WO2016197592A1 - 一种长链非编码rna hnf1a-as1在制备治疗人体恶性实体瘤药物中的应用 - Google Patents

一种长链非编码rna hnf1a-as1在制备治疗人体恶性实体瘤药物中的应用 Download PDF

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WO2016197592A1
WO2016197592A1 PCT/CN2016/000221 CN2016000221W WO2016197592A1 WO 2016197592 A1 WO2016197592 A1 WO 2016197592A1 CN 2016000221 W CN2016000221 W CN 2016000221W WO 2016197592 A1 WO2016197592 A1 WO 2016197592A1
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hnf1a
malignant solid
long
medicament
rna
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张新
丁晨虹
谢渭芬
文良志
陈斐
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中国人民解放军第二军医大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to the field of gene therapy and medical diagnosis.
  • the present invention relates to a long-chain non-coding RNA HNF1A-AS1 (Hepatocyte Nuclear Factor-1 ⁇ Antisense 1) for use in the preparation of a medicament for treating human malignant solid tumors, and provides a A new target for the treatment of malignant solid tumors.
  • HNF1A-AS1 Hepatocyte Nuclear Factor-1 ⁇ Antisense 1
  • Long noncoding RNA is a class of RNA molecules with a transcript length of more than 200 nt (nucleotide units). Because such RNA molecules lack a specific and complete open reading frame, they do not encode proteins. Current research suggests that only 2% of human genomic sequences are genes encoding proteins, and more than 60% of human genomic sequences can be transcribed into long-chain non-coding RNAs. Previous studies have shown that long-chain non-coding RNA can be widely involved in the regulation of gene expression and function, has a variety of biological functions, and has important functions in gene expression regulation and signal transduction.
  • RNA coding gene
  • mRNA coding gene
  • long-chain non-coding RNAs The number of long-chain non-coding RNAs is extremely large, and the genomic mapping forms are diverse and the classification is complex.
  • One of the long-chain non-coding RNAs (LincRNAs) located between two genes is a more common and functionally important class in the current research field because its gene position does not coincide with other genes.
  • Chain non-coding RNA certain long-chain non-coding RNAs occur only at specific developmental stages, or are specifically expressed in certain tissues or cells, with precise subcellular localization, suggesting that these long-chain non-coding RNAs may have important physiological functions. Studies have also shown that specific expression and/or expression changes of LncRNA are closely related to the development of malignant tumors (Lee, J.T.. Epigenetic regulation by long noncoding RNAs. Science. 2012. 338 (6113): 1435-1439.). Despite the large number of long-chain non-coding RNAs, the number of long-chain non-coding RNAs with well-defined functions is still very small.
  • the long-chain non-coding RNA HNF1A-AS1 (Hepatocyte Nuclear Factor-1 ⁇ Antisense 1) is a long-chain non-coding RNA specifically expressed in digestive system organs such as liver and gastrointestinal tract. Its gene is located in the coding gene hepatocyte nuclear factor 1 ⁇ (Hepatocyte). On the antisense strand upstream of Nuclear Factor 1 ⁇ ), it belongs to intergenic long-chain non-coding RNA (lincRNA).
  • HNF1A-AS1 may be elevated in esophageal cancer and lung cancer (Yang X, Song JH, Cheng Y, et al.
  • RNA HNF1A-AS1 Long non-coding RNA HNF1A-AS1regulates proliferation and migration in oesophageal adenocarcinoma cells. Gut, 2014, 63(6):881-90.)(Wu Y,Liu H,Shi X,Yao Y,Yang W,Song Y.The long non-coding RNA HNF1A-AS1regulates proliferation and metastasis in lung adenocarcinoma.Oncotarget.2015;6 :9160-72.), but its role in other malignancies and its complete basis No information has been reported.
  • HNF1A-AS1 is incomplete in both the literature and the database.
  • HNF1A-AS1 can be used to treat malignant tumors.
  • the object of the present invention is to find a long-chain non-coding RNA which can be effectively used for the treatment of malignant solid tumors in human body.
  • Another object of the present invention is to provide a novel medical use of a long-chain non-coding RNA HNF1A-AS1, in particular for the preparation of a medicament for treating human malignant solid tumors.
  • the present invention finds that its expression in HHC1A-AS1 is decreased in liver cancer; the full length of HNF1A-AS1 is cloned by molecular biological means, and the full-length HNF1A-AS1 obtained by the present invention is found to have significant inhibition on the malignant phenotype of hepatoma cells. Role is a potential therapeutic target for malignant tumors.
  • the main technical solution of the present invention is that the present invention firstly found that the HNF1A-AS1 gene/RNA can effectively inhibit the malignant phenotype of malignant solid tumor in vivo.
  • HNF1 ⁇ not only affects the proliferation of tumor cells, but also inhibits the migration and invasion of solid tumor cells and reduces the tumorigenicity of tumor cells. Therefore, HNF1A-AS1 has potential application prospects as the first proven gene/RNA that can effectively inhibit the malignant phenotype of malignant solid tumors.
  • the present inventors have completed the present invention, and provide a long-chain non-coding RNA gene HNF1A-AS1 gene and a transcription product thereof HNF1A-AS1 RNA, and HNF1A-AS1 gene/RNA, which inhibit the malignant phenotype of malignant solid tumor cells. Use in the treatment of solid tumors.
  • RNA HNF1A-AS1 Hepatocyte Nuclear Factor-1 ⁇ Antisense 1
  • the cDNA sequence of the long-chain non-coding RNA HNF1A-AS1 of the present invention is shown in SEQ ID NO: 1.
  • the gene sequence of the long-chain non-coding RNA HNF1A-AS1 of the present invention has a 330 bp extension at the 5' end compared to the HNF1A-AS1 gene sequence provided by the NCBI database (gene ID: NR_024345.1) (see the box in FIG. 4).
  • the sequence indicated), 2785 bp in length, is a long-chain non-coding RNA with no splicing form in a contiguous sequence.
  • the medicament for treating human malignant solid tumor of the invention can increase the expression level of the long-chain non-coding RNA gene HNF1A-AS1 gene or its transcription product HNF1A-AS1 RNA.
  • the drug for increasing the expression level of the long-chain non-coding RNA gene HNF1A-AS1 gene or its transcription product HNF1A-AS1 RNA includes, but is not limited to:
  • the medicament for treating a human malignant solid tumor according to the present invention may also be a pharmaceutical composition comprising the active ingredient of any of the above 1) to 3) and a pharmaceutically acceptable carrier or excipient.
  • the expression vector includes a recombinant plasmid, a recombinant virus, a recombinant viral vector, a non-viral vector and the like.
  • Recombinant virus preferably adenovirus, lentivirus, and the like.
  • Non-viral vectors preferably liposomes or other targeting media.
  • the human malignant solid tumor is selected from the group consisting of liver cancer, gastric cancer, intestinal cancer, pancreatic cancer, prostate cancer or gonad tumor.
  • the pharmaceutical composition is also useful for inhibiting the formation of solid tumors in vivo.
  • the human malignant solid tumor is liver cancer, including but not limited to liver tumor cell lines Huh7, Hep3B, MHCC and the like.
  • the HNF1A-AS1 is human HNF1A-AS1.
  • the drug is a lentiviral recombinant plasmid pCDH-HNF1A-AS1.
  • the pharmaceutical composition is in the form of an injection.
  • the pharmaceutical composition further comprises a chemotherapeutic agent.
  • a method of inhibiting a malignant solid tumor in a mammal comprising the step of administering HNF1A-AS1 RNA or an expression vector comprising the gene sequence to a mammalian subject in need of treatment.
  • the mammal is a human.
  • gene/RNA refers to a gene and/or RNA.
  • HNF1A-AS1 gene As used herein, the terms “HNF1A-AS1 gene", “HNF1A-AS1 RNA” are used interchangeably and refer to HNF1A-AS1 RNA. In a narrow sense, the term refers to human HNF1A-AS1; broadly speaking, the term includes not only human HNF1A-AS1, but also other mammalian HNF1A-AS1, especially primate HNF1A-AS1, such as ⁇ Or monkey HNF1A-AS1. The term also encompasses active fragments, active derivatives and analogs of HNF1A-AS1 RNA.
