CN113897435A - MiRNA for evaluating melanoma metastasis risk and application thereof - Google Patents
MiRNA for evaluating melanoma metastasis risk and application thereof Download PDFInfo
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- CN113897435A CN113897435A CN202111385245.7A CN202111385245A CN113897435A CN 113897435 A CN113897435 A CN 113897435A CN 202111385245 A CN202111385245 A CN 202111385245A CN 113897435 A CN113897435 A CN 113897435A
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- 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
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- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention relates to the field of biomedicine, and particularly relates to miRNA for evaluating melanoma metastasis risk and application thereof. The correlation between the miR-182-5p and melanoma metastasis is analyzed and known by detecting the expression level of the miR-182-5p in melanoma, on the basis, when a mimic capable of improving miR-182-5p expression, such as miR-182-5p imic, is transferred into a melanoma cell line, the migration capacity of the cell is enhanced, and when an inhibitor for inhibiting miR-182-5p expression, such as miR-182-5p inhibitor, the migration capacity of the cell is reduced, so that the result has important guiding significance for diagnosis and treatment of melanoma.
Description
Technical Field
The invention relates to the field of biomedicine, and particularly relates to miRNA for evaluating melanoma metastasis risk and application thereof.
Background
Melanoma is a neoplastic disease caused by uncontrolled and massive proliferation of melanocytes, mostly evolving from normal nevi and pigmented plaques. The disease is frequently seen on the skin, and the clinical manifestations mainly include skin bleeding, pruritus, tenderness, ulcer and the like. In the early stage of melanoma, more than 90% of patients can be cured by means of surgical resection, but if melanoma enters a rapid growth phase, it is extremely difficult to cure. Mainly, the melanoma metastasis rate in the period is high, and the melanoma can be transferred to various organs such as lymph, heart, liver, lung, kidney and the like in a short period of time. Therefore, the disease is extremely difficult to treat and extremely high in fatality rate, and is second only to pancreatic cancer.
The detection of the expression level of miRNA can provide reference for clinical diagnosis of cancer. The abnormal expression of miRNA directly causes the abnormal expression of genes related to cancer occurrence and metastasis, thereby inducing the cancer occurrence. In future clinics, miRNA can be used as a related marker for early diagnosis and cancer process of melanoma and a target site for treatment of melanoma.
Disclosure of Invention
In view of the above technical problems, one of the objects of the present invention is to provide a miRNA for assessing melanoma metastasis risk, wherein the miRNA is miR-182-5p and has a sequence shown in SEQ ID No. 1.
The invention also aims to provide application of the miRNA in preparing a diagnostic product for evaluating melanoma metastasis risk.
The invention also aims to provide application of a reagent for detecting the expression level of the miRNA in preparing a diagnostic product for evaluating the transfer risk of melanoma.
The fourth purpose of the invention is to provide a diagnostic product for evaluating the risk of melanoma metastasis, which comprises a related reagent for detecting the expression of the miRNA.
Further, the diagnostic product is in the form of an in vitro diagnostic product.
Still further, the diagnostic product is in the form of a diagnostic kit.
Furthermore, the diagnostic kit is a qRT-PCR detection kit and comprises a primer for detecting miR-182-5p by qRT-PCR.
Furthermore, the sequence of the upstream primer for detecting miR-182-5p by qRT-PCR is shown in SEQ ID NO. 10.
The fifth purpose of the invention is to provide a method for evaluating melanoma metastasis risk without diagnosis purpose, the diagnostic product is used for detecting the expression level of miR-182-5p in melanoma, and if the expression level of miR-182-5p in melanoma is remarkably increased, the risk of melanoma metastasis of a subject is judged to be high.
Compared with the prior art, the invention has the following beneficial effects:
1. the research result proves that miR-182-5p is related to the development of melanoma, and whether the risk of melanoma metastasis exists can be judged by detecting the expression level of miR-182-5 p.
2. The invention verifies that when an inhibitor capable of reducing miR-182-5p, such as miR-182-5p inhibitor, is transferred into a melanoma cell line, the migration capacity of the cell can be obviously reduced, and the result has important guiding significance for the treatment of melanoma.
Drawings
Figure 1A shows the expression of miR-182-5P in the various treatment fractions of cells, indicating P < 0.001. And B is the change of the expression quantity of the target gene and the downstream gene after miR-182-5p mature body mimic and inhibitor transfection. Denotes P <0.05, denotes P < 0.001.
FIG. 2 shows a cell scratch test, in which column A is a blank; column B is a miR-182-5p mimic group for transfection; the column C is a miR-182-5p inhibitor transfection group, each column comprises three groups of repeats, the upper graph in each group of repeats is the state of cell scratch before treatment, and the lower graph is the state of healing after cell treatment.
FIG. 3 is a result of cell scratching, in which: p <0.01, P <0.001, P < 0.0001.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.
Example 1
After the melanoma cells are transfected with miR-182-5p imic and inhibitor, the expressions of miR-182-5p and a target gene are detected by qRT-PCR.
1. Materials and cell culture
1) The B16F10 melanoma cells were passage 10 cells (available from the institute of Life sciences 113, university of Shanxi agriculture). Taking out melanocyte from liquid nitrogen, recovering in 37 deg.C water bath, adding equivalent amount of DMEM + 10% fetal calf serum, centrifuging at 1000r/mim for 10min, discarding supernatant, suspending melanocyte with melanocyte complete culture medium (DMEM + 10% fetal calf serum + 1% double antibody), and packaging in 6-well cell culture plate at 37 deg.C and 5% CO2Culturing in an incubator.
