13号所列核酸序列的 miRNA或它们的同源体的表 达水平。 在某些情况下， 可以检测至少一个 （包括例如至少 2， 3， 5， 7其中一 个） 具有序列号 14-20号所列核酸序列的 miRNA或它们的同源体的表达水平。 在某些情况下， 可以检测至少一个 （包括例如至少 2， 3， 5， 10， 13其中一个） 具有序列号 1-13号所列核酸序列的 miRNA或它们的同源体的表达水平和至少 一个 （包括例如至少 2， 3， 5， 7其中一个） 具有序列号 14-20号所列核酸序列 的 miRNA或它们的同源体的表达水平。
[0012] 在某些情况下，至少 15% (包括例如至少 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%或 95%) 的探针可以检测一个具有序列号 1-20号所列核酸序列 的 miRNA或它们的同源体。 在某些情况下， 此系统包含至少一个（包括例如至 少 2, 5, 10, 15, 20, 25, 30, 35和 40)探针可以检测一个具有序列号 1-20号所列核 酸序列的 miRNA或它们的同源体。
[0013] 在某些情况下， 至少一个 miRNA是 has-miR-133b或它的同源体。 在某些情况下， miRNA 包含 hsa-miR-133a, hsa-miR-133b, hsa-miR-140-3p, hsa-miR-143*, hsa-miR-145, hsa-miR-223, hsa-miR-99b, hsa-miR-221, hsa-miR-320a, hsa-miR-100, hsa-miR-199a-5p, hsa-miR-127-3p, hsa-miR-214或 它们的同源体。 在某些情况下， miRNA 包含 hsa-miR-203, hsa-miR-190, hsa-miR-200b, hsa-miR-200c, hsa-miR-200a, hsa-miR-31, hsa-miR-141或它们的同 源体。
[0014] 具体地讲， 探针可为 20条， 20条探针的核酸序列可以是如下 a) 或 b): a) 序列表中序列 1至序列 20所示的 20条核酸的完全互补序列； b) 在 a) 所述的 20条核酸的 5'端连接有 10-30个 T， 具体可以是 19个1\
[0015] 另一方面， 此发明提供了检测宫颈癌或宫颈上皮内瘤样病变样本的 方法， 包括： a) 使用包含大量探针的系统检测样本中的 miRNA表达水平， 这 些探针中至少 50%可以检测一个具有序列号 1-20号所列核酸序列的 miRNA或 它们的同源体； b) 将 miRNA表达水平与参考水平进行比对； c) 如果样本的 miRNA表达水平表现出特征性变化， 则对样本是癌症还是上皮内瘤样病变进行 分类。
[0016] 在某些情况下， miRNA表达水平的特征性变化包含至少一个具有 序列号 1-13号所列核酸序列的 miRNA或它们的同源体表达水平显著上升。 在 另一情况下， miRNA表达水平的特征性变化包含 has-miR-133b或它的同源体表 达水平显著上升。 还有一种情况， miRNA表达水平的特征性变化包含至少一个 具有序列号 14-20 号所列核酸序列的 miRNA或它们的同源体表达水平显著下 降。
[0017] 更进一步， 还有一种情况， miRNA表达水平的特征性变化包含至 少一个具有序列号 1-13号所列核酸序列的 miRNA或它们的同源体表达水平真 实上升， 以及至少一个具有序列号 14-20号所列核酸序列的 miRNA或它们的同 源体表达水平真实下降。 另一种情况， miRNA表达水平的特征性变化包含至少 三个具有序列号 1-13号所列核酸序列的 miRNA或它们的同源体表达水平真实 上升， 以及至少三个具有序列号 14-20号所列核酸序列的 miRNA或它们的同源 体表达水平真实下降。 还有一种情况， miRNA表达水平的特征性变化包含至少 五个具有序列号 1-13号所列核酸序列的 miRNA或它们的同源体表达水平真实 上升， 以及至少五个具有序列号 14-20号所列核酸序列的 miRNA或它们的同源 体表达水平真实下降。 在某些情况下， miRNA表达水平的特征性变化包含所有 具有序列号 1-13号所列核酸序列的 miRNA或它们的同源体表达水平真实上升， 以及所有具有序列号 14-20号所列核酸序列的 miRNA或它们的同源体表达水平 真实下降。
[0018] 这里还提供了检测一个样本是否是宫颈癌或宫颈上皮内瘤样病变 的方法，该方法包括使用包含大量探针的系统检测样本中至少一个 miRNA的遗 传状态， 这些探针中至少 50%可以检测一个具有序列号 1-20号所列核酸序列的 miRNA或它们的同源体， 其中 miRNA遗传状态的特征性变化暗示该样本是癌 或者上皮内瘤样病变。 在某种情况下， miRNA遗传状态的特征性变化包括至少 —个具有序列号 1-13号所列核酸序列的 miRNA或它们的同源体的扩增。 在另 一种情况下， miRNA遗传状态的特征性变化包括 has-miR-133b或它的同源体的 扩增。 还有一种情况下， miRNA遗传状态的特征性变化包括至少一个具有序列 号 14-20号所列核酸序列的 miRNA或它们的同源体的缺失。
[0019] 更进一步， 还有一种情况， miRNA遗传状态的特征性变化包括至 少一个具有序列号 1-13号所列核酸序列的 miRNA或它们的同源体的扩增， 和 至少一个具有序列号 14-20号所列核酸序列的 miRNA或它们的同源体的缺失。 另一种情况下， miRNA遗传状态的特征性变化包括至少三个具有序列号 1-13号 所列核酸序列的 miRNA或它们的同源体的扩增， 和至少三个具有序列号 14-20 号所列核酸序列的 miRNA或它们的同源体的缺失。 还有一种情况下， miRNA 遗传状态的特征性变化包括至少五个具有序列号 1-13号所列核酸序列的 miRNA 或它们的同源体的扩增，和至少五个具有序列号 14-20号所列核酸序列的 miRNA 或它们的同源体的缺失。
[0020] 另一方面， 此发明提供了宫颈癌或宫颈上皮内瘤样病变的诊断方 法， 该方法包括检测个体样本的 miRNA表达水平是否有特征性变化， 包括： a) 使用包含大量探针的系统检测样本中的 miRNA表达水平，这些探针中至少 50% 可以检测一个具有序列号 1-20号所列核酸序列的 miRNA或它们的同源体； b) 将 miRNA表达水平与参考水平进行比对； c)如果样本的 miRNA表达水平表现 出特征性变化， 则对样本是癌症或上皮内瘤样病变作出判断。
[0021] 这里进一步提供了一个宫颈癌或宫颈上皮内瘤样病变的诊断方法， 该方法包括检测个体样本的 miRNA的基因状态。该方法包括使用包含大量探针 的系统检测样本中至少一个 miRNA的基因状态，这些探针中至少 50%可以检测 一个具有序列号 1-20号所列核酸序列的 miRNA或它们的同源体， 其中 miRNA 基因状态的特征性变化暗示该样本是癌或者上皮内瘤样病变。
[0022] 另一方面， 该发明提供了一个为宫颈癌或宫颈上皮内瘤样病变患者 预后的方法， 该方法包括： a) 使用包含大量探针的系统检测样本中的 miRNA 表达水平， 这些探针中至少 50%可以检测一个具有序列号 1-20号所列核酸序列 的 miRNA或它们的同源体； b)将 miRNA表达水平与参考水平进行比对， miRNA 的表达水平的特征性变化暗示着个体的高或者低存活率。 在某些情况下， 该方 法还包含了针对该个体的适当的治疗方法。
[0023] 这里还提供了一个为宫颈癌或宫颈上皮内瘤样病变患者预后的方 法，该方法包括使用包含大量探针的系统检测样本中的至少一个 miRNA的基因 状态， 这些探针中至少 50%可以检测一个具有序列号 1-20 号所列核酸序列的 miRNA或它们的同源体， miRNA基因状态的特征性变化暗示着该个体的高或低 存活率。 在某些情况下， 该方法还包含了针对该个体的适当的治疗方法。
[0024] 这里还提供了一个为宫颈上皮内瘤样病变和 /或宫颈癌病人分类的 方法，该方法基于例如使用包含大量探针的系统检测 miRNA或其同源体的表达 水平， 这些探针中至少 50%可以检测一个具有序列号 1-20 号所列核酸序列的 miRNA或它们的同源体。
[0025] 这里进一步提供了确定个体宫颈上皮内瘤样病变和 /或宫颈癌分化 水平的方法， 包括使用包含大量探针的系统检测 miRNA或其同源体的表达水 平， 这些探针中至少 50%可以检测一个具有序列号 1-20 号所列核酸序列的 miRNA或它们的同源体， 在此处 miRNA的表达水平被用来作为确定该个体的 宫颈上皮内瘤样病变和 /或宫颈癌分化水平的基础。
[0026] 另一方面， 此发明为结肠直肠癌、 舌头鳞状细胞癌、 食管鳞状细胞 癌和胰腺导管癌的诊断提供了一个方法。 该方法包括检测个体样本中 miR-133b 表达水平是否有特征性变化， 检测方法包括： a) 检测样本中 miR-133b表达的 水平； b) 将样本 miR-133b的表达水平与参考水平进行比较； c) 如果样本中该 miRNA水平表现出特征性变化， 则确定该样本为癌症。
[0027] 这里还提供了结肠直肠癌、 舌头鳞状细胞癌、 食管鳞状细胞癌和胰 腺导管癌的诊断方法。 该方法包括检测个体样本中 miR-133b的基因状态， 如果 样本中该 miRNA的基因状态表现出特征性变化， 则暗示该样本是癌症。
[0028] 还有一方面， 该发明还为宫颈癌或宫颈上皮内瘤样病变个体提供了 治疗药物， 包含可以降低至少一个具有序列号 1-13 号所列核酸序列的 miRNA 或它们的同源体的表达水平的成分， 以及药学上可以接受的载体。 在某种情况 下，该药物还包括可以提高至少一个具有序列号 14-20号所列核酸序列的 miRNA 或它们的同源体的表达水平的成分。
[0029] 这里还为宫颈癌或宫颈上皮内瘤样病变个体提供了一个治疗药物， 包括可以提高至少一个具有序列号 14-20号所列核酸序列的 miRNA或它们的同 源体的表达水平的成分， 以及药学上可以接受的载体。
[0030] 这里还为宫颈癌或宫颈上皮内瘤样病变个体提供了治疗方案， 使用 包含可以降低至少一个具有序列号 1-13号所列核酸序列的 miRNA或它们的同 源体的表达水平的成分， 以及药学上可以接受的载体。
[0031] 这里还为宫颈癌或宫颈上皮内瘤样病变个体提供了治疗方案， 使用 包含可以提高至少一个具有序列号 14-20号所列核酸序列的 miRNA或它们的同 源体的表达水平的成分， 以及药学上可以接受的载体。
[0032] 另一方面， 该发明提供了扩增一个 RNA序列的一个寡核苷酸引物， 它包含了一个具有如下特点的核苷酸序列： a) 在高严谨条件下， 与一个核酸序 列或它的互补序列杂交， 如序列表所列序列； b) 与一个核酸序列或它的互补 序列， 如序列表所列序列， 具有至少 90%的相似性。 在某种情况下， 引物包含 了序列表所示的一个核酸序列或其互补序列。 另一情况下， 引物包括 DNA、
RNA, PNA或其衍生物。 另一种情况， 引物可以是标记的。 还有一种情况， 标 记物属于以下几类： 化学的、 酶学的、 免疫原的、 放射性的、 荧光的、 化学发 光光的以及 FRET (荧光共振能量转移） 标记。
[0033] 该发明还为此处描述的方法提供试剂盒。 图表说明
[0034] 图 1 提供的是利用微阵列显著性分析软件 （Significance Analysis of Microarrays, SAMs) 分析得到的差异表达 miRNA聚类图。 宫颈癌组织 （T1-T5 ) 和正常组织（N1-N5 ) 间有 20个差异表达的 miRNA。 每一列是一个样本， 每一 行是一个 miRNA。
[0035] 图 2 提供的是利用宫颈癌组织 cDNA 扩增的前体 miR-133b (pre-miR-133b)和前体 miR-133a的琼脂糖电泳图。 M: 20 bp DNA Ladder Marker (宝生物工程 （大连） 有限公司产品）； N: 阴性对照， 使用不含 hsa-miR-133b 基因的质粒作为扩增反应模板； P: 阳性对照， 使用含有 hsa-miR-133b基因的质 粒作为扩增反应模板； 1-5: 5例宫颈癌组织 cDNA作为扩增反应模板。 [0036] 图 3提供的是利用定量 RT-PCR验证 hsa-miR-133b在宫颈癌发生和 发展的不同阶段的表达水平；图中每个点代表一个宫颈组织样品中 hsa-miR-133b 的表达量； CIN 2: 宫颈上皮内瘤样病变 II期； CIN 3 : 宫颈上皮内瘤样病变 III。
[0037] 图 4展示的是用原位杂交验证 hsa-miR-133b在宫颈癌发生和发展的 不同阶段的表达。 H&E: 苏木精和伊红染色； Ki-67: Ki-67抗体免疫组化染色；
Hsa-miR-133b探针：地高辛标记的 hsa-miR-133b检测探针； Scramble-miR探针： 地高辛标记的随机序列探针（阴性对照）。地高辛标记的 hsa-miR-133b检测探针 和地高辛标记的随机序列探针均购自丹麦 Exiqon公司 （www.exiqon.com)。
[0038] 图 5 展示的是稳定表达 hsa-miR-133b 的 CaSki 细胞 (CaSki-miR-133b) 和阴性对照 CaSki 细胞 （CaSki-NC) 在严重联合免疫缺陷 小鼠 （SCID鼠） 皮下形成肿瘤的生长曲线图。
图 6展示的是稳定表达 hsa-miR-133b的 SiHa细胞 （SiHa-miR-133b) 和阴 性对照 SiHai细胞（SiHa-NC)注入到 SCID鼠体内 60天后在肺表面形成的转移 瘤数目统计结果。 发明的详细描述
[0039] 该发明部分基于 miRNA表达谱的研究， 该研究使用的成对组织样 本中包括 6例正常的宫颈组织， 11例 CIN2宫颈组织， 9例宫颈原位癌组织， 11 例侵润癌组织。对比宫颈癌组织样本与正常宫颈组织样本的 miRNA表达谱， 发 现了 20个相对于正常组织在癌组织样本中上调或下调的 miRNA。 利用实时荧 光 PCR和原位杂交确证了 has-miR-133b在正常宫颈组织， 宫颈上皮内瘤样病变 组织和宫颈癌组织中的表达水平。 细胞学与动物学实验结果表明 has-miR-133b 促进宫颈癌的形成和转移。
[0040] 据此， 一方面， 此发明为检测 miRNA的表达水平或其基因的遗传 状态提供了一个系统。 还提供了扩增 miRNA的寡核苷酸引物。
[0041] 另一方面，此发明为癌症患者尤其是宫颈上皮内瘤样病变和 /或宫颈 癌患者提供了基于 miRNA表达水平或其基因的遗传状态进行分类和预后的方 法。
[0042] 还有一个方面， 此发明提供了含有改变 miRNA表达水平成分的药 物和治疗方法。 定义
[0043] 除非另外注明， 此处使用的所有技术和科学术语均与本发明所属领 域内通常的技术人员所理解的意思一样。此处涉及到的所有专利、专利申请（公 布与未公布的） 以及其他发表物都在参考文献中全部列出。 如果在该部分使用 的定义与此处参考文献引用的专利、 申请、 公布的申请和其他发表物相反或不 一致， 则以此处列出的定义为准。
[0044] 除非另外注明， 此处使用的 "一个"、 "某个"可以表示单数或复数 (如可以表示一个或多个）。
[0045] 此处的一个 "个体"指的是脊椎动物、 哺乳动物或者人类。 哺乳动 物包括， 但不局限于， 畜牧动物、 运动动物、 宠物、 灵长类、 小鼠和大鼠。 在 某些情况下， 个体指的是人类。 在某些情况下， 个体是研究宫颈癌的动物模型。 可以理解， 当个体不是指人类时， miRNA指的是人类 miRNA相应的同源体或 直系同源体。
[0046] 此处的 "宫颈组织样本"指的是来源于宫颈的组织样本。 在某些情 况下， 组织样本是新鲜的。 在某些情况下， 组织样本是冷冻的。 在某些情况下， 组织样本是保存的。 在某些情况下， 组织样本是福尔马林保存的。 在某些情况 下， 组织样本是石蜡包埋的。 如下所述， 根据所使用的方法， 组织可以全部使 用， 或者使用该领域已知的各种方法将组织分离成小片、 细胞团或者单个细胞。