  • the RNA of the present invention may be natural RNA or synthetic RNA.
  • the RNA of the present invention may be a naturally purified product, or a chemically synthesized product, or produced in vitro.
  • fragment refers to an RNA fragment that substantially retains the same biological function or activity of the native HNF1A-AS1 RNA of the invention. These fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.
  • human HNF1A-AS1 RNA refers to an RNA having a wild-type HNF1A-AS1 sequence. The term also encompasses variant forms of wild-type sequences that have the same inhibitory function as human HNF1A-AS1 RNA.
  • Such variants include, but are not limited to, deletions, insertions and/or substitutions of one or more nucleotides, and the addition of one or several nucleotides at the 5' end and/or the 3' end.
  • deletions, insertions and/or substitutions of one or more nucleotides and the addition of one or several nucleotides at the 5' end and/or the 3' end.
  • the function of the RNA when substituted with similarly similar nucleotides, the function of the RNA is generally not altered.
  • the addition of one or several nucleotides at the 5' end and/or the 3' end will generally not alter the function of the RNA.
  • the term also includes active fragments of human HNF1A-AS1.
  • the invention also includes analogs of human HNF1A-AS1 RNA.
  • the difference between these analogs and the native human HNF1A-AS1 RNA may be a difference in nucleic acid sequence, a difference in the form of nucleotide modification that does not affect the sequence, or a combination thereof.
  • Analogs also include analogs having different bases than natural nucleotides, as well as analogs having non-naturally occurring or synthetic nucleotides.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded of.
  • the full length sequence of human HNF1A-AS1 nucleotide or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed based on the nucleotide sequence of human HNF1A-AS1, and amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the invention also relates to vectors comprising HNF1A-AS1 sequences, in particular viral vectors, and host cells genetically engineered with the vectors of the invention or HNF1A-AS1 sequences, and methods for producing the RNAs of the invention by in vitro transcription techniques .
  • the method of introducing a polynucleotide into a tissue or a cell comprises: injecting the polynucleotide directly into the tissue in vivo; or introducing the polynucleotide into the cell by a vector (such as a virus, a phage or a plasmid, etc.) in vitro. Then transplant the cells into the body and so on.
  • a vector such as a virus, a phage or a plasmid, etc.
  • Recombinant gene therapy vectors such as viral vectors, can be designed to express wild-type HNF1A-AS1 to increase the number and activity of HNF1A-AS1 in solid tumors.
  • Viral-derived expression vectors such as retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, parvoviruses and the like can be used to transfer the HNF1A-AS1 gene into cells.
  • a method for constructing a recombinant viral vector carrying the HNF1A-AS1 gene can be found in the literature (Sambrook, et al.).
  • recombinant human HNF1A-AS1 gene/RNA can be packaged into liposomes and then transferred into cells.
  • the HNF1A-AS1 RNA, HNF1A-AS1 DNA and vector of the present invention can inhibit the malignant phenotype of malignant solid tumor when administered (administered) to a mammalian subject such as a human.
  • these materials can be formulated in a non-toxic, inert, andpharmaceutically acceptable carrier medium (including aqueous carrier medium) to form a pharmaceutical composition.
  • the pH of the aqueous carrier medium will generally be from about 5 to about 8, preferably from about 6 to about 8, although the pH may vary depending on the nature of the substance being formulated and the condition to be treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used for the treatment of solid tumors, and representative examples include, but are not limited to, liver cancer, gastric cancer, intestinal cancer, pancreatic cancer, renal cancer, prostate cancer, and gonad tumor.
  • HNF1A-AS1 gene/RNA or pharmaceutical composition of the present invention other therapeutic agents such as cisplatin, TNF, etc. may be simultaneously or additionally used, and other genes such as HSK-TV gene, P53 gene or the like may be used or Chemotherapy combined with radiotherapy.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising (a) a safe and effective amount (e.g., 0.0001 to 99% by weight) of the HNF1A-AS1 RNA, HNF1A-AS1 DNA or vector of the present invention, and (b) a pharmaceutically acceptable carrier or Shape agent.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.
  • Other therapeutic agents such as chemotherapeutic agents, may also be included in the pharmaceutical compositions of the invention.
  • a pharmaceutical composition is used, a safe and effective amount of HNF1A-AS1 RNA, HNF1A-AS1 DNA or a vector is administered to a mammal.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the invention also provides a method for gene therapy of tumor cells (especially malignant solid tumors), which comprises introducing the HNF1A-AS1 gene into tumor cells for expression, wherein the method for introducing the HNF1A-AS1 gene into tumor cells comprises Transfection with plasmid, adenovirus or adeno-associated virus.
  • Rapid amplification of cDNA ends amplifies the 5' end (A) and 3' end (B) of HNF1A-AS1 cDNA.
  • Figure 4 Nucleotide sequence of the HNF1A-AS1 full-length gene obtained from the 5' end and 3' end extension of the HNF1A-AS1 cDNA.
  • FIG. 7 Green fluorescent expression of Huh7 cells infected with lentivirus lenti-Ctrl (A) or lentivirus lenti-HNF1A-AS1 (B) for 3 days.
  • FIG. 8 The expression level of HNF1A-AS1 gene was detected by Real-Time PCR 4 days after Lenti-HNF1A-AS1 lentivirus infection of Huh7 cells (A) and Hep3B cells (B).
  • FIG. 11 Exogenous introduction of HNF1A-AS1 can cause G2 phase cell arrest in Hep3B cells, where A is the control cell flow cytometry cycle detection map, and B is the HNF1A-AS1 overexpression group cell flow cytometry cycle detection map. C is a graph of cell cycle data of A and B graphs.
  • FIG. 13 In vivo inoculation tumor formation experiments of Huh7 cells infected with control lentivirus lenti-Ctrl or lentivirus lenti-HNF1A-AS1, respectively, where A is the tumor growth rate, B is the tumor formation in mice (top) and tumor In vitro (bottom), C is the expression level of HNF1A-AS1 gene in the tumor.
  • HNF1A-AS1 gene therapy for liver in situ tumor implantation in which A is a fluorescence-like image of the two groups before and after the injection of the virus; B is a mouse liver and tumor in vitro, with the arrow pointing to the tumor tissue. C is the expression level of HNF1A-AS1 gene in the tumor.
  • reagents and starting materials used in the present invention are either commercially available or can be prepared by literature methods. Not specified in the following examples Conditional experimental methods, usually in accordance with conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Guide (New York: Cold Spring Harbor Laboratory Press, 1989), or according to conventional conditions, or as recommended by the manufacturer conditions of. Percentages and parts are by weight unless otherwise stated.
  • Example 1 Real-Time PCR detection of HNF1A-AS1 gene expression in human liver tumor cell lines
  • liver tumor cell lines HepG2, Huh7, Hep3B, MHCC-L, MHCC-H, LM3, PLC, and Focus were inoculated into a six-well plate at 5 ⁇ 10 5 / dish to contain 10% fetal cattle. Serum was cultured in fresh medium, RNA was extracted on the next day, OD260 value was measured by spectrophotometer, and RNA integrity was detected by 1% agarose gel electrophoresis.
  • RNA RNA to 4 ⁇ l of 5 ⁇ PrimeScript RT master mix (reverse transcription kit), add DEPC water to make up the volume to 20 ⁇ l, react at 37 ° C for 15 min, and inactivate the reverse transcriptase at 85 ° C for 5 s to obtain reverse transcript.
  • 1 ⁇ l was used as a template for HNF1A-AS1 PCR amplification, and ⁇ -actin was used as an internal reference for PCR reaction under the same reaction conditions.
  • the reaction system was as follows:
  • the reaction conditions were 95 ° C, 30 sec; 95 ° C, 5 sec ⁇ 60 ° C, 34 sec, 40 cycles; 95 ° C, 15 sec ⁇ 60 ° C, 60 sec ⁇ 95 ° C, 15 sec.
  • the relevant primer sequences are shown in the table:
  • HNF1A-AS1 forward primer CAAGAAATGGTGGCTATGA (SEQ ID NO: 4);
  • Reverse primer TGGACTGAAGGACAAGGGT (SEQ ID NO: 5).
