CN106367415A - Use of human MIR135a-3p in preparation of preparations for diagnosing and treating prostate cancer - Google Patents
Use of human MIR135a-3p in preparation of preparations for diagnosing and treating prostate cancer Download PDFInfo
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- CN106367415A CN106367415A CN201510444471.6A CN201510444471A CN106367415A CN 106367415 A CN106367415 A CN 106367415A CN 201510444471 A CN201510444471 A CN 201510444471A CN 106367415 A CN106367415 A CN 106367415A
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Abstract
The invention belongs to the biotechnical field, and concretely relates to a new prostate cancer molecule marker microRNA 135a-3p (MIR135a-3p) and a use thereof in the preparation of preparations for diagnosing and treating the prostate cancer. The use comprises the following steps: screening prostate cancer high-risk populations, diagnosing the prostate cancer, designing prostate cancer drug target, monitoring the prostate cancer treatment condition, and carrying out prognosis detection on the prostate cancer. A method using the molecule marker and a diagnostic kit containing the molecule marker to diagnose the prostate cancer has the characteristics of simplicity in operation, convenience in material drawing, good safety, no wounds, high specificity, high sensitivity, and easiness in massive screening.
Description
Technical field
The invention belongs to biological technical field, a kind of specifically related to new molecular markers in prostate cancer thing mir135a-3p and its purposes in the preparation of preparation diagnosis and treatment carcinoma of prostate, including the application in the field such as screening, the diagnosis of carcinoma of prostate, the design of carcinoma of prostate drug targets, the monitoring of prostate cancer therapy situation, the monitoring of carcinoma of prostate direction of medication usage and carcinoma of prostate prognosis detection in carcinoma of prostate high-risk group by this biomolecular labeling thing.
Background technology
Prior art discloses liquid Biopsy (according to related content in " asco annual report: 2015 Clinical Oncology progress ") and be listed in one of therapeutic field of tumor coming decade trend.Research display, some molecular markers in blood are applied to the diagnosis of some diseases, and such as glutamate pyruvate transaminase is used for the diagnosis of hepatitis, and number of white blood cells is used for judging inflammation.Blood circulation tumor dna (circulating tumor dna, ctdna) it is a kind of extracellular free dna of acellular state, it is present in the body fluid such as blood, synovial fluid and cerebrospinal fluid, somatic cell dna is through coming off or when discharging into blood circulation after apoptosis, it is mainly made up of short single-stranded or double-stranded dna and the single-stranded mixture with double-strand dna, is existed with dna protein complex or two kinds of forms of free dna.Ctdna is derived from the somatic mutation of tumor cell, therefore, ctdna is a kind of distinctive new tumor biomarker, and also can be by qualitative, quantitation and tracking, the early diagnosiss of clinical tumor will be become, prognosis judges and follow the tracks of follow-up etc. to provide a series of convenient, fast, special, noinvasive or minimally invasive and molecular Biological Detection means, particularly with some do not have typical clinical symptom, check the tumor of no specificity and difficult diagnosis can avoid complexity, there is traumatic biopsy.To become " liquid biopsy " in conjunction with new-generation sequencing technology (ngs), and substitute intrusion sense of organization biopsy.
Mir135a-3p belongs to microrna, it is the small rna by source gene code for the class, not encoding proteins, size is 20～24 nucleotide, it is widely present in plant, animal and various virus, after rna silence and transcription, play very important effect in translational control.Each mirna can have multiple target genes, and each gene is likely to be regulated and controled by multiple mirna.This complicated regulating networks both can regulate and control the expression of multiple genes by a mirna it is also possible to by the combination of several mirna come the expression of certain gene of finely regulating.It is assumed that mirna adjusts the gene of one of trichotomy.The features such as microrna is because of the conservative of its height, Space-time speciality, stability and tissue specificity makes it be better than the other biological marks such as protein, dna fragment and play, at aspects such as disease incidence Mechanism Study, early diagnosiss, individualized treatment and prognosis, the effect becoming more and more important.
Carcinoma of prostate is one of common male reproductive system malignant tumor.In world wide, prostate-cancer incidence occupies second in all malignant tumor of male, alreadys exceed pulmonary carcinoma in the sickness rate of U.S.'s carcinoma of prostate, becomes the tumor of first harm men's health.The sickness rate of Asia carcinoma of prostate is well below American-European countries, but assumes ascendant trend in recent years, and increases rapider than European and American developed countries.Patients with prostate cancer is mainly elderly men, and typically at 50 years old with sequela, 95% betides the elderly men of more than 60 years old, and incidence rate constantly increases with age.Carcinoma of prostate early stage many no any symptoms, even if uncomfortable, are also not enough to cause the attention of patient, when tumor increases urethra, and often obscure with prostatic hyperplasia phase.Find that metastasis focus just finds carcinoma of prostate first in the patient of China about 80%.Now, pathological changes have reached late period, prognosis malas.