[0047] 宫颈癌包括， 但不局限于， 宫颈鳞状细胞癌或宫颈腺癌。
[0048] 这里使用的 "同源体"指的是通过对天然的核酸进行较小的修饰得 到的与天然核酸 （如 "原型"或 "野生型"核酸） 不同的核酸， 但是它保持了 与天然核酸相同的基本核苷酸构造。 这些改变包括， 但不局限于， 一个或多个 核苷酸的改变， 包括缺失、 插入和 /或替换。 与天然核酸相比较， 一个同源体可 以具有增强的、 减弱的或者与其基本相似的特征。 同源体核酸可以和天然核酸 互补或配对。 同源体可以通过该领域内已知的生产核酸的技术产生， 包括但不 局限于： DNA重组技术、 化学合成等等。
[0049] 此处使用的 "互补或配对"指的是两个核酸序列至少具有 50%相同 的序列。 更合适的是两个核酸序列至少具有 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% 或 100%相同的序列。 "互补或配对"还表示两个核酸序列在低、 中和 /或高严谨条件下可以杂交。
[0050] 此处使用的 "显著互补或显著配对" 指的是两个核酸序列至少具 有 90%相同的序列。 更合适的是两个核酸序列至少具有 95%, 96%, 97%, 98%, 99%或 100%相同的序列。 另外， "高度互补或高度配对"还表示两个核酸序列 在高严谨条件下可以杂交。
[0051] 总体而言， 影响杂交稳定性的是离子浓度和温度。 典型的情况是， 杂交反应在低严谨性条件下进行， 然后在不同但是更高严谨性的条件下清洗。 中等严谨性杂交反应指的是允许核酸分子， 如探针， 结合互补核酸分子的条件。 杂交的核酸分子通常具有至少 60%的相似性， 包括至少 70%, 75%, 80%, 85%, 90%, 或 95%相似性。 中等严谨性杂交条件等同于 50%甲酰胺、 5 x Denhardt's 溶液、 5 SSPE、 0.2%SDS, 42 °C反应， 然后在 0.2x SSPE、 0.2%SDS, 42 °C 清洗。 高严谨性杂交条件等同于 50%甲酰胺、 5χ Denhardt's溶液、 5χ SSPE、 0.2%SDS, 42 °C反应， 然后在 O. l x SSPE、 0.1%SDS, 65°C清洗。 低严谨性杂 交条件等同于 10%甲酰胺、 5 X Denhardt's溶液、 6 SSPE、 0.2%SDS, 22 °C反 应， 然后在 l x SSPE、 0.2%SDS, 37 °C清洗。 Denhardt's溶液包含 1%聚蔗糖，
1%聚乙烯吡咯烷酮和 1%牛血清白蛋白（BSA)。20x SSPE包含 3M氯化钠，0.2M 磷酸钠和 0.025M 乙二胺四乙酸。其他合适的中等严谨性和高严谨性杂交液和条 件都为具有该领域的普通技能的人所熟知，在别处也有所描述，例如 Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, N.Y. (1989); 禾 P Ausubel et al., Short Protocols in Molecular Biology, 4th ed" John Wiley & Sons (1999)。
[0052] 这里的 "基因状态"指的是 miRNA基因的结构、 拷贝数和在染色 体上的位置。 miRNA基因状态的 "特征性变化"指的是例如缺失或扩增， 拷贝 数的变化， 或者在染色体上的位置发生改变。
[0053] 此处的 miRNA表达水平的 "特征性变化"可以简单地理解为与参 考水平相比样本中一个 miRNA的表达水平显著上升或下降。特征性变化也可以 指多个 miRNA的表达水平显著上升或下降。 它还可以指一些 miRNA的表达水 平显著上升而另些 miRNA的表达水平显著下降。
[0054] 此处描述的方法中， "参考水平"指的是特定 miRNA被认为 "正常" 的水平。 在某些情况下， 参考水平基于同一个体中非癌宫颈上皮内或宫颈组织 中此 miRNA的表达水平。在某些情况下， 参考水平基于一个不患有宫颈上皮内 瘤样病变或宫颈癌的个体中此 miRNA的表达水平。在某些情况下， 参考水平基 于一群不患有宫颈上皮内瘤样病变或宫颈癌的个体中此 miRNA 的平均表达水 平。 在某些情况下， 参考水平来源于一个样本库， 包括被检测样本。 参考水平 可以事先测定也可以与被测样本同时测定。
[0055] 参考水平可以是另一个 miRNA的水平， 另一个 RNA的水平， 如 U6， 或另一个核酸的水平， 如 DNA。 miRNA的表达水平可以与同一样本或参 考样本的其他核酸表达水平相比较。 参考样本可以来源于同一组织或不同组织， 也可以来源于同一个体或不同个体。
[0056] 此处的 "参考值"可以是绝对值、 相对值、 一个具有上限和下限的 值、 一系列值、 平均值、 中值、 中间值或者是与特定对照或基准值比较的值。
[0057] 此处的 "显著"改变表示可以被此处所描述的方法检测到的变化， 或者是统计学上显著的变化。 此处的 "显著上升"指的是 miRNA水平至少上升 5%，包括例如至少上升 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%或更多。 同样地， "显著下降"指的是 miRNA水平至少下降 5%， 包括例如至少下降 6%,
7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%或更多。
[0058] 此处的探针指的是包括例如 DNA、 RNA、 PNA、 LNA、 它们的组 合和 /或它们的修饰物。 它们还可以包括修饰的寡核苷酸骨架。 在某些情况下， 这些探针包含与 miRNA完全或部分相同或互补的至少 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20或更多个连续的寡核苷酸。一个探针序列可以包含两个或多个这种 互补序列。 在某些情况下， 探针的 5'或 3'端可以连接有活性基团 （如氨基） 以 便探针与基质连接， 比如， 案例 1中提供的探针为 20条， 20条探针的核酸序列 是序列表中序列 1至序列 20所示的 20条核酸的完全互补序列， 在每个探针序 列的 5'端均连接有 19个 T， 探针的 5'端经氨基修饰。 探针既可以用作例如原位 杂交用的寡核苷酸， 也可以用作 PCR扩增反应的引物（如案例 2、 3中提供的引 物）。
[0059] 此发明的其他对象、 优势和特征将在下文的描述及相应的图中阐 明。 检测 miRNA表达水平和基因状态的系统 [0060] 此发明提供了多个系统用于检测宫颈上皮内瘤样病变或宫颈癌患 者中 miRNA表达水平的特征性变化。 还提供了检测 miRNA基因状态的系统。 这些系统可以用于多个目的， 包括例如宫颈上皮内瘤样病变或宫颈癌诊断， 宫 颈上皮内瘤样病变或宫颈癌患者分类， 以及为宫颈上皮内瘤样病变或宫颈癌患 者生存率预后。
[0061] 此处描述的 miRNA也可以用于以下一个或多个方面： 基于个体宫 颈上皮或宫颈组织样本的一个或多个 miRNA表达水平或基因状态，为宫颈上皮 内瘤样病变或宫颈癌患者分类， 对发展成宫颈上皮内瘤样病变或宫颈癌的风险 进行预测， 对宫颈上皮内瘤样病变或宫颈癌患者的肿瘤发展进行监测， 对宫颈 上皮内瘤样病变或宫颈癌患者的治疗进行监测。
[0062] 此处描述的系统包括检测 miRNA或其基因状态的探针。 下文的讨 论将集中在可以检测 miRNA表达水平的系统上，对于熟悉该领域的普通技术人 员而言， 很容易理解该描述的一些方面也适用于包含检测基因缺失、 扩增和 /或 miRNA基因拷贝数改变 （合起来称为 miRNA的基因状态） 的探针的系统。
[0063] 例如， 在某些情况下， 这里提供了一个包含大量探针的系统， 这些 探针可以检测样本中不同的 miRNA, 至少 15% (包括例如至少 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%或 95%) 的探针可以检测表 1中的一个 miRNA或它 们的同源体。在某些情况下， 该系统包含（包括必须包含或可以包含）至少 2, 5, 10, 20, 30, 40或 50个探针， 其中每个探针都可以检测表 1中的一个 miRNA或 它们的同源体。
表 1 宫颈上皮内瘤样病变和 /或宫颈癌患者样本中差异表达的 miRNA 序列 miRNA 表达水平 染色体位置
Ch 18: 19405659-19405746 [-]
序列表中序列 1 hsa-miR-133a ι¾ Ch 20: 61162119-61162220 [+]
序列表中序列 2 hsa-miR-133b l¾ Ch 6: 52013721-52013839 [+]
序列表中序列 3 hsa-miR-140-3p l¾ Ch 16: 69966984-69967083 [+]
序列表中序列 4 hsa-miR-143* l¾ Ch 5: 148808481-148808586 [+] 序列表中序列 5 hsa-miR-145 l¾ Ch 5: 148810209-148810296 [+] 序列表中序列 6 hsa-miR-223 Ch X: 65238712-65238821 [+] 序列表中序列 7 hsa-miR-99b l¾ Ch 19: 52195865-52195934 [+]
序列表中序列 8 hsa-miR-221 l¾ Ch X: 45605585-45605694 [-]
序列表中序列 9 hsa-miR-320a l¾ Ch 8: 22102475-22102556 [-]
序列表中序列 10 hsa-miR-100 l¾ Ch 11 : 122022937-122023016 [-]
Ch 19: 10928102-10928172 [-]
序列表中序列 11 hsa-miR-199a-5p
Ch 1 : 172113675-172113784 [-] 序列表中序列 12 hsa-miR-127-3p l¾ Ch 14: 101349316-101349412 [+] 序列表中序列 13 hsa-miR-214 Ch 1 : 172107938-172108047 [-] 序列表中序列 14 hsa-miR-203 低 Ch 14: 104583742-104583851 [+] 序列表中序列 15 hsa-miR-190 低 Ch 15: 63116156-63116240 [+]
序列表中序列 16 hsa-miR-200b 低 Ch 1 : 1102484-1102578 [+]
序列表中序列 17 hsa-miR-200c 低 Ch 12: 7072862-7072929 [+]
序列表中序列 18 hsa-miR-200a 低 Ch 1 : 1103243-1103332 [+]
序列表中序列 19 hsa-miR-31 低 Ch 9: 21512114-21512184 [-]
序列表中序列 20 hsa-miR-141 低 Ch 12: 7073260-7073354 [+]
[0064] 此处描述的系统可以包含检测同一个 miRNA的两个或多个探针。 例如， 当系统是微阵列的情况下， 探针在微阵列上是多 （例如 2， 3， 4， 5， 6， 7或更多） 拷贝的。 在某些情况下， 系统包含检测同一个 miRNA的不同探针。 例如， 这些探针可能结合 miRNA的不同区域 （重叠或非重叠）。
[0065] 可以检测 miRNA水平的任意探针都可以使用。 在某些情况下， 探 针可以是寡核苷酸。 可以理解， 为检测 miRNA, 部分序列上的变异是可以接受 的。 因此， 寡核苷酸序列 （或其互补序列）可以与此处描述的 miRNA序列略有 不同。 具有该领域的普通技能的人很容易理解这种序列变化不会显著影响寡核 苷酸检测 miRNA水平的能力。 例如， 当用标准的方法进行比对时， 这些寡核苷 酸分子的同源体或变异体拥有高度的序列相似性。 此发明包含的寡核苷酸序列 与此处描述的 miRNA具有至少 40%， 包括例如至少 50%, 60%, 70%, 80%, 90%, 95%或更高的序列同源性。 在某些情况下， 寡核苷酸包含检测 miRNA的部分和 另一部分。 另一部分可以用来诸如将寡核苷酸连接到基质上。 在某些情况下， 另一部分包含一个非特异的序列 （例如 polyT ) , 从而增加互补序列与基质表面 的距离。
[0066] 此处描述的寡核苷酸包括例如 DNA、 RNA、 PNA、 LNA、 它们的 组合和 /或它们的修饰物。它们还可以包括修饰的寡核苷酸骨架。在某些情况下， 这些寡核苷酸包含与 miRNA完全或部分相同或互补的至少 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20或更多个连续的寡核苷酸。 一个寡核苷酸序列可以包含两个或 多个这种互补序列。 在某些情况下， 寡核苷酸的 5'或 3 '端可以连接有活性基团 (如氨基） 以便寡核苷酸与基质连接。
[0067] 在某些情况， 系统是含有探针的微阵列。 此处使用的 "微阵列"和
"阵列"可以相互替换， 它们表面有阵列， 最好是有序的阵列， 具有与特征不 确定的生化样本 （靶标） 结合的预测位点 （如杂交）。 在某些情况下， 微阵列指 的是固定在基质特定位置上的独特的寡核苷酸探针集合。
[0068] 举个例子，在某些情况下，这里提供了一个含有大量探针的微阵列， 其中每个探针可以检测样本中一个不同的 miRNA,至少 15% (包括例如至少 20%:
30%, 40%, 50%, 60%, 70%, 80%, 90%或 95% ) 的探针可以检测一个具有表 1所 列的 miRNA或它们的同源体。
[0069] 在某些情况下， 此处提供了检测此处提到的 miRNA对应的基因的 遗传状态的微阵列。 检测基因遗传状态的微阵列在此领域内是已知的。 例如， 系统可以包含检测遗传状态的序列标记的倒置探针分子。
[0070] 阵列可以在纸、 玻璃、 塑料 （如聚丙烯、 尼龙、 聚苯乙烯）、 聚丙 烯酰胺、 硝化纤维、 硅、 光纤或其他合适的固体或半固体支持物制成的基质上 形成， 并且呈平面 （例如玻片、 硅片） 或三维 （例如针尖、 光纤、 珠子、 颗粒、 微孔、 毛细管） 的构象。
[0071] 在某些情况下， 探针是寡核苷酸。 寡核苷酸可以通过以下几种 （但 并不局限于） 方式连接到基质上形成阵列： （i)使用照相平版印刷技术原位合成 (如高密度寡核苷酸阵列）； （ii)在介质中低密度地点样于玻璃、尼龙或硝化纤维 上； （iii)掩模； 和 (iv)点阵于尼龙或硝化纤维杂交膜。 寡核苷酸也可以非共价地 固定在基质上， 如锚定杂交， 以磁珠或流动相形式包含在微孔或毛细管中。
[0072] 该领域内有几种广为人知的将核酸连接到固体基质如玻璃上的技 术。 一种方法是在扩增的核酸上嵌入修饰的碱基或者类似物， 它们包含可以连 接到固体基质上的基团， 例如胺一一胺的衍生物或其它带正电的基团。 然后扩 增产物与固体基质如玻片连接， 玻片可能包被有醛基或者是可以与扩增产物上 的活性基团形成共价连接的其他活性基团， 这样扩增产物与玻片间就形成了共 价连接。 包含扩增产物的微阵列可以通过 Biodot点样仪 (BioDot, Inc. Irvine, CA) 和醛基包被的玻片 (CEL Associates, Houston, TX)生产。扩增产物可以点到醛基包 被的玻片上， 然后根据已发表的程序加工 CSchena et al.， Proc. Natl. Acad. Sci. U.S.A. (1995), 93: 10614-10619)。 阵列也可以通过机械手点样到玻璃、 尼龙 (Ramsay, G Nature Biotechnol. (1998), 16:40-44)、 聚丙烯 (Matson, et al, Anal Biochem. (1995), 224(1): 110-6)和硅片 (Marshall and Hodgson, Nature Biotechnol.