  • Example 2 Real-Time PCR detection of HNF1A-AS1 expression in hepatocarcinoma tissues and their corresponding adjacent tissues
  • the cDNA of the liver cancer tissue specimen was obtained by reverse transcription according to the reverse transcription method of the RNA of Example 1. After the cDNA was diluted, the expression of HNF1A-AS1 in the human liver cancer tissue was detected according to the same method and conditions as in Example 1, and the ⁇ -actin gene was simultaneously used. Expression as an internal reference showed that HNF1A-AS1 was down-regulated in HCC tissues (Fig. 2).
  • Example 3 Rapid amplification of cDNA ends (RACE) to amplify the full length of HNF1A-AS1 gene
  • the 5' end specific amplification primer (5'GSP) AACTCGGACTGTTCTCCTTCCCACCCC (SEQ ID NO: 6) and the 3' end specific amplification primer (3'GSP) ACGGCTAGTAAACGGCAGAACGAGGC (SEQ ID NO) were designed according to the sequence known by HNF1A-AS1. :7), synthetic primers.
  • HNF1A-AS1 gene amplification of cDNA sequences using a commercially available kit CLONTECH's Marathon TM kit includes adapter primer (adaptorprimer) and prefabricated human liver cDNA, was amplified using the following PCR system.
  • the components were mixed, centrifuged, and placed in a PCR machine for amplification.
  • the 5' end and 3' end fragment of HNF1A-AS1 were obtained by PCR amplification, and the product was separated and identified by 1% agarose gel electrophoresis (Fig. 3).
  • the longest piece of gel was collected and placed in an Eppendorf tube, and the weight of the gel was weighed.
  • the ratio of each 100 mg of glue/200 ml of NT solution was added to the NT solution at 50 ° C for 5-10 minutes until the gel melted; the liquid was passed through the column, centrifuged at 13,000 rpm for 1 min, 700 ⁇ l of NT3 washing solution was added, centrifuged at 13,000 rpm for 1 min, and washed twice.
  • the purification column was placed on a clean Eppendorf tube, and the lid was opened for 1 min. 30 ⁇ l of double distilled water was added dropwise to the intra-column filter, allowed to stand for 2 min, centrifuged at 13,000 rpm for 1 min, and the column was eluted to obtain a DNA fragment, and the spectrophotometer was obtained. The concentration was measured. The 5' end of the purified HNF1A-AS1 or 4 ⁇ l of the 3' end fragment, 1 ⁇ l of the pMD-19T vector, and 5 ⁇ l of the solution I ligase mixture were respectively ligated at 16 ° C for 3 h. The ligation product was added to the conventional competent E.
  • coli DH5 ⁇ transformation plated with LB medium plate containing ampicillin, and kept at 37 ° C overnight, and single colony clones were picked and cultured in LB liquid medium containing ampicillin.
  • OD600 0.8-1.0, sent to Yingjun Biological Company for sequencing.
  • Reverse primer GACGGAGTTTCGTTCTTGTTCC (SEQ ID NO: 9);
  • the BamH I and EcoR I restriction sites were used, and the human liver cDNA was used as a template to PCR-amplify the HNF1A-AS1 sequence.
  • the product was identified by 0.7% gel electrophoresis. The identification results are shown in Figure 5. The correct size is the reagent. The cassette was purified to recover the PCR amplified fragment.
  • BamH I and EcoR I respectively digested the PCR amplified fragment of pCDH plasmid (purchased from System Biosciences, USA) and HNF1A-AS1, and identified and purified the recovered product by gel electrophoresis. Take 50-100 ng of linearized pCDH plasmid, 500-1000 ng BamH I and EcoR I digested HNF1A-AS1 fragment, 5 ⁇ l of solution I ligase mixture, mix and connect overnight at 16 °C. The ligation product was added to the lentiviral vector-specific competent E.
  • coli Stbl3 transformation plated with LB medium plate containing ampicillin, and kept at 37 ° C overnight, and single colony clones were picked and cultured in LB liquid medium containing ampicillin, and the appropriate amount was obtained.
  • the bacterial solution was sequenced by the company.
  • the lentiviral recombinant expression plasmid pCDH-HNF1A-AS1 was obtained by sequencing, and the recombinant expression plasmid pCDH-HNF1A-AS1 was digested with BamH I and EcoR I. As shown in Fig. 6, a fragment of about 7 kb was found as a vector fragment and another 3 kb.
  • the left and right fragments are HNF1A-AS1 cDNA fragments, indicating that the plasmid is correct (Fig. 6).
  • Example 5 Real-Time PCR detection of HNF1A-AS1 gene expression level after Lenti-HNF1A-AS1 infection of human liver tumor cell lines
  • Human liver tumor cell lines Huh7 and Hep3B were inoculated to a 35mm culture dish at a density of 3 ⁇ 10 5 cells/dish, and 500 ⁇ l lentivirus Lenti-Ctrl or Lenti-HNF1A-AS1 were added respectively. After 10h-48h, 10% fetal was replaced. The fresh DMEM medium of bovine serum was observed after 3 days of culture, and it was found that the cells infected by the control virus and HNF1A-AS1 virus showed obvious green fluorescence (Fig. 7), indicating that the virus successfully infected the cells. After 5 days, total RNA was extracted with a Trizol kit, and the OD260 value was measured with a spectrophotometer, and RNA integrity was detected by 1% agarose gel electrophoresis.
  • the cDNA was reverse transcribed according to the reverse transcription method of RNA in Example 1. After the cDNA was diluted, the expression of HNF1A-AS1 gene in Huh7 and Hep3B cells after Lenti-HNF1A-AS1 infection was detected according to the same method and conditions as in Example 1. At the same time, the expression of ⁇ -actin gene was used as an internal reference. The results showed that the expression of HNF1A-AS1 gene was significantly up-regulated in Huh7 and Hep3B cells after Lenti-HNF1A-AS1 infection (Fig. 8).
  • Example 6 Exogenous introduction of HNF1A-AS1 inhibits proliferation of liver tumor cells
  • Human liver tumor cell lines Huh7 and Hep3B were inoculated into 35 mm culture dishes at 2 ⁇ 10 5 and infected with control lentivirus Lenti-Ctrl (control group) or lentivirus Lenti-HNF1A-AS1 (experimental group) for 48 h, respectively. 3 ⁇ 10 3 cells were evenly divided into 96-well plates, and 3 replicate wells were tested every day for 7 days. On the next day, 10% CCK8 was used in serum-free medium, the original culture solution in 96 wells was aspirated, 100 ⁇ l of the configured CCK8 reagent was added, and the absorbance at 450 nm was measured at 37 ° C for 1 h to determine the number of active cells. .
  • Example 7 Exogenous introduction of HNF1A-AS1 inhibits colony forming ability of liver tumor cells
  • Human liver tumor cell lines Huh7 and Hep3B were inoculated into 35 mm culture dishes at 2 ⁇ 10 5 and infected with control lentivirus Lenti-Ctrl (control group) or lentivirus Lenti-HNF1A-AS1 (experimental group) for 48 h, respectively.
  • the cell density of 2 ⁇ 10 3 /dish was inoculated into a 35 mm culture dish, and the cells were changed every 3 days for 3-4 weeks until obvious clones were visible, 4% PFA was fixed, crystal violet staining, counting clones, and HNF1A-AS1 was observed for Huh7.
  • the ability of Hep3B cell clone formation ability The results showed that the number of clones formed by Huh7 and Hep3B cells after Lenti-HNF1A-AS1 infection was significantly reduced compared with the control group (Fig. 10).
  • Example 8 Exogenous introduction of HNF1A-AS1 causes G2 phase cell arrest in Hep3B cells
  • Hepatoma cell line Hep3B was inoculated into 12-well plates at 1 ⁇ 10 5 / dish, and cells were infected with equal volume lentivirus lenti-Ctrl or lenti-HNF1A-AS1, and fresh DMEM containing 10% fetal bovine serum was replaced after 24 hours. On the 3rd day, the fluorescence expression was observed. On the 4th day, the cells were collected. After washing with PBS, 1 ⁇ 10 6 cells were resuspended in 1 ml PBS mixture (containing 0.2% TritionX-100, 50 ⁇ g RNase, 5 ⁇ l propidium iodide). , 0.1% FBS), protected from light, incubated for 15 min at room temperature, the cell cycle was measured and statistical analysis was performed.