At present, the mode of clinical diagnosises carcinoma of prostate mainly has digital rectal examination, serum PSA (psa) detection, the detection of endorectal ultrasonography ripple, living tissue pathologic finding etc..Digital rectal examination is the simplest, most economical practical method, mainly touches prostate by the forefinger of doctor, in order to find that much asymptomatic patients with prostate cancer is it is possible to obtain the chance of early diagnosiss and radical cure.But all there is limitation in above method.The limitation of such as digital rectal examination is mainly at 4 aspects: when (1) patient prostate lump is little, easy to cause missed diagnosis；(2) enlargement of some patientss carcinoma of prostate is inconspicuous, but is already belonging to late period, is difficult to effect a radical cure；(3) patient's rectum is had and can not be detected using this during illness；(4) doctors experience may have when not enough fail to pinpoint a disease in diagnosis or mistaken diagnosis possibility.Psa in blood not high (not higher than 4ng/ml) under normal circumstances, when being in carcinoma of prostate and other prostatosis disease states, psa raises becomes the most sensitive tumor mark of current examination carcinoma of prostate, but it there is also certain limitation: (1) needs to take blood examination to survey, and has certain damage to patient；(2) psa increases also common and non-prostate cancer disease, such as prostatitis, prostate hyperplasia etc., is therefore difficult to make a definite diagnosis；(3) psa increases when making a definite diagnosis carcinoma of prostate, and often patient already belongs to middle and late stage, does not reach the purpose of early diagnosiss.Prostate ultrasound examination simple and direct-viewing operation, not damaged, provide positioning and qualitative sign and judge the property of pathological changes by showing the size of lump, number, position, density, edge, body, the halo of form, size, number, distribution and surrounding having or not calcification and calcification, skin change etc.；Its limitation is: (1) easily fails to pinpoint a disease in diagnosis to the little cancer of compactness；(2) sometimes it is not provided that clear and definite etiologic diagnosis；(3) because it can not show the internal structure of tumor and surrounding tissue so diagnostic accordance rate is very low；(4) the good Malignant mass of real property lacking typical sign to some has higher misdiagnosis rate.Living tissue pathologic finding is because it is traumatic, complexity cannot function as the means of primary dcreening operation, but its goldstandard that to be carcinoma of prostate make a definite diagnosis, general with additive method technical battery use.
Recent studies indicate that, mirna is closely related with carcinoma of prostate, they may participate in generation, development and the transfer of tumor, so may have corresponding effect to the pathogenesis of tumor, early diagnosiss, individualized treatment, the detection of transfer and prognosis etc..
Because of the present circumstance, present inventor intends providing new purposes in the diagnostic preparation of preparation diagnosis and treatment carcinoma of prostate for the mir135a-3p.
Content of the invention
It is an object of the invention to, the defect existing for prior art, mir135a-3p and its new purposes in the diagnostic preparation of preparation diagnosis and treatment carcinoma of prostate are provided.
The application experimental research find that, the content in prostate cancer tissue sample is lower than the content in prostate normal structure sample, points out mir135a-3p and the tumorigenesis of carcinoma of prostate to there is close dependency.
The present invention is based on the studies above basis, there is provided the application in carcinoma of prostate high-risk group screening, diagnosis, treatment condition monitoring, Prognosis scoveillance of a kind of new carcinoma of prostate molecular marker mir135a-3p related to carcinoma of prostate, diagnostic kit, the design of carcinoma of prostate drug targets and this biomarker.
The technical solution adopted in the present invention is as follows:
Mir135a-3p, as prostate cancer marker, is a kind of mirna, and its nucleotide sequence is 5 '-uauagggauuggagccguggcg-3 ' (seq id no.1).
Described carcinoma of prostate biomarker mir135a-3p can be used for preparing carcinoma of prostate high-risk group screening, diagnosis, treatment condition monitoring, in the test kit of Prognosis scoveillance, by detecting the content of the mir135a-3p in subjectss' tissue samples, blood and urine, and to carry out carcinoma of prostate high-risk group's screening, diagnosis, treatment condition monitoring, Prognosis scoveillance compared with normal level mir135a-3p content.
More specifically, the invention provides described carcinoma of prostate biomarker mir135a-3p is in preparation carcinoma of prostate high-risk group screening, diagnosis, treatment condition monitoring, the application in the diagnostic kit of Prognosis scoveillance.