(1998), 16:27-31)上。 其他形成阵列的途径包括电动的精细微吸管 (Marshall, and Hodgson, Nature Biotechnol. (1998), 16:27-31)和直接将多聚核苷酸点样于正电包 被的片子上。 那些使用氨基丙烷基硅表面化学修饰的方法在此领域内也有所报 道 ， 以 下 网 立占 上 有 所 描 述 ： www.cmt.corning.com 禾口 http ://cmgm. Stanford . edu/ pbrown/。
[0073] 制作微阵列的方法之一是形成高密度核酸阵列。 使用的技术有快速 沉积多聚核苷酸的技术 (Blanchard, et al., Biosensors & Bioelectronics, 11 :687-690) 也可以使用其他制作微阵列的方法，例如掩模 CMaskos and Southern, Nucleic. Acids. Res. (1992), 20: 1679-1684)。 原则上， 上述的任意一种阵列都可以 使用， 如尼龙杂交膜上的点阵。 然而， 正如能被熟悉该领域的人认可， 通常更 倾向于使用很小的阵列， 因为其杂交体积更小。 宫颈上皮内瘤样病变或宫颈癌诊断的 miRNA
[0074] 此发明发现了表达水平与宫颈上皮内瘤样病变或宫颈癌相关的 20 个 miRNA。 这些 miRNA列在表 1中。 表 1提供了 miRNA的名字、 序列、 染色 体位置。 关于 miRNA的信息可以在以下网站上找到 http://miRNA.sanger.ac.uk/ (Griffths- Jones, et al., Nucleic Acids Research, 2006, Vol. 34, Database issue)。 诊断 宫颈上皮内瘤样病变或宫颈癌的方法可以基于表 1所示的任意一个 miRNA的表 达水平或基因状态。 此处描述的系统可以用来检测表 1中的一个或多个 miRNA 的表达水平，并根据表 1中的一个或多个 miRNA的表达水平对宫颈上皮内瘤样 病变或宫颈癌进行诊断。
[0075] 虽然经过实践此处描述的方法检测一个 miRNA可以达到可接受的 灵敏度和特异性， 但是通常利用至少两个 miRNA可以促进此方法的有效性（如 灵敏度和 /或特异性）。在某些情况下，至少利用表 1中的 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20个 miRNA。
[0076] 具体说，检测至少 2个（例如至少 2, 3, 5, 10或更多个）序列号 1-13 的 miRNA的表达水平或基因状态。 具体说， 检测至少 2个（例如至少 2, 5, 7或 更多个） 序列号 14-20的 miRNA的表达水平或基因状态。 具体说， 检测至少 2 个 （例如至少 2, 3, 5, 10或更多个）序列号 1-13的 miRNA的表达水平或基因状 态， 和至少 2个 （例如至少 2, 5, 7或更多个）序列号 14-20的 miRNA的表达水 平或基因状态。 具体说， 检测表 1所示的所有 miRNA的水平或遗传状态。
[0077] 具体说，检测此处描述的 miRNA相应的同源体的水平。 MiRNA "相 应的同源体"指的是至少 50%序列 （包括例如至少 60%, 70%, 80%, 90%, 95%, 98%, 或 99% ) 与此处的 miRNA相同的 miRNA。 例如， 1号 miRNA的同源体 具有至少 50% (包括例如至少 60%, 70%, 80%, 90%, 95%, 98%, 或 99% )与其相 同的序列。
[0078] 具有至少 95%与参考序列 （例如 1号） 相同的 miRNA序列被认为 此 miRNA与参考序列相同。 如果此 miRNA序列每 100个核苷酸含有 5个与参 考序列不同的核苷酸则除外。 这 5 个不同的点可以是缺失、 替换、 插入， 可以 发生在序列的任意位置， 可以独立散布在参考序列中也可以形成一个或多个连 续的片段。
[0079] 对于结肠直肠癌、 舌头鳞状细胞癌、 食管鳞状细胞癌和胰腺导管癌 的诊断， 则检测 miR-133b的表达水平或基因状态。 癌症诊断， 尤其是宫颈上皮内瘤样病变和宫颈癌诊断的方法
[0080] 此发明一方面提供了为个体诊断宫颈上皮内瘤样病变和宫颈癌的 方法， 包括 a)检测怀疑有宫颈上皮内瘤样病变和宫颈癌的组织样本中至少一个 miRNA (例如至少一个表 1中的 miRNA或它们的同源体） 的表达水平； b ) 当 检测组织的 miRNA的表达水平与参考水平相比发生了特征性变化时，预示可能 有宫颈上皮内瘤样病变或宫颈癌。 在某些情况下， 该方法进一步包括从个体中 取样宫颈组织。 在某些情况下， 该方法进一步包括从组织样本中提取 miRNA。
[0081] 在某些情况下， 这里提供了为宫颈上皮内瘤样病变和宫颈癌诊断提 供信息的方法， 包括： a) 对怀疑有宫颈上皮内瘤样病变或宫颈癌的组织样本， 检测表 1中至少一个 miRNA或它们的同源体在该组织中的表达水平； b) 提供 诊断宫颈上皮内瘤样病变和宫颈癌的 miRNA的表达水平。 MiRNA的表达水平 是宫颈上皮内瘤样病变和宫颈癌的诊断基础，至少一个 miRNA的特征性变化预 示着宫颈上皮内瘤样病变或宫颈癌。
[0082] 在某些情况下，检测序列号 1-13中至少一个 (包括至少 2, 3, 5, 10, 13 个） miRNA的水平。 当至少一个被检测的 miRNA的水平显著上升时， 预示有 宫颈上皮内瘤样病变或宫颈癌。在某些情况下，检测序列号 14-20中至少一个 (包 括至少 2, 5, 7个） miRNA的水平。 当至少一个被检测的 miRNA的水平显著下 降时， 预示有宫颈上皮内瘤样病变或宫颈癌。 在某些情况下， 检测序列号 1-13 中至少一个 （包括至少 2, 3, 5, 10, 13个） miRNA的水平和序列号 14-20中至少 一个 （包括至少 2, 5, 7个） miRNA的水平。 当至少一个序列号 1-13的 miRNA 的水平显著上升和当至少一个序列号 14-20的 miRNA的水平显著下降时， 预示 有宫颈上皮内瘤样病变或宫颈癌。
[0083] 在某些情况下， 检测所有表 1所列的 miRNA的水平。 当至少一个 序列号 1-13的 miRNA的水平显著上升和当至少一个序列号 14-20的 miRNA的 水平显著下降时， 预示有宫颈上皮内瘤样病变或宫颈癌。 在某些情况下， 当至 少两个序列号 1-13 的 miRNA 的水平显著上升和当至少两个序列号 14-20 的 miRNA的水平显著下降时，预示有宫颈上皮内瘤样病变或宫颈癌。某些情况下， 当序列号 1-13 miRNA的水平显著上升和当序列号 14-20 miRNA的水平显著下 降时， 预示有宫颈上皮内瘤样病变或宫颈癌。
[0084] 组织样本中的 miRNA表达水平还可以反映 miRNA基因状态的变 化。 例如可以反映出 miRNA基因的缺失、 扩增或拷贝数的改变。
[0085] 因此， 在某些情况下， 这里提供了宫颈上皮内瘤样病变和 /或宫颈癌 的诊断方法， 包括分析怀疑有宫颈癌的个体的宫颈上皮或宫颈组织样本中至少 一个 miRNA基因 （例如至少一个对应表 1中的 miRNA的基因） 的遗传状态。 当 miRNA基因的遗传状态相对对照样本发生了特征性的变化则预示有宫颈上 皮内瘤样病变或宫颈癌。在某些情况下， 遗传状态的改变是由于 miRNA基因的 缺失或扩增。 在某些情况下， 遗传状态的改变是由于 miRNA基因拷贝数改变。
[0086] 具体说， 这里提供了宫颈上皮内瘤样病变和 /或宫颈癌的诊断方法， 包括分析怀疑有癌症的个体的宫颈上皮或宫颈组织样本中对应表 1 所示的至少 一个 miRNA基因的缺失或扩增状况。 当 miRNA的基因相对对照样本发生了缺 失或扩增则预示有宫颈上皮内瘤样病变或宫颈癌。 例如， 在某些情况下， 此方 法包含了分析至少一个序列号 1-13的 miRNA基因的扩增情况， 与对照样本相 比发生了扩增则表明有宫颈上皮内瘤样病变或宫颈癌。 在某些情况下， 此方法 包含了分析至少一个序列号 14-20的 miRNA基因的缺失情况， 与对照样本相比 发生了缺失则表明有宫颈上皮内瘤样病变或宫颈癌。 在某些情况下， 此方法进 一步包括从怀疑有癌变的个体取样宫颈上皮或宫颈组织。 在某些情况下， 该方 法进一步包括从宫颈上皮或宫颈组织中提取 DNA。
[0087] 具体说， 这里提供了宫颈上皮内瘤样病变和 /或宫颈癌的诊断方法， 包括检测怀疑有癌症的个体的宫颈上皮或宫颈组织样本中至少一个表 1 所列 miRNA 或其相应的同源体基因的拷贝数。 当位于常染色体或性染色体上的 miRNA基因拷贝数不是两个， 则预示有宫颈上皮内瘤样病变或宫颈癌。 例如， 在某些情况下，该方法包括检测个体样本中对应序列号 1-13的至少一个 miRNA 的基因的拷贝数， 当位于常染色体或性染色体上的 miRNA基因发生了多于两个 拷贝的变化则表明有宫颈上皮内瘤样病变或宫颈癌。 在某些情况下， 此方法包 括检测个体样本中对应序列号 14-20的至少一个 miRNA的基因的拷贝数， 当位 于常染色体或性染色体上的 miRNA基因发生了少于两个拷贝的变化则表明有 宫颈上皮内瘤样病变或宫颈癌。 在某些情况下， 此方法进一步包括从怀疑有癌 变的个体取样宫颈上皮或宫颈组织。 在某些情况下， 该方法进一步包括从宫颈 上皮或宫颈组织中提取 DNA。
[0088] 具体说， 宫颈上皮内瘤样病变和宫颈癌的诊断方法是基于检测 miRNA的表达水平。
[0089] 另一方面， 此发明为结肠直肠癌、 舌头鳞状细胞癌、 食管鳞状细胞 癌和胰腺导管癌诊断提供了一个方法， 包括： a) 检测怀疑有癌变的组织样本中 miR-133b表达的水平； b ) 样本的 miR-133b表达水平与参考水平比对， 当样本 中 miR-133b水平表现出特征性变化则表明有结肠直肠癌、 舌头鳞状细胞癌、 食 管鳞状细胞癌或胰腺导管癌。 [0090] 具体说， 这里为结肠直肠癌、 舌头鳞状细胞癌、 食管鳞状细胞癌和 胰腺导管癌诊断提供了一个方法， 包括分析怀疑有癌症的个体组织中的 miR-133b基因的遗传状态。当 miR-133b基因相对于对照样本有特征性变化时则 表明是结肠直肠癌、 舌头鳞状细胞癌、 食管鳞状细胞癌或胰腺导管癌。 在某些 情况下，检测基因遗传状态的变化是基于 miR-133b基因的缺失或扩增的基础上。 在某些情况下， 检测基因遗传状态的变化是基于 miR-133b基因拷贝数的变化的 基础上。
[0091] 此处的 "表达水平"和 "水平"可以互换， 均表示 miRNA分子或 其前体累积的量或比率。这个概念可以用来表示样本中 miRNA的绝对量（例如 杂交信号的强度）， 或者是相对于对照样本的比率 （例如该样本中杂交信号相对 于对照样本的比率）。 对照可以是同一样本中的另一个在宫颈上皮内瘤样病变或 宫颈癌组织中表达水平没有发生改变的 miRNA, 也可以是来源于不同样本 （例 如同一个体的非癌组织样本或另一个不患有宫颈上皮内瘤样病变或宫颈癌的个 体的组织样本） 的同一个 miRNA。
[0092] MiRNA分子的 "前体 "或者 " miRNA前体"指的是没有经过完整 加工的 miRNA基因转录物， 典型的是大约 70个碱基的 RNA转录物。 MiRNA 前体通常经过 RNA酶 （如 Dicer, Argonaut, 或 RNAase III) 消化得到一个活性 miRNA分子， 大约 19-25个碱基长度。