  • Example 9 Exogenous introduction of HNF1A-AS1 inhibits migration of human liver tumor cells
  • Human liver tumor cell lines Huh7, Hep3B, and MHCC-H were inoculated into 35mm culture dishes at 3 ⁇ 10 5 and infected with control lentivirus Lenti-Ctrl (control group) or lentivirus Lenti-HNF1A-AS1 (experimental group) for 48 hours.
  • the effect of -AS1 on the migration ability of human liver tumor cell lines, and the migration area by protein-plus software was used to analyze the effect of HNF1A-AS1 gene on the migration ability of liver cancer cell lines.
  • Example 10 Up-regulation of HNF1A-AS1 expression in human liver tumor cell line Huh7 inhibits tumorigenic ability in vivo
  • the Huh7 cells infected with the control lentivirus Lenti-Ctrl or Lenti-HNF1A-AS1 for 48h were inoculated into the armpits of nude mice by 2 ⁇ 10 6 cells, and the cells infected with the control virus lenti-Ctrl were inoculated on the left side.
  • the cells infected with lenti-HNF1A-AS1 were inoculated on the right side, and the tumor formation was observed in the body. When the tumor appeared, the size of the new tumor was measured with a vernier caliper every 3 days and the tumor growth curve of the control group and the experimental group was recorded.
  • the tumor growth rate of the nude mice inoculated with Huth7 cells of the virus lenti-HNF1A-AS1 was significantly lower than that of the control lentivirus lenti-Ctrl group (Fig. 13A), and the tumor formed by the control group was significantly larger and heavier than the experimental group (Fig. 13B).
  • Hepatoma cells Huh7 were infected with a lentivirus (pSin-EF2-LUC-Pur) expressing a firefly luciferase (LUC) gene, and a liver cancer cell line stably expressing LUC was obtained by puromycin.
  • a lentivirus pSin-EF2-LUC-Pur
  • LUC firefly luciferase
  • the luciferase gene-labeled hepatoma cells were directly injected into the liver of NOD/SCID mice (the number of cells was about 2 ⁇ 10 6 /piece), and the tumor growth was dynamically monitored by a living in vivo imaging system, after tumor formation (about 10- 14 days)
  • the mice were roughly divided into two groups according to the fluorescence intensity of the tumors, 10 mice in each group, and the lentivirus Lenti-HNF1A-AS1 or the control virus Lenti-Ctrl was injected through the tail vein. Tumor growth was monitored by a living in vivo imaging system every 5 days, and the survival time, growth state, and ascites volume of the mice were observed, and the mice were sacrificed after about 6-8 weeks.
  • the dynamic monitoring results of living body imaging showed that the fluorescence detection value of the tumor in the experimental group after injection of the virus was significantly lower than that of the control group (Fig. 14A). After the mice were sacrificed, it was found that the size of the tumor formed by the liver of the experimental group was significantly lower than that of the control group (Fig. 14B). The RNA of mouse tumor tissues was extracted, and the results of real-time PCR showed that the expression of HNF1A-AS1 in the tumor tissues of the experimental group was higher than that of the control group (Fig. 14C). The above results indicate that overexpression of HNF1A-AS1 can significantly inhibit the growth rate of hepatoma in situ in Huh7 cells, and up-regulation of HNF1A-AS1 has a good therapeutic effect on mouse liver cancer implantation model.

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Abstract

提供了一种长链非编码RNA HNF1A-AS1在制备治疗人体恶性实体瘤药物中的应用。通过调控恶性实体肿瘤细胞HNF1A-AS1基因表达，可有效地抑制恶性实体肿瘤的增殖，为临床治疗恶性实体肿瘤提供了一种新靶点。

Description

一种长链非编码RNA HNF1A-AS1在制备治疗人体恶性实体瘤药物中的应用 技术领域
本发明涉及基因治疗和医学诊断领域，具体地，本发明涉及一种长链非编码RNA HNF1A-AS1(Hepatocyte Nuclear Factor-1αAntisense 1)在制备治疗人体恶性实体瘤药物中的应用，提供了一种治疗恶性实体肿瘤的新靶点。
背景技术
恶性实体瘤的治疗是目前临床的难点之一，尤其对于手术无法切除的恶性实体瘤，临床尚缺乏有效的治疗手段。选择与肿瘤细胞发生发展密切相关的关键分子和基因进行特异性靶向调控是恶性实体瘤治疗的核心问题之一。
长链非编码RNA(long noncoding RNA，lncRNA)系一类转录本长度超过200nt(核苷酸单位)的RNA分子，由于这类RNA分子缺少特异完整的开放阅读框，因而不编码蛋白质。目前的研究认为仅有2％的人类基因组序列是编码蛋白的基因，而约60％以上的人类基因组序列可转录为长链非编码RNA。既往的研究显示长链非编码RNA可广泛参与基因表达和功能的调节，具有丰富多样的生物学功能，在基因表达调控和信号转导过程中具有重要功能。在肿瘤细胞中，长链非编码RNA也发挥重要的调控作用，与编码基因(mRNA)一样可以作为肿瘤诊断和预后判段的分子标志物，同时也是潜在分子靶向治疗的靶点，具有良好的临床应用价值。