The diagnostic kit of described diagnosis of prostate cancer includes:
(1) total rna extracts reagent in tissue samples, blood and urine
(2) Reverse Transcription
(3) quantitative pcr reagent
Compare as rnu6 used by aforementioned detection kit, its primer sequence is as follows:
Rnu6 upstream primer sequence: 5 ＇-cgcttcggcagcacatatactaa -3 ＇ (seq id no.2)
Rnu6 downstream primer sequence: 5 ＇-tatggaacgcttcacgaatttgc -3 ＇ (seq id no.3)；
In the present invention, carry out the experiment of mir135a-3p horizontal diagnosis of prostate cancer in tissue samples, by detecting the content of the mir135a-3p in subjectss' tissue samples, blood and urine, and compared with the high-load of mir135a-3p in normal control tissue sample, result shows, the mir135a-3p content in patients with prostate cancer tissue samples is relatively low.And generally less than the meansigma methodss of normal structure sample, normal sample meansigma methodss are about 5 times of the meansigma methodss of tumor sample, and difference is statistically significant.
Advantages of the present invention has:
The present invention is that the diagnosis of carcinoma of prostate provides a kind of new molecular marker mir135a-3p, and be applied to prepare in diagnostic kit by this molecular marker further, using this molecular marker and the diagnostic kit diagnosis of prostate cancer containing this molecular marker, simple to operate, draw materials conveniently, safe hurtless measure, and there is higher specificity, susceptiveness and the feature facilitating a large amount of examinations, this molecular marker is suitably applied the screening of carcinoma of prostate high-risk group, the identification of carcinoma of prostate, the monitoring of prostate cancer therapy situation, the field such as the monitoring of carcinoma of prostate direction of medication usage and carcinoma of prostate Prognosis scoveillance.
Specific embodiment
For making the present invention easier to understand, the present invention is expanded on further with reference to specific embodiment, but following embodiments are merely to illustrate the present invention and do not limit the scope of the invention, in example below, NM specific experiment method, is carried out according to normal experiment method.
Embodiment 1
By following key steps in the present embodiment:
1. in tissue samples total rna extraction
Obtain the tissue samples after Prostate Cancer after Radical corrective surgery, obtain the patient of Pulmonary resections simultaneously
, as comparison, the total rna extracting aforementioned tissues sample is in no dna and no 1.5 milliliters of centrifuge tubes of rna enzyme pollution for the tissue samples of excision；
The test kit extracting total rna in tissue samples is purchased from Beijing CoWin Bioscience Co., Ltd.；The total rna extracting, by using therm nanodrop2000c spectrophotometer, measures the concentration mensuration that the ratio of 260/280nm ultraviolet wavelength is carried out；
2. the microrna in quantitative determination tissue sample
1) to obtain cdna single-stranded for reverse transcription rna
Configure reverse transcription system according to table 1 below, configuration process is carried out on ice.The system having configured is in pcr instrument
On carry out reverse transcription；Reaction condition is
42 degrees Celsius 15 minutes, 85 degrees Celsius 5 seconds；
Table 1
| Component | Usage amount (microlitre) |
| 5xprimescript buffer | 2 |
| primescript rt enzyme mix i | 0.5 |
| Oligo dt primer(50 micromole) | 0.5 |
| Random 6 mers(100 Micromole) | 0.5 |
| Specific primer(2 micromole) | 0.5 |
| total rna | x |
| rnase free dh2o | 6-x |
| Total amount | 10 |
In table 1, x represents that the rna volume of addition to be determined by rna concentration, is x=500 sodium gram/rna concentration in this experiment.Aforementioned reverse transcription reagent box is purchased from Dalian precious biology company limited (takara)；
2) qpcr detection by quantitative
With cdna diluent for real-time quantitative pcr template, the final concentration of 200nm of primer, reaction cumulative volume is 10 μ l；Real-time quantitative pcr instrument uses abi 7900ht sequence detection system, completed with 384 orifice plates, each sample repeats two to three times, in the case that solubility curve substantially conforms to requirement no non-specific amplification, reference operation handbook, relative expression quantity is obtained using threshold cycle (ct) method, with rnu6 as internal reference；Specific procedure is referring to table 2；
Table 2
3. data analysiss are carried out using array tools 4.1.0
Target mirna and the ct value level with reference to mirna in sample can be recorded with preceding method and try to achieve target mirna relative amount in the tissue, be with reference to the amount correcting mirna with rnu6, the 2 of classics in being detected by qpcr- δ ctMethod represent the level (- δ ct is the difference of target mirna and the ct value compareing) of mirna in tissue samples；
4. mir135a-3p horizontal diagnosis of prostate cancer in tissue samples
Experimental result shows, compared with the high-load of mir135a-3p in normal control tissue sample, the mir135a-3p content in patients with prostate cancer tissue samples is relatively low.And generally less than the meansigma methodss of normal structure sample, normal sample meansigma methodss are about 5 times of the meansigma methodss of tumor sample, and difference is statistically significant.