[0093] "宫颈上皮或宫颈组织样本中的 miRNA水平"指的是组织样本中 的 miRNA水平。 大多数情况下宫颈上皮或宫颈组织样本中的 miRNA水平是通 过直接检测宫颈上皮或宫颈组织样本中的 miRNA水平得到的，然而宫颈上皮或 宫颈组织样本中的 miRNA水平也可以通过淋巴结样本（如最接近的淋巴结或淋 巴）、 血清、 血液或其它最接近的生物学流动样本如唾液中的 miRNA水平反映 出来。具体说， miRNA水平的检测是基于淋巴样本（如淋巴结片段或针吸样本） 的 miRNA水平基础上的。 具体说， miRNA的水平的检测是基于血液或血清中 的 miRNA水平基础上的。 具体说， miRNA的水平的检测是基于宫颈上皮或宫 颈组织刮片的 miRNA水平基础上的。 具体说， miRNA水平的测定是内窥镜取 样程序得到的样本中的 miRNA水平 （例如通过 RT-PCR分析）。 检测除了宫颈 上皮内瘤样病变或宫颈癌组织以外的样本中的 miRNA水平可以单独或联合使 用。 例如， miRNA的水平可以首先从血清中检测， 然后再分析可操作的区域性 的淋巴结中的水平。 这种多步骤分析可以提供更多信息并增加诊断的可信度。
[0094] miRNA水平可以在不同阶段检测， 例如， 可以在手术前、 手术中、 手术后、 肿瘤治疗前、 治疗中或治疗后检测。
[0095] 检测 miRNA水平的方法在该领域内也是已知的。 例如， 可以通过 Northern blot、 原位杂交、 RT-PCR和微阵列 (Einat, Methods Mol. Biol. (2006),
342: 139-157; Thompson, et al, Genes Dev. (2006), 20:2202-2207)等技术来检测 miRNA水平。
[0096] 根据一个示范性的方法， 总 RNA可以通过在核酸提取液中对细胞 进行匀浆， 然后离心， 再沉淀核酸。 通过 DNA酶消化去除 DNA后再沉淀。 根 据标准的技术通过琼脂糖凝胶电泳将 RNA分子分离， 然后通过诸如 Northern blotting技术转到硝化纤维滤膜上。 通过加热使得 RNA固定在滤膜上。 通过与 待测 RNA互补的适当标记的 DNA或 RNA探针对特定的 RNA进行检测和定量。 利用放射自显影检测与 miRNA杂交的探针可以通过将杂交后的膜曝光到胶片 上实现。 对曝光后的胶片进行浓度扫描可以提供准确的 RNA转录水平。 另外， RNA转录水平也可以通过照片处理软件对杂交点进行计算得到。
[0097] 除了 Northern和其他 RNA印迹杂交技术， miRNA转录本的水平可 以通过原位杂交技术获得。 该技术涉及将整个细胞或组织置于盖玻片上， 然后 在含有放射性或者标记的探针 （例如 cRNA探针） 的溶液中检测细胞或组织中 的核酸。
[0098] miRNA的水平也可以通过 RT-PCR检测。 miRNA的水平可以通过 与标准内参进行比较得到， 例如同一样本中的 "看家基因" 的 mRNA水平。 用 来当内参的合适的 "看家基因"包括肌球蛋白、 3-磷酸甘油醛脱氢酶 （G3PDH) 或人 U6。 定量 RT-PCR或其变体都是该领域内普通的技术人员所熟知的。 在某 些特定情况下， 实时定量 PCR (qRT-PCR) 可能比经典的组织切片染色检测早 期癌症中的 miRNA水平更灵敏。此处检测 miRNA水平的 qRT-PCR法可能为宫 颈上皮内瘤样病变或宫颈癌的诊断和预后提供了一个灵敏并且特异的工具。 此 发明提供了通过 RT-PCR检测个体（例如患病个体，如癌症患者）样本中 miRNA 水平的方法。 具体说， miRNA的水平通过 qRT-PCR检测。
[0099] 在某些情况下， miRNA 的水平通过微阵列 （例如此处描述的微阵 列） 来检测。 [00100]上述的一种或多种方法使用的探针可以通过该领域内已知的方法 如 DNA重组或化学合成得到。 另外， 杂交探针可以用不同的标签标记， 例如放 射性同位素、 荧光探针、 报告酶、 生物素或其它配体。 这种可检测的标签可以 通过与色谱的或光谱的指示剂偶联从而实现分光光度检测。 标记和检测此类探 针的方法在该领域内是已知的。
[00101]该领域内的技术人员可以很容易地摸索到用来检测样本中的 miRNA的核酸探针不同严谨性的杂交条件。 根据特定的检测实验， 该领域内的 技术人员可以改变严谨性以寻找检测特定样本中的某个特定的 miRNA最优化 的条件。
[00102]在某些情况下， miRNA 的水平可能来源于个体的不同时间点。 这 种 "序列" 的样本最适合于本发明中宫颈上皮内瘤样病变或宫颈癌个体的宫颈 上皮内瘤样病变或宫颈癌发展监视。 序列取样法在任意的时间序列进行， 例如 每半年、 每年、 每两年或更长时间。 检测水平与参考水平间的比较可以在每次 采样后进行， 也可以将数据保存到一定数量后进行分析。
[00103]与参考水平进行比较的 miRNA可以是表 1中的任意 1, 2, 3, 4, 5, 6, Ί, 8, 9, 10, 15, 18, 19或 20个 miRNA或其同源体。 miRNA水平与参考水平进行比 较的过程可以采取任意适合于检测到的 miRNA 的值的方式。 例如， 当使用 miRNA的杂交信号作为 miRNA的检测水平时， 可以通过视觉定性比较杂交信 号的强度。 对于定量检测， 可以通过比较观察的到的数据或代表性的数据 （例 如观察绘图表示法如柱状图或线条） 进行。 比较的过程可以是人工的 （例如该 方法的执业者的视觉观察） 或者自动的。
[00104]在某些情况下， 通过比较检测 miRNA的水平和参考水平之间的幅 度差异 （如比较检测 miRNA的水平和参考水平间的 "倍数"或百分比差异）进 行二者间的比较。 此处的 "倍数差异"指的是检测 miRNA的水平和参考值间幅 度差的数值代表。
[00105]表 1提供了所有的示范性的方法中检测到的变化 miRNA。 MiRNA 水平的特征性变化用作宫颈上皮内瘤样病变和宫颈癌的诊断基础。 例如， 在某 些情况下， 当至少一个序列号 1-13的 miRNA水平被检测时， 至少一个被检测 的 miRNA水平发生真实上升就预示着宫颈上皮内瘤样病变或宫颈癌。在某些情 况下， 当至少一个序列号 14-20 的 miRNA水平被检测时， 至少一个被检测的 miRNA水平发生真实下降就预示着宫颈上皮内瘤样病变或宫颈癌。 在某些情况 下， 当至少一个序列号 1-13的 miRNA和至少一个序列号 14-20的 miRNA水平 被检测时， 至少一个被检测的 1-13号 miRNA水平发生真实上升和至少一个被 检测的 14-20号 miRNA水平发生真实下降就预示着宫颈上皮内瘤样病变或宫颈 癌。
[00106]在某些情况下， 表 1所示的所有 miRNA的水平都检测， 至少一个 1-13号 miRNA水平发生真实上升和至少一个 14-20号 miRNA水平发生真实下 降就预示着宫颈上皮内瘤样病变或宫颈癌。 在某些情况下， 至少两个 1-13 号 miRNA水平发生真实上升和至少两个 14-20号 miRNA水平发生真实下降就预 示着宫颈上皮内瘤样病变或宫颈癌。
[00107]当利用多于一个 miRNA但是它们的水平并不一致地预示着宫颈上 皮内瘤样病变或宫颈癌时， 则考虑以 "大部分" 的结果为准。 例如， 当使用 5 个 miRNA时， 其中 3个预示宫颈上皮内瘤样病变或宫颈癌， 则认为结果预示该 个体是宫颈上皮内瘤样病变或宫颈癌。 然而， 在某些情况下， 宫颈上皮内瘤样 病变或宫颈癌的诊断需要至少一个或更多个特定的 miRNA发生特征性变化。例 如， 其中一个是 hsa-miR-133b， 在某些情况下， hsa-miR-133b水平的真实上升 可能是诊断宫颈上皮内瘤样病变或宫颈癌的先决条件。 基于 miRNA遗传状态的诊断方法
[00108]此处还提供了基于个体样本中至少一个表 1所示的 miRNA或其同 源体的遗传状态诊断宫颈上皮内瘤样病变或宫颈癌的方法。
[00109]在某些情况下， 通过分析样本中至少一个 miRNA基因的缺失或扩 增情况来评价遗传状态，如果 miRNA基因相对于对照样本是缺失或扩增则预示 着个体有宫颈上皮内瘤样病变或宫颈癌。
[00110] MiRNA基因的缺失或扩增可以通过检测被怀疑患有宫颈上皮内瘤 样病变或宫颈癌的个体的宫颈上皮或宫颈组织样本细胞中的基因结构或序列实 现， 并且与对照样本中的基因结构或序列进行比较。 适合检测基因结构或序列 改变的任何技术都可以用来实现当前的方法。例如， 检测 miRNA基因的缺失或 扩增， 可以利用针对 miRNA的特异的核酸探针， 通过 Southern blot对个体的基 因组 DNA进行杂交检测到。 也可以利用序列分析和单链构象多态性进行分析。 [00111] miRNA基因的缺失或扩增也可以通过 PCR扩展这些基因的片段检 测，然后通过测序或电泳分析从个体 DNA样本扩增到的片断的序列或长度是否 与对照 DNA样本相同。 MiRNA基因的缺失也可以通过检测 miRNA基因附近的 染色体标志的缺失实现。
[00112]个体细胞中 miRNA基因的状况也可以通过检测样本中至少一个基 因的拷贝数进行评价。当常染色体和性染色体上的 miRNA基因拷贝数不是二时 则预示着个体患有宫颈上皮内瘤样病变或宫颈癌。
[00113]任意可以检测基因拷贝数的方法都可以用来实现当前的方法， 包括 Southern blot 和 PCR 扩增技术。 另外一种检测宫颈上皮或宫颈组织样本中 miRNA基因拷贝数的方法依赖于很多 miRNA或基因簇都与染色体标志或其他 基因紧密相连。在一个个体中如果临近 miRNA基因的标志或基因是杂合子， 则 miRNA基因丢失一个拷贝数可以通过染色体标志或基因的杂合性丢失反映出 来。 检测染色体标志杂合性丢失的方法是该领域内的已知技术。
[00114] "对照样本"可以是来源于不患有宫颈上皮内瘤样病变或宫颈癌个 体的组织样本， 也可以是从一个群体中收集来的组织样本。
[00115]表 1中的至少 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18或 20 个 miRNA或其 同源体的遗传状态可以用来检测。在某些情况下， 当多于一个 miRNA的遗传状 态被使用但并不都暗示宫颈上皮内瘤样病变或宫颈癌， 则以 "大部分"结果为 准。例如， 当使用 5个 miRNA时，其中 3个暗示宫颈上皮内瘤样病变或宫颈癌， 则认为结果暗示该个体是宫颈上皮内瘤样病变或宫颈癌。 然而， 在某些情况下， 宫颈上皮内瘤样病变或宫颈癌的诊断需要至少一个或更多个特定的 miRNA基 因发生特征性变化。
[00116]有多种技术可以用来检测 miRNA基因遗传状态。 包括例如微阵列 上的等位基因特异引物扩增， PCR/LDR通用阵列， 基于微球体的单链扩增， 序 列标记分子倒置探针， 以及组合杂交测序。
[00117]这里还提供了基于个体样本中 miR-133b或其同源体的遗传状态诊 断结肠直肠癌、 舌头鳞状细胞癌、 食管鳞状细胞癌和胰腺导管癌的方法。 在某 些情况下， 遗传状态通过分析样本中的 miR-133b基因的缺失或扩增进行评价， 当 miR-133b基因相对于对照样本缺失或扩增时则暗示着个体是结肠直肠癌、舌 头鳞状细胞癌、 食管鳞状细胞癌或胰腺导管癌。 为宫颈上皮内瘤样病变或宫颈癌患者生存率预后的方法
[00118]此发明另一方面为宫颈上皮内瘤样病变或宫颈癌患者提供了预后 的方法， 包括为宫颈上皮内瘤样病变或宫颈癌个体进行生存率预后。 此发明的 预后方法对于宫颈上皮内瘤样病变或宫颈癌个体决定合适的治疗方法很有应用 价值。 例如， 生存预后可以帮助决定采用更保守的还是更激进的治疗方法， 或 者需要采取组合的治疗方式。 此外， 这种预后可以帮助决定使用帮助存活的药 物 （例如此处提到的药物） 是否有必要和 /或是否有效。
[00119]在某些情况下， 这里为宫颈上皮内瘤样病变或宫颈癌个体提供了生 存率预后的方法， 包括： （a) 检测个体宫颈上皮内瘤样病变或宫颈癌组织样本 中的至少一个 miRNA的水平； （b ) 将该样本的 miRNA水平与阈值进行比较， 其中 miRNA水平相对阈值与个体生存率正相关或负相关。 此处的 "正相关"指 的是相对阈值 miRNA低水平暗示宫颈上皮内瘤样病变或宫颈癌个体的低生存 率， 反之亦然。 此处的 "负相关"指的是相对阈值 miRNA高水平暗示宫颈上皮 内瘤样病变或宫颈癌个体的低生存率， 反之亦然。