长链非编码RNA数量极大、基因组定位形式多种多样，分类复杂。其中一类位于两个基因之间的长链非编码RNA(Large Intergenic Noncoding RNA，lincRNA)，由于其基因位置不与其他基因存在重合，是目前研究领域中较常见及功能较为重要的一类长链非编码RNA。此外，某些长链非编码RNA仅在特定的发育阶段出现，或特异性地表达于某些组织或细胞，具有精确的亚细胞定位，提示这些长链非编码RNA可能具有重要的生理功能。也有研究表明，LncRNA的特异性表达和/或表达变化与恶性肿瘤的发生发展密切相关(Lee，J.T..Epigenetic regulation by long noncoding RNAs.Science.2012.338(6113)：1435-1439.)。尽管长链非编码RNA数量庞大，但目前功能明确的长链非编码RNA数量还是极少。
长链非编码RNA HNF1A-AS1(Hepatocyte Nuclear Factor-1αAntisense 1)为一仅在消化系统器官如肝、胃肠道等特异表达的长链非编码RNA，其基因位于编码基因肝细胞核因子1α(Hepatocyte Nuclear Factor 1α)上游的反义链上，属于基因间长链非编码RNA(lincRNA)。尽管有研究认为HNF1A-AS1可能在食道癌、肺癌中表达升高(Yang X，Song JH，Cheng Y，et al.Long non-coding RNA HNF1A-AS1regulates proliferation and migration in oesophageal adenocarcinoma cells.Gut，2014，63(6)：881-90.)(Wu Y，Liu H，Shi X，Yao Y，Yang W，Song Y.The long non-coding RNA HNF1A-AS1regulates proliferation and metastasis in lung adenocarcinoma.Oncotarget.2015；6：9160-72.)，但其在其他恶性肿瘤中的作用及其完整的基 因信息均未见报道。
目前HNF1A-AS1无论文献还是数据库所公布的序列均不完整。
目前尚无文献报道HNF1A-AS1可用于治疗恶性肿瘤。
发明内容
本发明的目的在于寻找到能够有效用于人体恶性实体瘤治疗的一种长链非编码RNA。
本发明的另一目的在于提供一种长链非编码RNAHNF1A-AS1的新的医药用途，具体是在制备治疗人体恶性实体瘤药物中的应用。
本发明在研究HNF1A-AS1过程中发现其在肝癌中表达下降；利用分子生物学手段克隆HNF1A-AS1的全长，发现本发明得到的全长HNF1A-AS1对肝癌细胞的恶性表型具有显著抑制作用，是恶性肿瘤潜在的治疗靶点。
本发明的主要技术方案是：本发明首次发现了HNF1A-AS1基因/RNA可有效地在体内抑制恶性实体瘤的恶性表型。实验表明，HNF1α不仅对肿瘤细胞增殖有影响，更可抑制实体瘤细胞的迁移和侵袭、降低肿瘤细胞的成瘤性。因此HNF1A-AS1作为首次被证实的可有效地在抑制恶性实体瘤恶性表型的关键基因/RNA，具有潜在的应用前景。本发明人在此基础上完成了本发明，提供一种抑制恶性实体瘤细胞恶性表型的长链非编码RNA基因HNF1A-AS1基因及其转录产物HNF1A-AS1RNA，以及HNF1A-AS1基因/RNA在治疗实体瘤中的用途。
本发明的第一方面，提供了一种长链非编码RNA HNF1A-AS1(Hepatocyte Nuclear Factor-1αAntisense 1)在制备治疗人体恶性实体瘤药物中的应用。
本发明所述的长链非编码RNA HNF1A-AS1的基因cDNA序列如SEQ ID NO：1所示。
本发明所述的长链非编码RNA HNF1A-AS1的基因序列与NCBI数据库提供的HNF1A-AS1基因序列(基因ID：NR_024345.1)相比较，5’末端有330bp的延伸(如图4中方框所标序列)，全长为2785bp，是一连续序列无其他剪切形式的长链非编码RNA。
本发明所述的治疗人体恶性实体瘤药物，可以提高长链非编码RNA基因HNF1A-AS1基因或其转录产物HNF1A-AS1RNA的表达量。
所述的提高长链非编码RNA基因HNF1A-AS1基因或其转录产物HNF1A-AS1RNA的表达量的药物，包括但不限于：
1)HNF1A-AS1RNA；
2)HNF1A-AS1基因；
3)含有HNF1A-AS1基因的表达载体。
本发明所述的治疗人体恶性实体瘤药物，也可以是药物组合物，所述的药物组合物包含以上1)至3)任一活性成份以及药学上可接受的载体或赋形剂。
所述的表达载体，包括重组质粒、重组病毒、重组病毒载体、非病毒载体等。
重组病毒，优选腺病毒、慢病毒等。
非病毒载体，优选脂质体或其他靶向介质。
在本发明的一个优选例中，所述的人体恶性实体瘤选自：肝癌、胃癌、肠癌、胰腺癌、前列腺癌或生殖腺肿瘤等。
在本发明的另一个优选例中，所述的药物组合物还用于体内抑制实体瘤的形成。
在本发明的另一个优选例中，所述的人体恶性实体瘤为肝癌，包括但不限于肝肿瘤细胞株Huh7、Hep3B、MHCC等。
在本发明的另一个优选例中，所述的HNF1A-AS1是人的HNF1A-AS1。
在本发明的另一个优选例中，所述的药物为慢病毒重组质粒pCDH-HNF1A-AS1。
在本发明的另一个优选例中，所述的药物组合物的剂型为注射剂。
在本发明的另一个优选例中，所述的药物组合物还含有化疗剂。
在本发明的第二方面，提供了一种在哺乳动物中抑制恶性实体瘤的方法，它包括步骤：给需要治疗的哺乳动物对象施用HNF1A-AS1RNA或含所述基因序列的表达载体。
在本发明的另一个优选例中，所述的哺乳动物是人。
术语解释
如本文所用，术语“基因/RNA”指基因和/或RNA。
如本文所用，术语“HNF1A-AS1基因”、“HNF1A-AS1RNA”可互换使用，都指HNF1A-AS1RNA。狭义上，所述术语指人的HNF1A-AS1；广义上，所述术语不仅包括人的HNF1A-AS1，还包括其他哺乳动物的HNF1A-AS1，尤其是灵长类动物的HNF1A-AS1，如猿或猴的HNF1A-AS1。该术语还包括HNF1A-AS1RNA的活性片段、活性衍生物和类似物。
本发明RNA可以是天然RNA、合成RNA。本发明的RNA可以是天然纯化的产物，或是化学合成的产物，或体外转录产生。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明的天然HNF1A-AS1RNA相同的生物学功能或活性的RNA片段。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。例如，在本发明中，术语“人HNF1A-AS1RNA”指具有野生型HNF1A-AS1序列的RNA。该术语还包括具有与人HNF1A-AS1RNA相同的抑制实体瘤功能的、野生型序列的变异形式。这些变异形式包括(但并不限于)：一个或多个核苷酸的缺失、插入和/或取代，以及在5′末端和/或3′末端添加一个或数个核苷酸。例如，在本领域中，用性能相近核苷酸进行取代时，通常不会改变RNA的功能。又比如，在5′末端和/或3′末端添加一个或数个核苷酸通常也不会改变RNA的功能。同样，该术语还包括人HNF1A-AS1的活性片段。
本发明还包括人HNF1A-AS1RNA的类似物。这些类似物与天然人HNF1A-AS1RNA的差别可以是核酸序列上的差异，也可以是不影响序列的核苷酸修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然核苷酸碱基的类似物，以及具有非天然存在的或合成的核苷酸的类似物。
本发明的多核苷酸可以是DNA形式或RNA形式。
DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链 的。
在本发明中，人HNF1A-AS1核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据人HNF1A-AS1的核苷酸序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
本发明也涉及包含HNF1A-AS1序列的载体(尤其是病毒载体)，以及用本发明的载体或HNF1A-AS1序列经基因工程产生的宿主细胞，以及经体外转录技术产生本发明所述RNA的方法。
将多核苷酸导入组织或细胞内的方法包括：将多聚核苷酸直接注入到体内组织中；或在体外通过载体(如病毒、噬菌体或质粒等)先将多聚核苷酸导入细胞中，再将细胞移植到体内等。重组的基因治疗载体(如病毒载体)可设计成表达野生型的HNF1A-AS1，以增加实体瘤中HNF1A-AS1的数量和活性。来源于病毒的表达载体如逆转录病毒、腺病毒、腺病毒相关病毒、单纯疱疹病毒、细小病毒等可用于将HNF1A-AS1基因转移至细胞内。构建携带HNF1A-AS1基因的重组病毒载体的方法可见于已有文献(Sambrook，et al.)。另外重组人HNF1A-AS1基因/RNA可包装到脂质体中，然后再转入细胞内。