The present invention has been shown and described above be not restricted to the described embodiments; merely illustrating the principles of the invention described in above-described embodiment and description; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements both fall within scope of the claimed invention.Claimed scope is by appending claims and its equivalent thereof.
sequence listing
<110>
Fudan University；Shanghai Ji Yuan bio tech ltd
<120>purposes in the preparation of preparation diagnosis and treatment carcinoma of prostate for the people mir135a-3p
<130>
1
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3
<170>
patentin version 3.3
<210>
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rna
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homo sapiens
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uauagggauu ggagccgugg cg 22
<210>
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<212>
dna
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Artificial sequence
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cgcttcggca gcacatatac taa 23
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dna
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Artificial sequence
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tatggaacgc ttcacgaatt tgc 23
Claims (5)
1. a kind of carcinoma of prostate biomarker mir135a-3p it is characterised in that: described mir135a-3p is a kind of mirna, and the nucleotides sequence of mir135a-3p is classified as: seq id no.1:5 '-uauagggauuggagccguggcg-3 '.
2. purposes in preparation carcinoma of prostate high-risk group screening, diagnosis, drug targets design, treatment condition monitoring, the test kit of Prognosis scoveillance and in Drug therapy target for the carcinoma of prostate biomarker mir135a-3p described in claim 1.
3. purposes according to claim 2, it is characterized in that: the content of mir135a-3p in subjectss' serum, urine and tissue samples is detected by described test kit, and compared with normal level mir135a-3p content, carry out carcinoma of prostate high-risk group's screening, diagnosis, treatment condition monitoring and Prognosis scoveillance.
4. purposes according to claim 2 it is characterised in that: described test kit includes:
(1) total rna extracts reagent in tissue samples, blood and urine,
(2) Reverse Transcription,
(3) quantitative pcr reagent, the forward and reverse primer of specificity that described quantitative pcr reagent includes rnu6, respectively the rnu6 upstream primer sequence of seq id no.2: the rnu6 downstream primer sequence of 5 ＇-cgcttcggcagcacatatactaa -3 ＇ and seq id no.3: 5 ＇-tatggaacgcttcacgaatttgc -3 ＇.
5. purposes according to claim 3 it is characterised in that: in described subjectss' serum, urine and tissue samples, the content of mir135a-3p is extracted by total rna, reverse transcription, quantitative pcr are detected.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012138691A2 (en) * | 2011-04-04 | 2012-10-11 | Children's Medical Center Corporation | Diagnosis and treatment of taxane-resistant cancers |
| WO2014085906A1 (en) * | 2012-12-03 | 2014-06-12 | St. Michael's Hospital | Microrna biomarkers for prostate cancer |
-
2015
- 2015-07-24 CN CN201510444471.6A patent/CN106367415A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012138691A2 (en) * | 2011-04-04 | 2012-10-11 | Children's Medical Center Corporation | Diagnosis and treatment of taxane-resistant cancers |
| WO2014085906A1 (en) * | 2012-12-03 | 2014-06-12 | St. Michael's Hospital | Microrna biomarkers for prostate cancer |
Non-Patent Citations (6)
| Title |
|---|
| AURIANE KROISS 等: "Androgen-regulated microRNA-135a decreases prostate cancer cell migration and invasion through downregulating ROCK1 and ROCK2", 《ONCOGENE》 * |
| EUN AH SHIN 等: "Upregulation of microRNA135a-3p and death receptor 5 plays a critical role in Tanshinone I sensitized prostate cancer cells to TRAIL induced apoptosis", 《ONCOTARGET》 * |
| LAGOS-QUINTANA M 等: "accession number:MIMAT0004595", 《MIRBASE》 * |
| 汪宁 等: "MiR-135b对前列腺癌增殖与转移功能及分子机制学影响的研究探讨", 《浙江省医学会男科学泌尿外科学学术年会》 * |
| 赵明宇: "miR-135a通过抑制STAT6表达及其磷酸化调控激素非依赖前列腺癌细胞增殖的研究", 《DOC88.COM》 * |
| 黄烨清: "Hsa-miR-135a通过抑制TIAM1的表达调控去势抵抗性前列腺癌转移及侵袭能力的研究", 《东南大学临床医学硕士学位论文》 * |
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Application publication date: 20170201 |