[00120]具体说， miRNA基因位于 6号、 18号和 20号染色体中的任意一条 上。 具体说， 至少一个 miRNA是 hsa-miR-133b。
[00121]在某些情况下， 这里为宫颈上皮内瘤样病变或宫颈癌个体提供了生 存率预后的方法， 包括： （a) 检测个体宫颈上皮内瘤样病变或宫颈癌组织样本 中的至少一个 miRNA的水平； （b ) 将该样本的 miRNA水平与阈值进行比较， 其中 miRNA水平相对阈值与个体生存率负相关， 并且其中至少一个 miRNA是 hsa-miR-133b或与它相应的同源体。
[00122]此处描述的 miRNA 水平也可以反映 miRNA (如此处描述的 miRNA) 基因遗传状态的变化。 具体说， 这里为宫颈上皮内瘤样病变或宫颈癌 个体提供了生存率预后的方法， 包括分析至少一个 miRNA 基因 （例如 hsa-miR-133b或它的同源体的 miRNA基因） 的遗传状态， 当遗传状态相对于对 照样本发生改变则暗示着高或低生存率。 例如， 在某些情况下， 这里为宫颈上 皮内瘤样病变或宫颈癌个体提供了生存率预后的方法， 包括分析 hsa-miR-133b 或与它的同源体对应的至少一个 miRNA基因的扩增情况， 当 miRNA基因相对 对照样本发生扩增则个体生存率低。 在某些情况下， 这里为宫颈上皮内瘤样病 变或宫颈癌个体提供了生存率预后的方法， 包括分析 hsa-miR-133b或与它的同 源体对应的至少一个 miRNA基因的拷贝数， 当 miRNA基因多于两个拷贝则个 体生存率低。 使用探针检测 miRNA水平
[00123]这里还提供了使用检测 miRNA水平（或 miRNA基因遗传状态）的 探针或使用包含一个或多个探针的系统为病人进行生存预后的方法。 例如， 具 体说， 这里提供了使用一个或多个探针 （或包含一个或多个探针的系统） 为宫 颈上皮内瘤样病变或宫颈癌个体生存预后的方法， 其中探针可以检测样本中的 miRNA, 并且 miRNA的水平相对于阈值与个体生存率正相关或负相关。在某些 情况下， 这里提供了使用一种或多种探针为宫颈上皮内瘤样病变或宫颈癌个体 生存预后的方法， 其中 miRNA水平相对阈值与个体生存率负相关， 并且其中至 少一个 miRNA是 hsa-miR-133b或它的同源体。
[00124]具体说， 这里提供了使用一个或多个探针生产一个为宫颈上皮内瘤 样病变或宫颈癌个体生存预后的试剂 （或系统） 的方法， 其中探针可以检测样 本中的 miRNA, 并且 miRNA的水平相对于阈值与个体生存率正相关或负相关。 具体说， 这里提供了使用一个或多个探针生产一个为宫颈上皮内瘤样病变或宫 颈癌个体生存率预后的试剂（或系统） 的方法， 其中 miRNA水平相对阈值与个 体生存率负相关， 并且其中至少一个 miRNA是 hsa-miR-133b或它的同源体。
[00125]此处讨论的生存可以是无疾病的生存或总的生存。 "无疾病的生存" 指的是确诊后的病人没有肿瘤复发和 /或扩散条件下的生存， 例如一个没有复发 肿瘤的存活病人。 "总的生存"指的是确诊病人无论肿瘤复发与否的总的生存。 阈值
[00126]此处的一些方法和使用涉及基于 miRNA 水平为生存预后， 而 miRNA水平是相对阈值水平而言的。
[00127]阈值可以通过很多方法测定。 假设得到的阈值可以准确地提供一个 miRNA水平， 高于此水平的一组病人生存率不同于低于此水平的另一组病人的 生存率。
[00128]阈值可以通过例如非癌宫颈上皮内瘤样病变或宫颈癌组织样本确 定。阈值也可以通过分析一群宫颈上皮内瘤样病变或宫颈癌病人的 miRNA水平 确定。 这可以通过例如柱状图分析完成， 图中包括被检测过的群体的所有个体， 其中一条轴表示 miRNA水平， 另一条轴表示个体生存率。 根据不同的 miRNA 水平将一个群体分成两个或多个独立的群体。 然后确定能最好地区分这些群体 的 miRNA水平阈值。 例如， 在某些情况下， 阈值可以基于具有高生存率群体的 miRNA水平平均值和具有低生存率群体的 miRNA水平的平均值。 阈值也可以 代表两个或多个 miRNA水平。两个或多个 miRNA的水平可以通过每个 miRNA 水平的比值表现。
[00129]阈值可以是适用于每个宫颈上皮内瘤样病变或宫颈癌个体的一个 数值， 也可以根据特定的人群设定不同的值。 例如， 老年妇女的阈值可以与年 轻妇女不同。 进一步， 也可以为每个个体设定一个阈值。 例如， 阈值可以是宫 颈上皮内瘤样病变或宫颈癌组织中某个 miRNA水平与同一个体中的非癌组织 中的该 miRNA水平的比值。
[00130]可以使用单变量或多变量分析验证阈值水平。 这些方法可以确定一 个或多个变量与结果之间的相关性。 在特定的情况下， 该方法可以确定 miRNA 水平与癌症患者无疾病生存或总的生存之间的关联。 这些分析方法中的任意一 个都为熟悉该领域内的常规技术的人所知道， 并可以使用这些方法进行分析。 单变量分析的例子是 Kaplan-Meir分析法或 Cox比例风险回归模型。
[00131]可以利用足够大的一个群体样本来确定阈值， 例如通过柱状图分析 将群体分成两个或多个具有不同 miRNA水平的患者小组。 这种群体包含至少
25个患者， 包括例如至少 50, 75, 100, 125, 150或 200个患者。 类似地， 对得到 的阈值进行确认也可以含至少 25个患者， 包括例如至少 50, 75, 100, 125, 150或 200个患者。
[00132]一个阈值可以将两组患者分开。 进一步， 多个阈值可以将患者区分 为多组。 例如， 两个阈值可以将患者分成 miRNA高水平、 中等水平、 低水平三 个小组。 不同阈值的数据可以可以绘制一条曲线， 如连续的曲线， 根据患者的 miRNA水平来描述患者的无疾病的或总的生存率的可能性。 根据这种曲线建立 的 "连续的" miRNA水平， 患者的无疾病或总的生存率的可能性与该患者的 miRNA水平成比例。 这种曲线可以代表两个或多个 miRNA的水平。
[00133]在某些情况下，此发明中用于为癌症患者生存率预后的 miRNA (如 此处描述的 miRNA) 可以组合使用。 两个或多个 miRNA的组合可以增加预后 的显著性或可信度。
[00134] MiRNA 的水平也可以与另一个指标联合使用。 这个指标对预测宫 颈上皮内瘤样病变或宫颈癌患者无疾病或总生存率具有统计学意义。 这样的指 标包含例如病理的指标 （例如年龄、 肿瘤大小、 肿瘤组织学、 临床分期、 家族 史等等）。 例如， 癌症的临床分期是统计学上显著的预测无疾病或总生存率的指 标。 因此， 当作为另一个宫颈上皮内瘤样病变或宫颈癌患者无疾病或总生存率 的指标使用时， miRNA的阈值可以不同。
[00135]在某些情况下， Kaplan-Meier分析被用来检测生存率与 miRNA水 平间的关系。
[00136]具体说， 此方法包括： （a) 检测个体宫颈上皮内瘤样病变或宫颈癌 组织中至少一个 miRNA水平； （b) 根据 miRNA水平将宫颈上皮内瘤样病变或 宫颈癌患者进行分类。 高 miRNA水平组患者比低 miRNA水平组患者生存率更 低， 其中至少一个 miRNA是 hsa-miR-133b。
[00137]当患者样本中的一个或多个 miRNA水平检测完并与阈值比较后， 将患者分到一个群组中。 个体的无疾病生存率或总生存率通过对群组的无疾病 生存率或总生存率的评估得出。
[00138]例如， 一个样本可能被检测为 miRNA低水平。 此患者将被分到 miRNA低水平的群组。 因为已知 miRNA低水平群组的无疾病或总生存率可能 性较高， 所以该患者则具有较高的无疾病或全面生存率。
[00139]此处描述的方法可能进一步含有针对个体决定合适的治疗方法的 步骤。 通常认为， 早期阶段癌症病人的生存率与晚期阶段癌症病人的生存率不 同。 例如， I期的预后可能指示癌症的持续增长和 /或转移， 而 IV期的预后可能 指示癌症治疗方法的有效性。 决定合适的治疗方式将考虑这些因素。 宫颈癌或宫颈上皮内瘤样病变个体治疗药物及治疗方法
[00140]在某些情况下， 这里提供了一个治疗药物， 其中包含可以降低 miRNA 水平的组分和药学上可接受的载体， 其中至少一个 miRNA 是 hsa-miR-133b, hsa-miR-104-3p, hsa-miR-143*。在某些情况下， 至少一个 miRNA 是 hsa-miR-133b。 在某些情况下， 至少一个 miRNA是 hsa-miR-104-3p。 在某些 情况下， 至少一个 miRNA是 hsa-miR-143 *。在某些情况下， 该组分是双链 RNA (例如短的或小干扰 RNA， 或" siRNA") , 反义核酸， 或具有酶活性的 RNA分 子如核酶。 改善生存率的方法和药物将在下文详述。
[00141]这里还提供使用药物降低特定 miRNA 水平， 如 hsa-miR-133b, hsa-miR-100, hsa-miR-104-3p, hsa-miR-143 * , ±曾加宫颈上皮内瘤样病变或宫颈 癌患者的生存率。
[00142]任意可以降低 miRNA水平的药物组分都可以在此发明的方法中使 用。 抑制 miRNA基因表达的合适的组分包括， 但不局限于， 双链 RNA (例如 短的或小干扰 RNA，或 " siRNA") , 反义核酸， 具有酶活性的 RNA分子如核酶， 小分子复合物， 以及蛋白质。 这些组分可以单独使用也可以与其他组分 （如此 处描述的其他组分）组合使用。 这些组分可以直接（如通过抑制 miRNA表达或 其功能） 或间接地 （如作用于 miRNA基因的遗传状态） 降低 miRNA水平。
[00143]例如，一个特定的 miRNA基因可以通过诱导 RNA干扰抑制其表达。 此方法通过一个与该 miRNA基因产物的一部分具有至少 70%， 包括例如至少 75%, 80%, 85%, 90%, 95%, 98%, 99%或 100%的序列同源性的双链 RNA
(" dsRNA")分子进行。具体说， dsRNA分子是短的或小干扰 RNA (" siRNA") .
[00144]可以有效用于当前这些方法的 siRNA可以是 10-30个核苷酸长度的 短双链 RNA (包括例如， 约 12-28, 14-26, 16-24, 或 18-22核苷酸）。 siRNA可 以含有一条正义 RNA链和一条互补配对的反义 RNA链，二者按照 Watson-Crick 碱基互补配对原则通过退火形成双链。正义链含有与靶标 miRNA基本相同的核 酸序列。 siRNA的正义链和反义链可以由互补配对的两条单链 RNA组成， 也可 以由一个分子互补配对的两个部分通过单链的 "发卡"结构连在一起组成。
[00145]通过一个或多个核苷酸的插入、缺失、替换和 /或转换， siRNA可能 与自然形成的 RNA不同。这些改变包括非核苷酸物质的加入（如加在 siRNA的 末端或内部）， 导致 siRNA能抵抗核酶消化的修饰， 或者将 siRNA中的一个或 多个核苷酸替换为脱氧核苷酸。 在某些情况下， siRNA 的一条或两条链可以包 含 3' 突出末端。 siRNA可以通过化学或生物学的方法得到， 或者从重组的质粒 或病毒载体表达得到， 这将在下文进行阐述。
[00146]一个特定的 miRNA的表达也能被反义核酸抑制。在此， "反义核苷 酸"指的是能够通过 RNA-RNA或 RNA-DNA相互作用的方式结合目标 RNA， 从而改变目标 RNA活性的核酸分子。适用于当前方法的反义核苷酸可以是包含 与 miRNA毗邻序列互补的单链核酸（如 RNA， DNA, RNA-DNA嵌合体， PNA 和 LNA)。 在某些情况下， 反义核酸包含与 miRNA毗邻序列至少有 70% (例如 至少 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 或 100% )互补配对的核酸序 列。在某些情况下，反义核酸的长度大约为 10-30核苷酸 (包括例如约 12-28, -26.