本发明HNF1A-AS1RNA、HNF1A-AS1DNA及载体，当施用(给药)于哺乳动物对象(如人)时，可抑制恶性实体瘤的恶性表型。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的载体介质(包括水性载体介质)，形成药物组合物。水性载体介质的pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、肌内、腹膜内、静脉内、皮下、皮内、或局部给药。
本发明的药物组合物可直接用于诱导实体瘤的治疗，代表性的例子包括(但并不限于)：肝癌、胃癌、肠癌、胰腺癌、肾癌、前列腺癌及生殖腺肿瘤等。在使用本发明HNF1A-AS1基因/RNA或药物组合物时，还可同时或辅助使用其他治疗剂，如顺铂、TNF等，还可与其他基因如HSK-TV基因、P53基因等其他基因或化疗与放疗联合治疗。
本发明还提供了一种药物组合物，它含有(a)安全有效量(如0.0001-99wt％)的本发明HNF1A-AS1RNA、HNF1A-AS1DNA或载体以及(b)药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。诸如片剂和胶囊之类的药物组合物，可通过常规方法进行制备。药物组合物如针剂、溶液、片剂和胶囊宜在无菌条件下制造。本发明药物组合物中还可含有其他治疗剂，如化疗药物。使用药物组合物时，是将安全有效量的HNF1A-AS1RNA、HNF1A-AS1DNA或载体施用于哺乳动物。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明还提供了一种肿瘤细胞(尤其是恶性实体瘤)基因治疗的方法，它包括将HNF1A-AS1基因导入肿瘤细胞，使之表达，其中所述将HNF1A-AS1基因导入肿瘤细胞的方法包括用质粒转染、腺病毒或腺相关病毒介导。
本发明的有益效果如下：
(a)筛选并证实长链非编码RNA HNF1A-AS1对恶性实体瘤的抑制作用。
(b)体内外证实HNF1A-AS1治疗恶性实体瘤的可行性及其对临床研究的潜在意义。
附图说明
图1.Real-Time PCR检测HNF1A-AS1在肝癌细胞株中的表达。
图2.Real-Time PCR检测HNF1A-AS1在肝癌组织及其对应癌旁组织中的表达。
图3.cDNA末端快速扩增技术(rapid amplification of cDNA ends，RACE)扩增HNF1A-AS1cDNA的5′末端(A)和3′末端(B)。
图4.根据HNF1A-AS1cDNA的5′末端和3′末端延伸获得的HNF1A-AS1全长基因核苷酸序列。
图5.PCR方法扩增获得全长HNF1A-AS1的cDNA。
图6.酶切鉴定重组慢病毒载体pCDH-HNF1A-AS1。
图7.对照慢病毒lenti-Ctrl(A)或慢病毒lenti-HNF1A-AS1(B)分别感染Huh7细胞3天后绿色荧光表达情况。
图8.lenti-HNF1A-AS1慢病毒感染Huh7细胞(A)和Hep3B细胞(B)4天后Real-Time PCR检测HNF1A-AS1基因的表达水平。
图9.外源导入HNF1A-AS1抑制肝癌细胞Huh7(A)和Hep3B(B)的增殖。
图10.外源导入HNF1A-AS1抑制肝癌细胞Huh7(A)和Hep3B(B)的克隆形成能力。
图11.外源导入HNF1A-AS1可使Hep3B细胞发生G2期细胞阻滞，其中A为对照细胞流式细胞仪周期检测图，B为HNF1A-AS1过表达组细胞流式细胞仪周期检测图，C为A、B图细胞周期数据统计图。
图12.外源导入HNF1A-AS1抑制肝癌细胞Huh7(A)、Hep3B(B)、MHCC-H(C)的迁移能力。
图13.分别感染对照慢病毒lenti-Ctrl或慢病毒lenti-HNF1A-AS1的Huh7细胞的体内接种成瘤实验，其中A为肿瘤生长速率，B为小鼠成瘤在体图(上)及肿瘤离体图(下)，C为瘤体HNF1A-AS1基因表达水平。
图14.HNF1A-AS1基因治疗肝脏原位种植瘤实验，其中A为两组小鼠注射病毒前后活体呈像荧光检测图；B为小鼠肝脏及瘤体离体图，其中箭头指向为肿瘤组织；C为瘤体HNF1A-AS1基因表达水平。
具体实施方式
现结合实施例和附图，对本发明作详细描述，但本发明的实施不仅限于此。
本发明所用试剂和原料均市售可得或可按文献方法制备。下列实施例中未注明具体 条件的实验方法，通常按照常规条件如Sambrook等人《分子克隆：实验室指南》(New York：Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1：Real-Time PCR检测人肝肿瘤细胞株HNF1A-AS1基因的表达情况
1.将市售的常规肝肿瘤细胞株HepG2、Huh7、Hep3B、MHCC-L、MHCC-H、LM3、PLC、Focus均以5×105/皿接种于六孔板，以含10％胎牛血清的新鲜培养液培养，第二天抽提细胞RNA，分光光度计测定OD260值，1％琼脂糖凝胶电泳检测RNA完整性。
2.Real-Time PCR：
取1μg RNA加入4μl 5×PrimeScript RT master mix(逆转录试剂盒)，另加入DEPC水补足体积至20μl，37℃反应15min；85℃反应5s灭活逆转录酶，即可得到逆转录产物。将逆转录产物稀释后取1μl为模板进行HNF1A-AS1PCR扩增，同时以β-actin作为内参照在相同反应条件下进行PCR反应，反应体系如下：
Figure PCTCN2016000221-appb-000001
反应条件为95℃，30sec；95℃，5sec→60℃，34sec，40个循环；95℃，15sec→60℃，60sec→95℃，15sec。相关引物序列见表：
β-actin正向引物：CATCCTGCGTCTGGACCT(SEQ ID NO：2)；
反向引物：GTACTTGCGCTCAGGAGGAG(SEQ ID NO：3)；
HNF1A-AS1正向引物：CAAGAAATGGTGGCTATGA(SEQ ID NO：4)；
反向引物：TGGACTGAAGGACAAGGGT(SEQ ID NO：5)。
Real-Time PCR检测各肝癌细胞株HNF1A-AS1的基因表达水平。结果如图1所示，在HepG2、MHCC-L细胞中HNF1A-AS1的基因表达水平相对较高，而Huh7、Hep3B、MHCC-H细胞中HNF1A-AS1的基因表达水平居中，而PLC、LM3、Focus中HNF1A-AS1的基因表达水平相对较低。
Figure PCTCN2016000221-appb-000002
Figure PCTCN2016000221-appb-000003
实施例2：Real-Time PCR检测HNF1A-AS1在肝癌组织及其对应癌旁组织中的表达
取53例肝癌患者术后肝癌组织(样本来源：东方肝胆外科医院)，采用Trizol法抽提其RNA，分光光度计测定OD260值，1％琼脂糖凝胶电泳检测RNA完整性。
按照实施例1中RNA逆转录方法逆转录得到肝癌组织标本的cDNA，将cDNA稀释后按照实施例1上述同样方法和条件检测人肝癌组织中HNF1A-AS1的表达情况，同时以β-actin基因的表达情况作为内参照，结果表明HNF1A-AS1在肝癌组织中表达下调(图2)。
实施例3：cDNA末端快速扩增技术(rapid amplification of cDNA ends，RACE)扩增HNF1A-AS1基因全长
根据HNF1A-AS1已知的序列分别设计5’末端特异性扩增引物(5’GSP)AACTCGGACTGTTCTCCTTCCCACCCC(SEQ ID NO：6)和3’末端特异性扩增引物(3’GSP)ACGGCTAGTAAACGGCAGAACGAGGC(SEQ ID NO：7)，合成引物。
采用市售CLONTECH的MarathonTM试剂盒扩增HNF1A-AS1基因的cDNA序列，试剂盒中包括接头引物(adaptorprimer)及预制好的人肝脏cDNA，采用以下PCR体系进行扩增。
PCR扩增体系：
Figure PCTCN2016000221-appb-000004
混匀各组分，离心后放入PCR仪中进行扩增反应。
反应条件：94℃，5min；94℃30sec，70℃30s，72℃1min，40个循环；72℃7min，4℃∞
PCR扩增分别得到HNF1A-AS1的5’末端和3’末端片段，1％琼脂糖凝胶电泳分离鉴定产物(图3)，选择最长片段割胶回收置入Eppendorf管内，称取胶重量，按每100mg胶/200ml NT液的比例加入NT液，50℃，5-10min至凝胶熔化；将液体过柱，13,000rpm离心1min，加入700μl NT3洗涤液，13,000rpm离心1min；洗涤2次。将纯化柱置于干净的Eppendorf管上，开盖晾置1min，滴加30μl双蒸水至柱内滤膜上，静放2min，13,000rpm离心1min，过柱洗脱得到DNA片段，分光光度计测定其浓度。分别取纯化后的HNF1A-AS1的5’末端或3’末端片段4μl、pMD-19T载体1μl、solution I连接酶混合液5μl，16℃连接3h。将连接产物加入常规的感受态大肠杆菌DH5α转化，用含氨苄青霉素的LB培养基平皿铺板，37℃恒温过夜，挑取单菌落克隆至含氨苄青霉素的LB液体培养基中培养，待菌液浓度 OD600＝0.8-1.0，送至英骏生物公司测序。
测序结果表明HNF1A-AS1基因与NCBI数据库提供的序列(基因ID：NR_024345.1)5’末端有330bp的延伸(图4中方框中所标序列)，全长为2785bp，是一连续序列无其他剪切形式的长链非编码RNA。其基因全长序列为SEQ ID NO：1。
实施例4：构建HNF1A-AS1基因过表达慢病毒
1.构建HNF1A-AS1基因过表达慢病毒载体
根据新得到的HNF1A-AS1的基因序列设计并合成引物：
正向引物：GGAACAGCCGGACATGGTAG(SEQ ID NO：8)；
反向引物：GACGGAGTTTCGTTCTTGTTCC(SEQ ID NO：9)；
同时分别带有BamH I和EcoR I酶切位点，以人肝脏cDNA为模板，PCR扩增HNF1A-AS1序列，产物0.7％凝胶电泳鉴定，鉴定结果如图5所示，大小正确则采用试剂盒纯化回收PCR扩增片段。
BamH I和EcoR I分别酶切pCDH质粒(购自美国System Biosciences公司)和HNF1A-AS1的PCR扩增片段，凝胶电泳鉴定并纯化回收酶切后产物。取50-100ng线性化的pCDH质粒、500-1000ng BamH I和EcoR I酶切后的HNF1A-AS1片段，5μl solution I连接酶混合液，混匀后16℃连接过夜。将连接产物加入慢病毒载体专用感受态大肠杆菌Stbl3转化，用含氨苄青霉素的LB培养基平皿铺板，37℃恒温过夜，挑取单菌落克隆至含氨苄青霉素的LB液体培养基中培养，送适量菌液至公司测序。测序正确即得到慢病毒重组表达质粒pCDH-HNF1A-AS1，选择BamH I和EcoR I酶切鉴定重组表达质粒pCDH-HNF1A-AS1，如图6所示出现一个7kb左右片段为载体片段，另一3kb左右片段为HNF1A-AS1cDNA片段，表明质粒正确(图6)。
2.慢病毒的包装
按4×106个HEK293T细胞/皿的密度接种至10mm培养皿12-24h后，用
Figure PCTCN2016000221-appb-000005
将2500ng对照质粒pCDH-Ctrl或慢病毒重组质粒pCDH-HNF1A-AS1分别与1875ng psPAX2、625ng pMD2.G包装质粒共转染HEK293T细胞，5％CO2培养箱中培养12h后换成含有10％胎牛血清(FBS)的杜氏培养基(DMEM)，24h后收集上清储存于4℃，重新加入10ml上述培液继续培养24h，收集上清，混合两次收集的上清，1250rpm离心5min后取上清即得到对照慢病毒Lenti-Ctrl或携带有HNF1A-AS1基因的慢病毒Lenti-HNF1A-AS1。
实施例5：Real-Time PCR检测Lenti-HNF1A-AS1感染人肝肿瘤细胞株后HNF1A-AS1基因的表达水平
人肝肿瘤细胞株Huh7和Hep3B均以3×105个细胞/皿的密度接种至35mm培养皿，分别加入500μl慢病毒Lenti-Ctrl或Lenti-HNF1A-AS1，24h-48h后更换含10％胎牛血清的新鲜DMEM培液，培养3天后观察荧光表达情况，发现对照病毒和HNF1A-AS1病毒感染后的细胞均可看到明显绿色荧光(图7)，表明病毒成功感染细胞。5天后以Trizol试剂盒抽提总RNA，并采 用分光光度计测定OD260值，同时1％琼脂糖凝胶电泳检测RNA完整性。
按照实施例1中RNA逆转录方法逆转录得到cDNA，将cDNA稀释后按照实施例1上述同样方法和条件检测感染慢病毒Lenti-HNF1A-AS1后Huh7和Hep3B细胞中HNF1A-AS1基因的表达情况，同时以β-actin基因的表达情况作为内参照，结果表明感染慢病毒Lenti-HNF1A-AS1后Huh7和Hep3B细胞中HNF1A-AS1基因的表达均出现明显上调(图8)。
实施例6：外源导入HNF1A-AS1抑制肝肿瘤细胞增殖
人肝肿瘤细胞株Huh7和Hep3B均以2×105接种于35mm培养皿，分别感染对照慢病毒Lenti-Ctrl(对照组)或慢病毒Lenti-HNF1A-AS1(实验组)48h后，计数每孔3×103个细胞均匀分至96孔板，每天检测3复孔连续检测7天。第二天采用无血清培养基配置10％的CCK8，吸去96孔中原有培液，加入100μl配置好的CCK8试剂，37℃孵箱放置1h后检测450nm波长的吸光度以判断有活性细胞的数量。连续检测6天450nm吸光值，并绘制生长曲线。结果表明，感染慢病毒Lenti-HNF1A-AS1后人肝肿瘤细胞Huh7(图9A)和Hep3B(图9B)的增殖能力较对照组均受到明显抑制(图9)。
实施例7：外源导入HNF1A-AS1抑制肝肿瘤细胞的克隆形成能力
人肝肿瘤细胞株Huh7和Hep3B均以2×105接种于35mm培养皿，分别感染对照慢病毒Lenti-Ctrl(对照组)或慢病毒Lenti-HNF1A-AS1(实验组)48h后，计数分别取2×103/皿的细胞密度接种于35mm培养皿，每3天换液，培养3-4周，直至可见明显克隆，4％PFA固定，结晶紫染色，计数克隆，观察HNF1A-AS1对Huh7和Hep3B细胞克隆形成能力的影响。结果表明，感染慢病毒Lenti-HNF1A-AS1后Huh7和Hep3B细胞形成的克隆数较对照组明显减少(图10)。
实施例8：外源导入HNF1A-AS1使Hep3B细胞发生G2期细胞阻滞
将肝癌细胞株Hep3B以1×105/皿接种于12孔板，分别加入等体积慢病毒lenti-Ctrl或lenti-HNF1A-AS1感染细胞，24h后更换含10％胎牛血清的新鲜DMEM培液，第3天观察荧光表达情况，第4天收集细胞，PBS清洗后，重悬1×106个细胞至1ml PBS混合液中(含0.2％TritionX-100，50μg RNA酶，5μl碘化丙啶，0.1％FBS)，避光，室温孵育15min后，上机检测细胞周期变化并进行统计学分析。
结果表明在病毒感染72h后，HNF1A-AS1可使Hep3B细胞周期明显阻滞在G2期(图11)。
实施例9：外源导入HNF1A-AS1抑制人肝肿瘤细胞的迁移能力
人肝肿瘤细胞株Huh7、Hep3B、MHCC-H均以3×105接种于35mm培养皿，分别感染对照慢病毒Lenti-Ctrl(对照组)或慢病毒Lenti-HNF1A-AS1(实验组)48h后，计数每孔1×105个细胞均匀分至聚碳酸酯膜小室(Corning公司产品)的上层小室内，每组3复孔，48h后PBS清洗小室，结晶紫染色20min，镜下拍照观察HNF1A-AS1对人肝肿瘤细胞株迁移能力的影响，同时采用protein-plus软件统计迁移面积，分析HNF1A-AS1基因对肝癌细胞株迁移能力 的影响。
结果表明，感染HNF1A-AS1病毒后人肝肿瘤细胞株Huh7(图12A)、Hep3B(图12B)、MHCC-H(图12C)的迁移能力较对照组明显受到抑制。上调HNF1A-AS1可明显降低肝肿瘤细胞株的迁移能力(图12)。
实施例10：上调人肝肿瘤细胞株Huh7中HNF1A-AS1的表达可抑制其体内成瘤能力
分别取感染对照慢病毒Lenti-Ctrl或慢病毒Lenti-HNF1A-AS1 48h后的肝癌细胞Huh7按2×106的细胞数接种于裸鼠腋下，左侧接种感染对照病毒lenti-Ctrl的细胞，右侧接种感染lenti-HNF1A-AS1的细胞，观察体内成瘤情况，待肿瘤出现，每隔3天用游标卡尺测量新生肿瘤的大小并记录绘制对照组与实验组的肿瘤生长曲线。
结果表明感染对照病毒的Huh7细胞在接种后第18天检测出肿瘤生长，而感染慢病毒lenti-HNF1A-AS1的Huh7细胞则在第21天才检测到有肿瘤生成；在后续观察时间中，感染慢病毒lenti-HNF1A-AS1的Huh7细胞接种的裸鼠肿瘤生长速率明显低于对照慢病毒lenti-Ctrl组(图13A)，且对照组所形成的肿瘤较实验组明显较大、较重(图13B)，分离肿瘤组织，real-time PCR检测结果显示实验组肿瘤组织中HNF1A-AS1的表达水平高于对照组肿瘤组织(图13C)，上述结果表明HNF1A-AS1过表达可明显抑制Huh7细胞的体内成瘤能力。
实施例11：HNF1A-AS1对实验性肝癌的治疗作用
用可表达荧光素酶(firefly luciferase，LUC)基因的慢病毒(pSin-EF2-LUC-Pur)感染肝癌细胞Huh7，嘌呤霉素筛选获得可稳定表达LUC的肝癌细胞株。将荧光素酶基因标记的肝癌细胞直接注入NOD/SCID小鼠肝内(细胞数量约2×106/只)，并用活体生物体内成像系统动态监测肿瘤生长情况，待成瘤后(约10-14天)根据监测到瘤体的荧光强度将小鼠大致均匀分为2组，每组10只，通过尾静脉分别注射慢病毒Lenti-HNF1A-AS1或对照病毒Lenti-Ctrl。每隔5天用活体生物体内成像系统监测肿瘤生长情况，观察小鼠存活时间、生长状态及腹水量等，约6-8周后处死小鼠。
活体成像动态监测结果表明，注射病毒后实验组肿瘤的荧光检测值明显低于对照组(图14A)。处死小鼠后，可见实验组小鼠肝脏所成肿瘤的大小明显低于对照组小鼠(图14B)。抽提小鼠肿瘤组织的RNA，real-time PCR检测结果表明实验组小鼠肿瘤组织中HNF1A-AS1表达量高于对照组(图14C)。上述结果表明HNF1A-AS1过表达可明显抑制Huh7细胞肝脏原位种植瘤的生长速率，上调HNF1A-AS1对小鼠肝癌种植模型具有良好的治疗效果。
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可作出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。

Claims (10)

  1. 一种长链非编码RNA HNF1A-AS1在制备治疗人体恶性实体瘤药物中的应用。