16-24, 或 18-22核苷酸）。
[00147]反义核酸也可以包含对核酸骨架或糖和碱基 （或它们的等价物） 的 修饰， 从而增强靶标特异性、 抵抗核酸酶降解能力、 运输或其他功效相关的特 征。 这些修饰包括胆固醇家族分子， 双螺旋插入物如吖啶， 或者一个或多个具 有核酸酶抗性的组分。
[00148]反义核酸可以通过化学或生物学的方法得到， 或者从重组的质粒或 病毒载体表达得到， 这将在下文进行阐述。
[00149]一个特定的 miRNA的表达也能被具有酶活性的核酸抑制。 在此， "具有酶活性的核酸"指的是含有底物结合区域的核酸， 该区域与 miRNA的毗 邻序列互补配对， 可以特异性地剪切 miRNA。 在某些情况下， 具有酶活性的核 酸结合区域与 miRNA毗邻序列有 50-100%的互补性 （包括例如 75-95%互补性 或 95-100%互补性）。一个酶活性的核酸也可以在碱基、糖、磷酸组分上有修饰。 一个典型的可以用于当前方法的具有酶活性的核酸就是核酶。
[00150]具有酶活性的核酸可以通过化学或生物学的方法得到， 或者从重组 的质粒或病毒载体表达得到， 这将在下文进行阐述。
[00151]当前有很多方法可以将核酸分子导入到细胞中， 包括癌细胞。 这些 方法包括显微注射， 电穿孔， 脂质体转染， 磷酸钙介导的转染， DEAE 葡聚糖 介导的转染， 微粒子轰击法， 通过胶态分散体运输 （例如大分子复合物， 凝胶 颗粒， 水油乳化剂， 胶态离子， 混合胶态离子和脂质体）， 以及与抗体、 短杆菌 肽、 人造病毒外壳或其它细胞内载体如 TAT。
[00152]核酸药剂也可以通过文献中已知的载体导入到体外或体内的哺乳 动物细胞中。 合适的载体有病毒载体和非病毒载体， 如质粒载体。 这些载体在 提供具有治疗效果剂量的反义 RNA或 siRNA等药物上很有帮助。
[00153]基于病毒的系统的优势在于能够相对高效地导入异源的核酸到各 种各样的细胞中。 导入核酸的合适的病毒载体有单纯疱疹病毒载体， 牛痘病毒 载体， 细胞巨化病毒载体， 鼠莫洛尼氏白血病毒载体， 腺病毒载体， 腺关联病 毒载体， 逆转录病毒载体和慢病毒。 病毒载体的趋向性可以通过使用其它病毒 的包膜蛋白或表面抗原来控制。 例如， 腺关联病毒载体可以假借口腔小泡病毒， 狂犬病毒， 埃博拉病毒， Mokola等病毒的表面蛋白来实现这些病毒的趋向性。
[00154]此发明中的核酸或载体中可以含有任意一种可诱导的启动子或增 强子，从而可以通过剌激或添加分子诱导反义 RNA或 siRNA的表达。这些诱导 系统包括诸如四环素诱导系统， 重金属诱导的金属硫蛋白启动子， 蜕皮激素或 相关的类固醇如鼠 酮应答的昆虫类固醇激素， 类固醇如糖皮质激素和雌激素 诱导的小鼠乳腺肿瘤病毒 （MMTV), 以及温度改变诱导的热激启动子。
[00155]如果一个药物的剂量能够足以改变其靶标 miRNA 的水平 （如降 低）， 那么这个剂量可以说是此药物的有效剂量。 在某些情况下， 一种药物可以 将靶标 miRNA的水平至少降低至与阈值间差异的 10%, 20%, 30%, 40%或 50%。 此处提供的药物 （如核酸药物） 的典型剂量包括 0.1-3000 mg/kg体重， 10-2000 mg/kg体重， 50-1000 mg/kg体重， 以及 100-500 mg/kg体重， 但并不局限于这些 范围。 在某些情况下， 药物 （如核酸药物） 的剂量是 100-500 mg/g肿瘤重量，
20-300 mg/g肿瘤重量， 50-200 mg/g肿瘤重量， 或 100-150 mg/g肿瘤重量。
[00156]该领域内的技术人员可以很容易地确定给个体一种或多种药物的 合适剂量。 典型的给药频率包括但不局限于， 至少每三周一次， 每两周一次， 每周一次， 每周两次， 每周三次， 每周四次， 每周五次， 每周六次， 或每天一 次。 在某些情况下， 两次给药的时间间隔可以少于一周， 例如少于每六、 五、 四、 三、 二或一天。 在某些情况下， 两次给药的时间间隔是固定的。 例如， 可 以每天， 每两天， 每三天， 每四天， 每五天， 每六天或每周给药一次。 在某些 情况下， 可以每天给药两次， 三次或更频繁。
[00157]—种药物的给药时间可以是长期的， 诸如从大约一个月到三年。 例 如一种给药方式可以长达 2、 3、 4、 5、 6、 7、 8、 9、 10、 11、 12、 18、 24、 30 或 36个月。 在某些情况下， 在给药期间不能停药。 在某些情况下， 每两次给药 的间隔不能长于一周。
[00158]此处描述的药物可以通过此领域内的任意途径给个体给药， 包括但 不局限于， 静脉内， 腹腔内， 眼内， 动脉内， 肺内， 口服， 肺泡内， 肌肉， 呼 吸管， 皮下， 脑脊髓膜内， 跨皮肤， 跨胸膜， 局部的， 吸入 （如喷剂）， 跨粘膜 (如通过鼻粘膜）， 通过肠胃， 关节内， 尿道内， 心室内， 直肠内（如通过栓剂）， 阴道内 （如通过阴道栓剂）， 颅骨内， 肝内， 以及肿瘤内。 在某些情况下， 可以 全身性的给药。 在某些情况下， 可以局部给药。
[00159]这里还提供了药学上可接受的载体和能降低 miRNA水平的试剂组 成的药物。 在某些情况下， 药物包含一种成分， 此成分可以降低某个 miRNA的 水平， 该 miRNA是 hsa-miR-133b或它的同源体的一个。在某些情况下， 至少一 个 miRNA是 hsa-miR-133b。 在某些情况下， 该成分是 siRNA。 在某些情况下， 该成分是反义 RNA。 在某些情况下， 该成分是核酶。
[00160]具体说， 药物是无菌的。 具体说， 药物是无热源的。
[00161]合适的药学上可接受的载体有水、 水溶液、 标准盐溶液， 0.4%的盐 溶液， 0.3%的甘氨酸和透明质酸。 药物还可以包含传统的药物赋形剂和 /或添加 齐 lj。 合适的药物赋形剂包括稳定剂、 抗氧化剂、 渗透性调节剂、 缓冲液、 pH调 节剂。 合适的添加剂包括， 例如生理学上不排斥的缓冲液， 螯合剂 （如 DTPA 和 DTPA双酰胺） 以及钙螯合复合物（如钙 DTPA和 CaNaDTPA双酰胺）， 钙盐 或钠盐 （如氯化钙、 抗坏血酸钙、 葡萄糖酸钙和乳酸钙）。 此发明中的药物可以 是液体包装， 也可以是冻干品。
[00162]对于此发明中的固体药物， 可以使用制药学上可接受的传统的无毒 固体载体。 药学上可接受的固体载体包括制药级别的甘露醇、 乳糖、 淀粉、 硬 脂酸镁、 糖精钠、 滑石、 纤维素、 葡萄糖、 蔗糖、 碳酸镁等等。
[00163]此发明还提供了提高宫颈上皮内瘤样病变或宫颈癌患者生存率的 方法。 具体说， 这里提供了提高宫颈上皮内瘤样病变或宫颈癌个体生存率的方 法， 包括对该个体给予可以降低 miRNA水平的有效剂量的药物， 此 miRNA水 平相对于阈值与个体的生存率呈负相关性。 具体说， 这里为药物生产商提供了 降低 miRNA水平、 提高宫颈上皮内瘤样病变或宫颈癌患者生存率的药物， 此 miRNA水平相对于阈值与患者的生存率呈负相关性。
[00164]在某些情况下， 这里提供了提高宫颈上皮内瘤样病变或宫颈癌个体 生存率的方法， 包括对该个体给予可以降低 miRNA水平的有效剂量的药物， 此 miRNA选自于包含 hsa-miR-133b, hsa-miR-140-3p以及它们的同源体的 miRNA 组。 在某些情况下， 这里为药物生产商提供了提高宫颈上皮内瘤样病变或宫颈 癌个体生存率的药物， 此药物可以降低 miRNA水平， 此 miRNA选自于包含 hsa-miR-133b, hsa-miR-140-3p以及它们的同源体的 miRNA组。
[00165]此处描述的方法可以进一步包含为使用此药物前的个体生存率进 行预后 （例如， 通过此处描述的方法） 的步骤。
[00166]在某些情况下， 多于一个 miRNA的水平有下降。 这时就可以使用 可以降低两个或多个 miRNA水平的成分。此外， 可以使用两个或多个成分来降 低两个或多个 miRNA水平。 例如， 在某些情况下， 这里提供了提高宫颈上皮内 瘤样病变或宫颈癌个体生存率的方法， 包括对该个体给予一种或多种可以降低 至少两个 miRNA 水平的有效剂量的药物， 这些 miRNA 选自于包含 hsa-miR-133b, hsa-miR-140-3p, hsa-miR-143*以及它们的同源体的 miRNA组。 在某些情况下， 这里为药物生产商提供了提高宫颈上皮内瘤样病变或宫颈癌个 体生存率的一个或多个药物， 这些药物可以降低至少两个 miRNA水平， 这些 miRNA选自于包含 hsa-miR-133b, hsa-miR-143 *， hsa-miR-140-3p以及它们的同 源体的 miRNA组。在某些情况下， 这里提供了提高宫颈上皮内瘤样病变或宫颈 癌个体生存率的方法， 包括对该个体给予一种或多种可以降低 hsa-miR-133b, hsa-miR-143*, hsa-miR-140-3p以及它们的同源体水平的药物。 在某些情况下， 这里为药物生产商提供了提高宫颈上皮内瘤样病变或宫颈癌个体生存率的一个 或多个药物， 这些药物可以降低 hsa-miR-133b, hsa-miR-143* , hsa-miR-140-3p 以及它们的同源体的水平。 扩增 miRNA的寡核苷酸引物
[00167]另一方面， 该发明提供了扩增一个 RNA序列的一个寡核苷酸引物， 它包含了一个具有如下特点的核苷酸序列： a) 在高严谨条件下， 与一个核酸序 列或它的互补序列杂交， 如序列表所列序列所示； b) 与一个核酸序列或它的 互补序列， 如序列表所列序列， 具有至少 90%的相似性。
[00168]当前的引物可以包含任意合适的核酸， 例如 DNA、 RNA、 PNA或 它们的衍生物。 含有一个序列表所示的核酸序列或者它们的互补序列的引物更 好。 标记的引物也更好， 例如化学的、 酶学的、 免疫学的、 放射性的、 荧光的、 发冷光的以及 FRET标记。
[00169]寡核苷酸引物可以通过任意合适的方法生产。 例如， 引物可以通过 化学合成 （参考 Ausubel (Ed.) Current Protocols in Molecular Biology, 2.11. Synthesis and purification of oligonucleotides, John Wiley & Sons, Inc. (2000)), 从 天然来源中分离， 重组产生或者联合这些方法得到。 合成寡核苷酸也可以参考 以下文献中的方法 Matteucci et al., J. Am. Chem. Soc, 3:3185-3191 (1981)。 此夕卜， 自动合成可能更好， 例如， 采用氰乙基亚磷酰胺化学在 DNA合成仪上合成。 化 学合成引物的方法是首选的。
[00170]用于合成寡核苷酸引物的碱基可以选择天然碱基， 例如腺嘌吟、 胞 嘧啶、 鸟嘌吟、 尿嘧啶和胸腺嘧啶。 也可以选择非天然的或 "合成的"碱基， 例如 8-氧鸟嘌吟、 6-巯基鸟嘌吟、 4-乙酰基胞嘧啶、 5- (羧基羟基乙烷基）尿苷、 2'-0-甲基胞苷、 5-羧基甲基氨基 -甲基 -2-胸苷、 5-羧基甲基氨基甲基尿苷、 二氢 尿苷、 2'-0-甲基假尿苷、 β -D-半乳糖基环戊烯、 2'-甲氧基鸟苷、 次黄嘌吟、 N6-异戊烯基腺苷、 1-甲基腺苷、 1-甲基假尿苷、 1-甲基鸟苷、 1-甲基肌苷、 2， 2-双甲基鸟苷、 2-甲基腺苷、 2-甲基鸟苷、 3-甲基胞苷、 5-甲基胞苷、 N6-甲基腺 苷、 7-甲基鸟苷、 5-甲基氨基甲基尿嘧啶、 5-甲氧基氨甲基 -2-硫尿苷、 β -D-甘 露糖基环戊烯、 5-甲氧基羰基甲基尿苷、 5-甲氧基尿苷、 2-硫代甲基 -N6-异戊烯 基腺苷、 N- ( (9- β -D-呋喃基 -2-甲基硫代嘌吟 -6) 氨基甲酰） 苏氨酸、 N- ( (9- β -D-呋喃基嘌吟 -6) N甲基氨基甲酰） 苏氨酸、 尿苷 -5-羟乙酸甲基酯、 尿苷 -5- 羟乙酸、 假尿苷、 环戊烯、 2-硫代胞苷、 5-甲基 -2-硫代尿苷、 2-硫代尿苷、 5-甲 基尿苷、 2'-0-甲基 -5-甲基尿苷、 2'-0-甲基尿苷和 3- (3-氨基 -3-羧基丙烷基）鸟 苷。
[00171]同样地， 也可以使用寡核苷酸类似物 （例如磷酸二酯键被修饰的寡 核苷酸， 例如甲基磷酸酯、 磷酸三酯、 硫代磷酸酯、 二硫代磷酸酯、 或氨基磷 酸酯）。 通过 "3'末端加帽"可以保护引物不降解， 通过该技术将核酶抑制连接 替代寡核苷酸 3'末端的磷酸二酯（Shaw et al., Nucleic Acids Res., 19:747 (1991))。 氨基磷酸酯、 硫代磷酸酯和甲基磷酸酯连接功能相同。 对磷酸二酯骨架更为广 泛的修饰可以增加寡核苷酸的稳定性， 促进寡核苷酸的亲和性和寡核苷酸的细 胞渗透性 （Milligan et al., J. Med. Chem., 36: 1923 (1993))。 为了用新的连接替代 整个磷酸二酯骨架已经使用过很多不同的化学方法。 骨架类似物包括甲基磷酸 酯、 磷酸三酯、 硫代磷酸酯、 二硫代磷酸酯、 氨基磷酸酯、 硼基磷酸酯。 硫代 磷酸酯和甲基磷酸酯修饰的寡核苷酸可以通过自动寡核苷酸合成获得， 因此更 受欢迎。 寡核苷酸可以是 "肽核酸"， 如以下文献所述 （Milligan et al., J. Med. Chem., 36: 1923 (1993) ) 0唯一的要求就是寡核苷酸引物必须含有一段序列，该序 列至少一部分能够结合靶标 RNA序列的一部分。 试剂盒
[00172]此发明还提供了用于此处描述的各种方法的试剂盒。
[00173]例如， 在某些情况下， 这里提供了一个试剂盒， 内含此处描述的检 测 miRNA水平的系统 （例如微阵列）。 在某些情况下， 试剂盒还可以额外包括 用于检测的试剂。 试剂盒还包括详细描述此处提到的操作方法的说明书， 和 /或 提供具有此类说明的网址。
[00174]在某些情况下， 这里提供了一个试剂盒， 内含此处描述的用于宫颈 上皮内瘤样病变或宫颈癌诊断的系统 （例如微阵列）。 此外还可以包括一个或多 个对照样本来决定参考水平， 和 /或如何得到参考水平的信息。 在某些情况下， 试剂盒还可以包括使用此试剂盒进行宫颈上皮内瘤样病变或宫颈癌诊断的说明 书。
[00175]在某些情况下， 这里提供了一个试剂盒， 内含此处描述的宫颈上皮 内瘤样病变或宫颈癌患者的分类系统 （例如微阵列）。 此外还可以包括一个或多 个对照样本来决定个体分类， 和 /或关于对照样本的信息， 以及在某些情况下， 含有个体分类的试剂盒使用说明。
[00176]在某些情况下， 这里提供了一个试剂盒， 内含此处描述的用于结肠 直肠癌、 舌头鳞状细胞癌、 食管鳞状细胞癌和胰腺导管癌诊断的系统 （例如微 阵列）。此外还可以包括一个或多个对照样本来决定参考水平， 和 /或如何得到参 考水平的信息。 在某些情况下， 试剂盒还可以包括使用此试剂盒进行结肠直肠 癌、 舌头鳞状细胞癌、 食管鳞状细胞癌和胰腺导管癌诊断的说明书。
[00177]在某些情况下， 这里提供了一个试剂盒， 内含此处描述的结肠直肠 癌、 舌头鳞状细胞癌、 食管鳞状细胞癌和胰腺导管癌患者的分类系统 （例如微 阵列）。此外还可以包括一个或多个对照样本来决定个体分类， 和 /或关于对照样 本的信息， 以及在某些情况下， 含有个体分类的试剂盒使用说明。
[00178]在某些情况下， 这里提供了为宫颈上皮内瘤样病变或宫颈癌患者生 存率预后的试剂盒。 这种试剂盒包含例如检测 miRNA的探针。 在某些情况下， 试剂盒可以包含决定阈值的对照样本， 和 /或如何获得阈值的信息。 在某些情况 下， 还包括用此试剂盒为患者预后的使用说明。 在某些情况下， 试剂盒可以包 括降低 miRNA水平的试剂， 或包含这种试剂的药物， 帮助提高生存率。
[00179]此处提到的试剂盒还可以包括一些试剂， 包括但不局限于底物， 标 记物， 弓 I物， 标记 miRNA的试剂， 分离 miRNA的试剂， 杂交和检测的阴性或 阳性对照， 管子和 /或其它附件， 收集组织样本的试剂， 缓冲液， 杂交盒， 盖片 等等， 还有可能包含软件包（如使用此处提到的统计学方法分析 miRNA水平和 /或 miRNA水平特征性变化），和用于获取数据库信息的任意一个密码和 /或用户 名。
[00180]在某些情况下，这里提供了一个试剂盒，可以包含降低一个 miRNA 水平的药物， 及其用它提高颈上皮内瘤样病变或宫颈癌患者生存率的使用说明。 在某些情况下， 试剂盒还可以包括用来输送药物复合物的一个或多个载体或其 它试剂。 在某些情况下， 试剂盒还包括药物复合物的使用说明。 例子
[00181] 以下例子是用来阐释该但并不限制该发明。
案例 1
样本准备以及利用 miRNA微阵列进行 miRNA水平分析
病人和样本
[00182]利用 5对宫颈癌组织和相应的正常宫颈组织。 这些组织样本来源于 新疆医科大学第一附属医院 2006-2008年的患者，经过患者知情同意。所有组织 样本都来源于没有经过治疗便进行手术的患者，样本在提取 miRNA之前都经过 福尔马林固定、 石蜡包埋（FFPE)。 肿瘤组织经过病理医生确认， 肿瘤细胞所占 比例也经过病理医生确认。 该研究经过新疆医科大学第一附属医院医学道德规 范委员会批准。
MiRNA微阵列制作
[00183] MiRNA微阵列包括 509条成熟的 miRNA序列，其中包括网站登记 的 （网址 http：〃 microma.sanger.ac.uk) 435条人成熟 miRNA (包括报道的 122条 预测 miRNA序列 (Xie et al.， 2005))， 196条大鼠成熟 miRNA, 261条小鼠成熟 miRNA。此外，我们设计了 8条与已知的任意 RNA序列均无同源性的寡核苷酸， 并用 Ambion的 miRNA引物构建试剂盒（Cat. No.1550; Ambion, Inc., Austin, TX) 通过体外转录合成了它们相应的 miRNA。 这些合成的 miRNA作为内参以不同 量在分析前加入到人 miRNA样本中。
[00184]所有的 miRNA探针序列都与它们相应的成熟的 miRNA的全长序列 完全互补配对 （也即制作的 20个探针的序列分别与序列表中序列 1至序列 20 的 20条序列完全互补）。 为了使探针易于固定到醛基修饰的玻片上 （CapitalBio Corp., Beijing, China), 每个探针序列都设计为约 40 nt 寡核苷酸 （3'末端为 miRNA序列， 5'末端为 19聚 polyT, 5'端 C6氨基修饰）。寡核苷酸探针在 MWG Biotech公司合成，用 EasyArray™点样液（CapitalBio Corp. )溶解，浓度为 40μΜ。 使用 SmartArray™ microarrayer (CapitalBio Corp. ) 点制芯片， 每个探针有三个 重复点。
目标 RNA标记
[00185]根据以前报道的方法（Varnholt et al., 2008)从石蜡包埋组织中提取 总 RNA。 简单地说， 将组织切片 （20 μιη厚度） 用二甲苯处理去掉石蜡， 并用 100%的乙醇清洗。然后用蛋白酶 K 55°C消化样本 12h。采用酚 /氯仿提取总 RNA， 然后用异丙醇沉淀。 水溶解后， 用 NanoDrop D- 1000分光光度计 （Nano-Drop Technologies, Wilmington, DE)检测浓度。根据 Thomson的方法（Thomson et al.， 2004) 用 T4 RNA连接酶标记法标记目标 RNA。 简单地说用 2单位 T4 RNA连 接酶 （New England Biolabs, Beijing, China) 将 4 g小分子量 RNA用 500 ng的 5'-磷酸-胞嘧啶-脲嘧啶 -cy3-3， (Dharmacon; Lafayette, CO) 标记。 标记反应在 16°C进行 4小时。 标记的 RNA用 0.3M的醋酸钠和 2.5体积乙醇沉淀， 乙醇清 洗、 空气干燥后， 用含有 3xSSC， 0.2% SDS和 15%的甲醛的杂交液 15 μΐ重悬 标记的 RNA。
玻片杂交
[00186]标记后的 RNA 力 B到 miRNA 芯片上并用 LifterSlip™ ( Erie; Portsmouth, H) 盖玻片覆盖。 杂交在置于三阶段倾斜的混合仪 BioMixe™ (CapitalBio Corp. )中的杂交盒内进行， 使杂交液在整个玻片表面分布均匀， 避 免边缘效应。此操作的效率在我们的 mRNA表达谱平台上得到了证明。 50°C杂 交过夜。 然后将微阵列芯片连续清洗两次： 第一次在 0.2% SDS, 2xSSC的清洗 液中 50°C清洗 5分钟， 第二次在 0.2% SSC的清洗液中室温清洗 5分钟。 然后 用共聚焦扫描仪 LuxScan™对阵列进行扫描， 得到的图片用 LUxSCan TM3.0TM软 件分析 （都来源于 CapitalBio Corp. )。
数据分析
[00187]每个 miRNA重复点的平均值都进行了背景消除、 校正后再做进一 步分析。 校正的方法是利用每张芯片的中值进行校正。 芯片数据再进行过滤， 去除在所有样本中信号都低于 500 的基因， 然后通过微阵列显著性分析软件
( SAM ) (网址 www-stat.stanford.edu/~tibs/SAM/index.html ) 找出差异表达的 miRNA。 SAM软件根据表达变化与所有检测的标准偏差为每一个基因打分。 使 用平均数联结法和 Pearson相关系数进行等级聚类。 差异表达的 miRNA聚类结 果见图 1。 与正常宫颈组织相比， 宫颈癌组织中有 13个 miRNA的表达发生了 明显的上调， 7个 miRNA的表达发生了明显的下调。 案例 2
RT-PCR区分 hsa-miR-133a与 hsa-miR-133b在宫颈癌组织中的表达
[00188]案例 1的结果表明， hsa-miR-133a与 hsa-miR-133b是宫颈癌组织中 表达量发生显著上升的两个 miRNA。成熟的 hsa-miR-133a与 hsa-miR-133b序列 仅在 3'-端相差一个核苷酸，如表 1所示。这种差别在使用微阵列芯片进行 miRNA 表达谱分析时是区分不开的。 但是， hsa-miR-133a前体（pre-hsa-miR-133a)序 列与 hsa-miR-133b前体 （pre-hsa-miR-133b)序列的差别很大， 可根据两个前体 的序列设计特异性的 RT-PCR 引物区分细胞或组织中 hSa-miR-133a 与 hsa-miR-133b的表达。关于 pre-hsa-miR-133a与 pre-hsa-miR-133b的序列信息可 以在以下网站上找到 http：〃 miRNA. sanger. ac.uk/ (Griffths- Jones, et al, Nucleic Acids Research, 2006, Vol. 34, Database issue)。
[00189]用于扩增 pre-hsa-miR-133a与 pre-hsa-miR-133b的引物如表 2所示。 6个正常宫颈和 6个宫颈癌的 FFPE组织的总 RNA提取如案例 1所述。 逆转录 反应包含 10 ng/μΐ 总 RNA, 25 nM逆转录引物， 1 x 逆转录缓冲液， 0.25 mM dNTP, 7.5 U ThermoScript™逆转录酶和 0.25 U/ml RNA酶抑制剂 (Invitrogen, Carlsbad, CA)。 20 μΐ的反应体系在 MJ Research PTC-225 Thermocycler中 60 °C 孵育 30 min， 85°C孵育 5 min， 然后停在 4°C。
[00190] 50 μΐ MiRNA前体的扩增反应体系包括 200 nM dNTP, 1 PCR缓 冲液， 15 nM前向引物和 15 nM 反向引物， 2 μΐ逆转录产物， 1.25 U HotStar® Taq DNA聚合酶 （Qiagen)。 反应条件为 95°C孵育 10 min, 然后是 40个循环 的 95°C 孵育 15 s, 70 °C孵育 20 s。 反应后的扩增产物各取 5 μΐ进行 1.5%的琼 脂糖电泳， 结果如图 2。 Pre-hsa-miR-133a与 pre-hsa-miR-133b的扩增结果表明， 宫颈癌组织中主要表达 pre-hsa-miR-133b。 因此， 宫颈癌组织 hsa-miR-133的升 高主要是 hsa-miR-133b的升高引起的。 表 2 扩增 pre-hsa-miR-133a与 pre-hsa-miR-133b的引物
Figure imgf000039_0001
案例 3
RT-PCR分析 miRNA水平
[00191]来源于 FFPE组织的总 RNA提取如案例 1所述。逆转录反应试剂盒 购自 Exiqon公司。 10 μΐ反应体系中包含 10 ng总 RNA, 2 μΐ逆转录引物， 1 RT 缓冲液， 0.2 mM dNTP, 0.5 μΐ逆转录酶和 0.5 μΐ RNA酶抑制剂。 反应体系在 MJ Research PTC-225 Thermocycler中 50 °C孵育 30 min, 85°C孵育 5 min, 然后停 在 4°C。 所有逆转录反应， 包括无模板对照， 都做两次重复。 实时荧光 PCR使 用 miRCURY LNA™ microRNAPCR System试剂盒（Exiqon, Vedbaek, Denmark) 禾口 LightCycler仪器 (Roche Diagnostics, Mannheim, Germany)进行。 20 μΐ PCR体 系包括 4 μΐ稀释 10倍后的逆转录产物， 10 μΐ SYBR® Green master mix, 1 μΐ LN™ PCR引物和 1 μΐ Universal PCR引物。 反应条件为 95°C孵育 10 min, 然后 是 60个循环的 95°C 孵育 10 s, 60°C 孵育 20 s。
[00192]所有实时荧光 PCR反应，包括无模板对照，都做两次重复。 MiRNA 的相对表达水平通过拐点的循环数计算得到。 使用人 U6 基因表达水平作为内 参。 结果使用 LightCycler软件 3.5版（Roche Diagnostics)进行分析。 实时 PCR 扩增产物通过溶解曲线分析， 并用琼脂糖凝胶电泳进行验证。 引物序列如表 3 所示， 结果如图 3 所示。 随着宫颈癌的发展， 从宫颈上皮内瘤样病变 II期， 宫 颈上皮内瘤样病变 III到侵润癌， hsa-miR-133b的表达量逐渐升高。 表 3 RT-PCR引物序列
Figure imgf000040_0001
案例 4
原位杂交验证 hsa-miR-133b在宫颈癌组织中表达上升
[00193]原位杂交实验按 Exiqon 公司 提供 的操作流程进行
(http://www. exiqon. com/uploads/LN A_52-_FFPE_miRNA_in_situ_protocol.pdf)。杂 交反应液中含 50 nM miRCURY LNA™ miR-133b探针或 Scramble-miR探针 (Exiqon), 45 °C 反应过夜。 实验结果如图 4。 H&E染色与 Ki-67免疫组化结 果清楚的显示了宫颈组织中正常上皮， 上皮内瘤样病变 III期和侵润癌组织。 原 位杂交结果显示正常上皮中仅基底层的一些细胞表达 hsa-miR-133b， 而上皮内 瘤样病变 III期和侵润癌组织细胞中均高表达 hsa-miR-133b。 案例 5
严重联合免疫缺陷 （severe combined immuno-deficiencv ， SCID) 鼠皮下成瘤实 验验证 hsa-miR-133b促进肿瘤的形成
[00194]为研究 hsa-miR-133b表达水平的变化对宫颈癌形成的影响，利用引 物 5' CTGACAGGATCCGTAAGAGGACATTCTGGACAAGGCAAGC 3'禾口 5' CGCACGAATTCATTCCTGGGAGCATAAGAATATGGTGAAA 3 '扩增离体的人 基因组 DNA中的 miR-133b基因。 扩增后的产物通过 BamHI和 EcoRI消化后， 克隆到 pcDNA 3.1 -neomycin载体 （Invitrogen, Carlsbad, CA) 中。 重组质粒经测 序验证。 含有 miR-133b基因的重组质粒和空载体质粒 （阴性对照） 转染 CaSki (购自中国医学科学院基础医学研究所基础医学细胞中心） 后， 用 800 μ§/ιη1 G418筛选稳定细胞株。稳定细胞株中 miR-133b基因的过量表达经实时荧光 PCR 验证。
[00195] 5 X 106个稳定表达 hsa-miR-133b的 CaSki细胞 （CaSki-miR-133b) 或阴性对照 CaSki细胞（CaSki-NC)接种到 SCID鼠腋窝皮下。每种细胞分别接 种 8只鼠。 接种后的 SCID鼠在恒温 （25 °C±2°C )、 恒湿 （45%〜50%)、 无菌净 化屏障系统内饲养。 每 3 天测量一次肿瘤的体积， 实验结果如图 5。 高表达 hsa-miR-133b 的 CaSki细胞形成的肿瘤 6天后就与阴性对照 CaSki细胞形成的 肿瘤有明显差异。随着时间的推移，肿瘤大小的差异越来越大，说明 hsa-miR-133b 具有明显的促进肿瘤形成的作用。 案例 6
SCID鼠肺转移瘤形成实验验证 hsa-miR-133b促进转移瘤的形成
[00196]首先构建含有抗嘌吟霉素基因的 miR-133b表达质粒。 构建过程如 下：第一步使用 Bgl II和 Pvu II酶消化案例 5中的质粒 pcDNA3.1 -neomycin和含 有 miR-133b 基因的质粒。 消化后的产物经 1%琼脂糖电泳后切胶纯化小片段 DNA。第二步用 EcoR I消化 pSIREN-RetroQ载体（Clontech, Mountain View, CA) 后， 再用 T4 DNA polymerase处理消化后产物， 使之平端化。 然后用 Bgl II消化 载体， 消化后的产物经 1%琼脂糖电泳后切胶纯化大片段。 第三步使用 T4 DNA 链接酶链接第一步的小片段 DNA和第二步的大片段 DNA。 转化大肠杆菌后筛 选重组质粒， 并经测序验证。含有 miR -133b基因的重组质粒和空载体质粒（阴 性对照） 转染 SiHa细胞 （购自中国科学院上海生命科学研究所细胞资源中心） 后， 用 5 μ§/ιη1 嘌吟霉素筛选稳定细胞株。 稳定细胞株中 miR-133b基因的过量 表达经实时荧光 PCR验证。