  2. 根据权利要求1所述的一种长链非编码RNA HNF1A-AS1在制备治疗人体恶性实体瘤药物中的应用，其特征在于，所述的长链非编码RNA HNF1A-AS1的基因cDNA序列如SEQ ID NO：1所示。
  3. 根据权利要求1或2所述的一种长链非编码RNA HNF1A-AS1在制备治疗人体恶性实体瘤药物中的应用，其特征在于，所述的治疗人体恶性实体瘤药物为提高长链非编码RNA基因HNF1A-AS1基因或其转录产物HNF1A-AS1RNA的表达量的试剂。
  4. 根据权利要求1或2所述的一种长链非编码RNA HNF1A-AS1在制备治疗人体恶性实体瘤药物中的应用，其特征在于，所述的治疗人体恶性实体瘤药物包括以下任一：
    A)HNF1A-AS1RNA；
    B)HNF1A-AS1基因；
    C)含有HNF1A-AS1基因的表达载体。
  5. 根据权利要求4所述的一种长链非编码RNA HNF1A-AS1在制备治疗人体恶性实体瘤药物中的应用，其特征在于，所述的表达载体，包括重组质粒、重组病毒、重组病毒载体、非病毒载体。
  6. 根据权利要求4所述的一种长链非编码RNA HNF1A-AS1在制备治疗人体恶性实体瘤药物中的应用，其特征在于，所述的治疗人体恶性实体瘤药物包括慢病毒重组质粒pCDH-HNF1A-AS1。
  7. 根据权利要求4至6任一所述的一种长链非编码RNA HNF1A-AS1在制备治疗人体恶性实体瘤药物中的应用，其特征在于，所述的治疗人体恶性实体瘤药物为：包括A)至C)任一活性成份以及药学上可接受的载体或赋形剂组成的药物组合物。
  8. 根据权利要求7所述的一种长链非编码RNA HNF1A-AS1在制备治疗人体恶性实体瘤药物中的应用，其特征在于，所述的药物组合物的剂型为注射剂。
  9. 根据权利要求1或2所述的一种长链非编码RNA HNF1A-AS1在制备治疗人体恶性实体瘤药物中的应用，其特征在于，所述的人体恶性实体瘤选自：肝癌、胃癌、肠癌、胰腺癌、前列腺癌，或生殖腺肿瘤。
  10. 根据权利要求9所述的一种长链非编码RNA HNF1A-AS1在制备治疗人体恶性实体瘤药物中的应用，其特征在于，所述的人体恶性实体瘤为肝癌细胞株Huh7、肝癌细胞株Hep3B，或肝癌细胞株MHCC。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110358834A (zh) * 2019-07-12 2019-10-22 深圳大学 一种lncRNA的应用以及试剂盒和药物
CN110951884A (zh) * 2019-12-31 2020-04-03 北京泱深生物信息技术有限公司 Linc02166对胃癌诊断治疗的新用途
US10851376B2 (en) 2018-12-28 2020-12-01 The Florida International University Board Of Trustees Long noncoding RNAs in pulmonary airway inflammation

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105079821A (zh) * 2015-06-11 2015-11-25 中国人民解放军第二军医大学 一种长链非编码rna hnf1a-as1在制备治疗人体恶性实体瘤药物中的应用
GB201608907D0 (en) * 2016-05-20 2016-07-06 Ucl Business Plc Means for modulating gene expression
CN107038350B (zh) * 2016-07-11 2020-06-05 中国科学院数学与系统科学研究院 一种药物的长非编码rna靶点预测方法和系统
CN107022625B (zh) * 2017-05-09 2020-06-19 中南大学 一种人肝细胞癌发生发展相关的长链非编码rna、扩增检测方法及应用
CN109486816B (zh) * 2018-11-08 2021-04-06 浙江大学 一种用于肿瘤治疗的多聚核苷酸及其应用
CN113201540B (zh) * 2021-04-16 2023-10-10 重庆医科大学 非编码rna、包含非编码rna的rna序列及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105079821A (zh) * 2015-06-11 2015-11-25 中国人民解放军第二军医大学 一种长链非编码rna hnf1a-as1在制备治疗人体恶性实体瘤药物中的应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102475893B (zh) * 2010-11-25 2013-11-13 中国人民解放军第二军医大学 肝细胞核因子1α治疗人体恶性实体瘤
CN102286535B (zh) * 2011-06-17 2013-06-05 中国人民解放军第二军医大学 一种成纤维细胞转分化为肝干细胞的方法
AU2013320165A1 (en) * 2012-09-21 2015-04-02 Institut National De La Sante Et De La Recherche Medicale (Inserm) A new method for classification of liver samples and diagnosis of focal nodule dysplasia, hepatocellular adenoma, and hepatocellular carcinoma

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105079821A (zh) * 2015-06-11 2015-11-25 中国人民解放军第二军医大学 一种长链非编码rna hnf1a-as1在制备治疗人体恶性实体瘤药物中的应用

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DING, CHENHONG: ""MOLECULAR MECHANISM OF HNFL A IN INHIBITING THE DEVELOPMENT HEPATIC CARCINOMA", MED PUB HEALTH, 15 May 2015 (2015-05-15), pages E072, XP008184160 *
KLADI-SKANDALI.A.: "LONG NONCODING RNAS IN DIGESTIVE SYSTEM MALIGNANCIES: A NOVEL CLASS OF CANCER BIOMARKERS AND THERAPEUTIC TARGETS?", GASTROENTEROLOGY RESEARCH AND PRACTICE, vol. 2015, 29 April 2015 (2015-04-29), pages 1 - 18, XP055352865 *
WU.Y.: "THE LONG NON-CODING RNA HNF1A-AS1 REGULATES PROLIFERATION AND METASTASIS IN LUNG ADENOCARCINOMA", ONCOTARGET, vol. 6, no. 11, 26 March 2015 (2015-03-26), pages 9160 - 9172, XP055352833 *
YANG.X.: "LONG NON-CODING RNA HNF1A-AS1 REGULATES PROLIFERATION AND MIGRATION IN ESOPHAGEAL ADENOCARCINOMA CELLS", GUT, vol. 63, no. 6, 2 September 2013 (2013-09-02), pages 881 - 890, XP055352815 *
YIN,YANTAO: "LNCRNAEXPRESSION AND ITS WORKING MECHANISM IN CANCER",", CHIN JOUR. OF BIOCHEM. AND MOL BIOL, vol. 31, no. 4, 30 April 2015 (2015-04-30), pages 352 - 359, XP008183836 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10851376B2 (en) 2018-12-28 2020-12-01 The Florida International University Board Of Trustees Long noncoding RNAs in pulmonary airway inflammation
CN110358834A (zh) * 2019-07-12 2019-10-22 深圳大学 一种lncRNA的应用以及试剂盒和药物
CN110951884A (zh) * 2019-12-31 2020-04-03 北京泱深生物信息技术有限公司 Linc02166对胃癌诊断治疗的新用途

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