[00197] 2 X 106个稳定表达 hsa-miR-133b的 SiHa细胞（ SiHa-miR-133b)或 阴性对照 SiHa细胞 （SiHa-NC) 通过尾静脉注入到 SCID鼠体内。 每种细胞分 别接种 7只鼠。 接种后的 SCID鼠在恒温 （25 °C±2°C )、 恒湿 （45%〜50%)、 无 菌净化屏障系统内饲养。 实验过程中， SiHa-miR-133b 接种的鼠死亡两只。 60 天后， 处死所有鼠， 取出肺， 用 Bouin's液固定后计数肺表面的转移瘤数目， 实 验结果如图 6。 高表达 hsa-miR-133b的 SiHa细胞比阴性对照 SiHa细胞在 SCID 鼠的肺内形成了更多的转移瘤，说明 hsa-miR-133b具有促进转移瘤形成的作用。 参考文献
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Claims

权利要求书
1. 一种用于检测 microRNA或其前体的系统，该系统中包含有多种探针， 每个探针能检测样品中不同的 miRNA，其中至少 50%的探针能检测序列号 1-20 所示的任意一个核酸或它们相应的同源物。
2. 根据权利要求 1所述的系统， 其特征是至少 50%的探针能检测至少 5 个 miRNA, 这 5个 miRNA具有序列号 1-20所示的核酸序列或它们相应的同源 物的核酸序列。
3. 根据权利要求 1所述的系统，其特征是至少 50%的探针能检测至少 10 个 miRNA, 这 10个 miRNA具有序列号 1-20所示的核酸序列或它们相应的同 源物的核酸序列。
4. 根据权利要求 1所述的系统， 其特征是至少 50%的探针能检测至少 1 个具有序列号 1-13所示的核酸序列或它们相应的同源物的核酸序列的 miRNA,和 1个具有序列号 14-20所示的核酸序列或它们相应的同源物的核酸 序列的 miRNA。
5. 根据权利要求 1所述的系统， 其特征是至少 50%的探针能检测所有具 有序列号 1-20所示的核酸序列或它们相应的同源物的核酸序列的 miRNA。
6. 根据权利要求 1-5中任意一个所述的系统， 其特征是至少一个 miRNA 是 hsa-miR-133b或它的同源物。
7. 根据权利要求 1-5中任意一个所述的系统，其特征是探针长度至少包 含 10个核苷酸或至少包含 20个核苷酸。
8. 根据权利要求 1-7中任意一个所述的系统，其特征是探针固定在固相 基质上。
9. 根据权利要求 1-8中任意一个所述的系统， 其特征是探针为 20条， 20条探针的核酸序列是如下 a) 或 b)
a) 序列表中序列 1至序列 20所示的 20条核酸的完全互补序列； b) 在 a) 所述的 20条核酸的 5 ' 端连接有 10-30个！1， 优选是 19个 T。
10. 一种检测宫颈上皮内瘤样病变和 /或宫颈癌样品的方法，其特征是上 述方法包含以下步骤： a) 利用权利要求 1-9中任一所述的系统测定个体样品 中 miRNA表达水平， b) 比较测定样品中的 miRNA水平与参考 miRNA水平， c) 如果测定样品中 miRNA水平同参考 miRNA水平比较发生了特征性改变， 说明 个体有宫颈上皮内瘤样病变和 /或宫颈癌。
11. 根据权利要求 10所述的方法， miRNA表达水平的特征性变化包含至 少一个具有序列号 1-13所示的核酸序列或它们相应的同源物的核酸序列的 miRNA的表达水平发生了明显的上升。
12. 根据权利要求 11所述的方法， miRNA表达水平的特征性变化包含 hsa-miR-133b或它的同源物的表达水平发生了明显的上升。
13. 根据权利要求 10所述的方法， miRNA表达水平的特征性变化包含至 少一个具有序列号 14-20所示的核酸序列或它们相应的同源物的核酸序列的 miRNA的表达水平发生了明显的下降。
14. 根据权利要求 10所述的方法， miRNA表达水平的特征性变化包含至 少 1个具有序列号 1-13所示的核酸序列或它们相应的同源物的核酸序列的 miRNA的表达水平发生了明显的上升， 和至少 1个具有序列号 14-20所示的 核酸序列或它们相应的同源物的核酸序列的 miRNA的表达水平发生了明显的 下降。
15. 根据权利要求 10所述的方法， miRNA表达水平的特征性变化包含至 少 3个具有序列号 1-13所示的核酸序列或它们相应的同源物的核酸序列的 miRNA的表达水平发生了明显的上升， 和至少 3个具有序列号 14-20所示的 核酸序列或它们相应的同源物的核酸序列的 miRNA的表达水平发生了明显的 下降。
16. 根据权利要求 10所述的方法， miRNA表达水平的特征性变化包含至 少 5个具有序列号 1-13所示的核酸序列或它们相应的同源物的核酸序列的 miRNA的表达水平发生了明显的上升， 和至少 5个具有序列号 14-20所示的 核酸序列或它们相应的同源物的核酸序列的 miRNA的表达水平发生了明显的 下降。
17. 根据权利要求 10所述的方法， miRNA表达水平的特征性变化包含所 有具有序列号 1-13所示的核酸序列或它们相应的同源物的核酸序列的 miRNA 的表达水平发生了明显的上升， 和所有具有序列号 14-20所示的核酸序列或 它们相应的同源物的核酸序列的 miRNA的表达水平发生了明显的下降。
18. 根据权利要求 10-17任意一个所述的方法， 样品的类型包括宫颈组 织， 淋巴结， 血液或血清。
19. 根据权利要求 18所述的方法，样品来源于怀疑患有宫颈上皮内瘤样 病变或宫颈癌的个体。
20. 根据权利要求 10-19任意一个所述的方法， 其特征是 miRNA的表达 水平通过 Nortern blot, 原位杂交或定量 RT-PCR确定。
21. 根据权利要求 10-19任意一个所述的方法， 其特征是 miRNA的表达 水平通过微阵列分析确定。
22. 根据权利要求 10-21任意一个所述的方法， 其特征是宫颈癌是鳞状 细胞癌或腺癌。
23. 一种检测宫颈上皮内瘤样病变或宫颈癌样品的方法。 其特征是上述 方法包括利用权利要求 1-9中任一所述的系统确定样品中至少一个 miRNA基 因的遗传状态。 如果 miRNA基因的遗传状态发生了特征性的改变， 说明个体 有宫颈上皮内瘤样病变和 /或宫颈癌。
24. 根据权利要求 23所述的方法， miRNA基因遗传状态的特征性变化包 含至少一个具有序列号 1-13所示的核酸序列或它们相应的同源物的核酸序 列的 miRNA基因发生了扩增。
25. 根据权利要求 24所述的方法， miRNA基因遗传状态的特征性变化包 含 hsa-miR-133b或它的同源物的基因发生了扩增。
26. 根据权利要求 23所述的方法， miRNA基因遗传状态的特征性变化包 含至少一个具有序列号 14-20所示的核酸序列或它们相应的同源物的核酸序 列的 miRNA基因发生了缺失。
27. 根据权利要求 23所述的方法， miRNA基因遗传状态的特征性变化包 含至少 1个具有序列号 1-13所示的核酸序列或它们相应的同源物的核酸序列 的 miRNA基因发生了扩增， 和至少 1个具有序列号 14-20所示的核酸序列或 它们相应的同源物的核酸序列的 miRNA基因发生了缺失。
28. 根据权利要求 23所述的方法， miRNA基因遗传状态的特征性变化包 含至少 3个具有序列号 1-13所示的核酸序列或它们相应的同源物的核酸序列 的 miRNA基因发生了扩增， 和至少 3个具有序列号 14-20所示的核酸序列或 它们相应的同源物的核酸序列的 miRNA基因发生了缺失。
29. 根据权利要求 23所述的方法， miRNA基因遗传状态的特征性变化包 含至少 5个具有序列号 1-13所示的核酸序列或它们相应的同源物的核酸序列 的 miRNA基因发生了扩增， 和至少 5个具有序列号 14-20所示的核酸序列或 它们相应的同源物的核酸序列的 miRNA基因发生了缺失。
30. 根据权利要求 23-29任意一个所述的方法， 其特征是样品包含宫颈 组织， 淋巴结， 血液或血清。
31. 根据权利要求 30所述的方法，样品来源于怀疑患有宫颈上皮内瘤样 病变或宫颈癌的个体。
32. 根据权利要求 23-31任意一个所述的方法， miRNA基因的遗传状态 通过 Southern blot, FISH, 杂合性丢失分析或测序的方法确定。
33. 根据权利要求 23-31任意一个所述的方法， miRNA基因的遗传状态 通过微阵列分析确定。
34. 根据权利要求 23-33任意一个所述的方法， 其特征是宫颈癌是鳞状 细胞癌或腺癌。
35. 一种诊断个体患有宫颈上皮内瘤样病变或宫颈癌的方法， 其特征是 该方法包含利用权利要求 10-22任意一个所述的方法确定个体样品中 miRNA 表达水平的特征性改变。
36. 一种诊断个体患有宫颈上皮内瘤样病变或宫颈癌的方法， 其特征是 该方法包含利用权利要求 23-34任意一个所述的方法确定个体样品中 miRNA 基因的遗传状态。
37. 一种为宫颈上皮内瘤样病变或宫颈癌患者进行生存预后的方法。 其 特征是该方法包含以下步骤：
a)利用权利要求 1-9中任一所述系统确定个体样品中 miRNA的表达水平； b)将 miRNA水平与参考水平进行比较， 如果 miRNA的表达水平发生了特 征性改变则预示患者具有高或低的生存率。
38. 根据权利要求 37所述的确定宫颈上皮内瘤样病变或宫颈癌患者生 存预后的方法， 其特征是生存为总生存。
39. 根据权利要求 37所述的确定宫颈上皮内瘤样病变或宫颈癌患者生 存预后的方法， 其特征是生存为无疾病生存。
40. 根据权利要求 37-39任意一种确定宫颈上皮内瘤样病变或宫颈癌患 者生存预后的方法， 进一步确定对患者的合适处理方法。
41. 一种为宫颈上皮内瘤样病变或宫颈癌患者进行生存预后的方法。 其 特征是利用权利要求 1-9中任一所述的系统确定样品中至少 1个 miRNA基因 的遗传状态， 如果 miRNA的基因遗传状态发生了特征性的改变则预示患者具 有高或低的生存率。
42. 根据权利要求 41所述的确定宫颈上皮内瘤样病变或宫颈癌患者生 存预后的方法， 其特征是生存为总生存。
43. 根据权利要求 41所述的确定宫颈上皮内瘤样病变或宫颈癌患者生 存预后的方法， 其特征是生存为无疾病生存。
44. 根据权利要求 41-43任意一种确定宫颈上皮内瘤样病变或宫颈癌患 者生存预后的方法， 进一步确定对患者的合适处理方法。
45. 一种用于治疗宫颈上皮内瘤样病变或宫颈癌患者的药物制剂， 这种 药物制剂包含一种成分降低至少一个具有序列号 1-13所示的核酸序列或它 们相应的同源物的核酸序列的 miRNA的表达水平； 同时， 这种药物制剂包含 药学上可接受的载体成分。
46.权利要求 45所述的药物制剂的成分进一步包含一种成分升高至少一 个具有序列号 14-20所示的核酸序列或它们相应的同源物的核酸序列的 miRNA的表达水平。
47. 一种用于治疗宫颈上皮内瘤样病变或宫颈癌患者的药物制剂， 这种 药物制剂包含一种成分升高至少一个具有序列号 14-20所示的核酸序列或它 们相应的同源物的核酸序列的 miRNA的表达水平； 同时， 这种药物制剂包含 药学上可接受的载体成分。
48. 根据权利要求 45-47任意一个所述的药物制剂， 其特征是至少一个 miRNA是 hsa-miR-133b或它的同源物。
49. 根据权利要求 45-48任意一个所述的药物制剂， 其特征是制剂成分 是反义 RNA。
50. 根据权利要求 45-49任意一个所述的药物制剂， 其特征是制剂成分 是 siRNA。
51. 一种利用权利要求 45-50任意一个所述的药物制剂治疗宫颈上皮内 瘤样病变或宫颈癌的方法。
52. 一种使用药物制剂治疗宫颈上皮内瘤样病变或宫颈癌的方法， 该方 法的特征是药物制剂包含一种成分用于升高至少一个具有序列号 14-20所示 的核酸序列或它们相应的同源物的核酸序列的 miRNA的表达水平； 同时， 这 种药物制剂包含药学上可接受的载体成分。
53. 用于扩增 RNA序列的寡核苷酸引物， 其特征是寡核苷酸探针的序列 具有如下特点：
a)能在高严谨度的条件下与序列表所列序列或序列表所列序列的互补序 列杂交。
b )与序列表所列序列或序列表所列序列的互补序列具有至少 90%的相同 性。
54. 根据权利要求 53所述的寡核苷酸引物具有序列表所列的序列或序 列表所列序列的互补序列。
55. 根据权利要求 53所述的寡核苷酸引物由 DNA， RNA, PNA或相应的衍 生物组成。
56. 根据权利要求 53所述的寡核苷酸引物是标记的引物。
57. 根据权利要求 56所述的标记可以是化学的， 酶学的， 免疫学的， 放 射性的， 荧光的， 发冷光的或 FRET标记。
58. —种诊断结肠直肠癌， 舌头鳞状细胞癌， 食管鳞状细胞癌和胰腺导 管癌的方法。 其特征是该方法包含以下步骤：
a) 利用权利要求 1所述的系统确定样品中的 miRNA表达水平；
b ) 比较 miRNA水平与参照水平；
c ) 如果样品的 miRNA水平表现出特征性的变化则预示样品是癌组织。
59. 根据权利要求 58所述的方法， 其特征是 hsa-miR-133b或它的同源 物的表达水平发生显著性的改变。
60. 根据权利要求 58-59任意一个所述的方法， 样品类型包括组织， 淋 巴结， 血液或血清。
61. 根据权利要求 58-60任意一个所述的方法， 其特征是 miRNA的表达 通过 Northern blot, 原位杂交， 或定量 RT-PCR方法确定。
62. 根据权利要求 58-60任意一个所述的方法， 其特征是 miRNA的表达 通过微阵列方法确定。
63. 一种诊断结肠直肠癌， 舌头鳞状细胞癌， 食管鳞状细胞癌和胰腺导 管癌的方法， 其特征是利用权利要求 1-9中任一所述的系统确定 hsa-miR-133b的基因遗传状态， 如果 hsa-miR-133b的基因遗传状态发生特 征性改变则预示样品是癌症样品。
64. 根据权利要求 63所述的方法， miRNA的基因遗传状态通过 Southern blot, FISH, 杂合性的丢失分析或测序确定。
65. 根据权利要求 63所述的方法， miRNA的基因遗传状态通过微阵列分 析确定。
66. 一种为个体诊断结肠直肠癌， 舌头鳞状细胞癌， 食管鳞状细胞癌和 胰腺导管癌的方法， 该方法的特征是利用权利要求 58-62任意一个所述的方 法确定个体样品中 miRNA的表达水平。
67. 一种为个体诊断结肠直肠癌， 舌头鳞状细胞癌， 食管鳞状细胞癌和 胰腺导管癌的方法， 该方法的特征是利用权利要求 63-65任意一个所述的方 法确定个体样品中 miRNA的基因遗传状态。
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CN115040530A (zh) * 2022-04-20 2022-09-13 大连大学 hsa-miR-320a在抑制肿瘤细胞迁移药物中的应用

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