2. Preparation before transfection with transfection reagent: the concentration of the diluted miR-182-5p and miR-182-5p inhibitor is 300xng/mL, the diluted transfection reagent is 100 times, DMEM is used as the diluent, the used reagents are respectively diluted, the mixture is kept stand for 20min at room temperature, and miR-182-5p inhibitor diluent are uniformly mixed with the diluted transfection reagent and are placed for 5min to serve as transfection solution.
3. And taking out the cultured cells, discarding the culture solution, adding a corresponding transfection solution into each hole of the cells by using a serum-free cell culture solution (DMEM + 1% double antibody) as the cell culture solution, placing the cell culture plate in a cell culture box for culturing for 4 hours, and then replacing the complete culture medium of the melanocytes for the cells to continue culturing.
4. After 48h, cells were removed from the incubator and harvested, and the experiment was performed as a blank control with untransfected B16F10 cells as a control.
5. RNA extraction
Extracting RNA by a Trizol-chloroform extraction method and detecting the quality and the concentration of the RNA by a nucleic acid detector.
6. Reverse transcription of RNA
RNA reverse transcription bodyThe reaction solution was mixed and centrifuged instantaneously as follows (Table 1); reaction in a PCR instrument, and setting conditions are as follows: 60min at 37 ℃; 5min at 85 ℃; storing at 4 ℃. After the reaction system is completed, RNase-Free ddH is added2And obtaining miRNA reverse transcription products from O to 100 mu L. The reverse transcription product was stored at-20 ℃ until use.
TABLE 1 miRNA first Strand Synthesis reaction System
b. Reverse transcription of Total RNA to synthesize cDNA
Table 2 shows the reaction system of total RNA reverse transcription and DNA enzyme removal, the system is added according to the table 3 and then is blown and beaten by a pipette and mixed evenly, the temperature is kept at 42 ℃ for 2min, and the temperature is kept at 4 ℃. Corresponding reagents (DNA reverse transcription kit by Takara, reverse transcription primer set provided in the kit) were added to the reverse transcription reaction system shown in Table 3, and the reaction was carried out at 37 ℃ for 1 hour. The reaction product is stored at minus 20 ℃ for later use.
TABLE 2 RNA genome removal reaction System
TABLE 3 RNA reverse transcription System
7. qRT-PCR reaction and detection
Mitf, Tyr, Tyrp2 gene sequences were downloaded at the NCBI website and the gene primers were designed at the Premier 3.0 website. Wherein the internal reference of miR-182-5p is U6, and the downstream primers of miR-182-5p and U6 are provided by a reverse transcription kit. And 18S is selected as internal reference genes of Mitf, Tyr, Tyrp2 and the like, experimental components are a blank group, a miR-182-5p mimic transfection group and a miR-182-5p inhibitor transfection group, and a reaction system and an amplification program are shown in the following tables 5 and 6. Wherein all gene annealing temperatures were set at 60 ℃.
TABLE 4 primer sequences for genes of interest
TABLE 5 qRT-PCR reaction System for genes of interest
TABLE 6 reaction procedure for qRT-PCR of genes of interest
Quantitative data were analyzed by SPASS16.0, and the relative expression level of the target gene was 2-ΔΔCT. The result is shown in figure 1, after miR-182-5p imimic and inhibitor are transfected, miR-182-5p target gene expression in melanoma is not changed greatly, but key downstream gene expression is inhibited.
Example 2
Effect of miR-182-5p on melanoma cell migration ability
1. Cell culture and transfection
The experimental setup groups include a blank group, a transfection miR-182-5p mimic group and a transfection miR-182-5pinhibitor group, and the cell culture and transfection methods are the same as those in example 1.
2. Cell scoring
And performing scratch test on each group of cells, sucking away the original culture solution, marking a vertical line (along a straight ruler) at the center of the cells by using a 200-microliter sterile gun head, flushing the cells twice (the operation is gentle) by using PBS after marking the vertical line, adding a new cell culture medium, placing the cells under a microscope for photographing and recording, continuously culturing for 24 hours, and taking out the cells under the microscope again for photographing and recording. The effect of each component on cell migration was judged by the degree of healing between the scratches.
3. Cell scoring results
Integrating scratch test results, and obtaining a graph 3 by quantifying data in the graph as shown in a figure 2, wherein in the graph 2, a column A is a blank group, a column B is a transfection miR-182-5p mimic group, and a column C is a transfection miR-182-5p inhibitor group. Group a had healing and the degree of healing was very high, with column B having slightly higher mobility than column a, but not significant. While the cells in group C had very little to no healing. I.e., the healing degree of the groups A and B is significantly higher than that of the group C. The results show that when an inhibitor capable of reducing miR-182-5p, such as miR-182-5p inhibitor, is transformed into a B16F10 cell line, the migration capacity of the cells is remarkably reduced.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
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<120> miRNA for evaluating melanoma metastasis risk and application thereof
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Claims (9)
1. The miRNA for evaluating the melanoma metastasis risk is characterized in that the miRNA is miR-182-5p, and the sequence is shown in SEQ ID NO. 1.
2. Use of a miRNA according to claim 1 for the preparation of a diagnostic product for assessing the risk of melanoma metastasis.
3. Use of a reagent for detecting the expression level of the miRNA of claim 1 for the preparation of a diagnostic product for assessing the risk of melanoma metastasis.
4. A diagnostic product for assessing the risk of melanoma metastasis comprising an agent related to the detection of the expression of the miRNA of claim 1.
5. The diagnostic product of claim 4, in the form of an in vitro diagnostic product.
6. The diagnostic product of claim 5, in the form of a diagnostic kit.
7. The diagnostic product of claim 6, wherein the diagnostic kit is a qRT-PCR assay kit comprising primers for qRT-PCR detection of miR-182-5 p.
8. The diagnostic product of claim 7, wherein the sequence of the upstream primer for detecting miR-182-5p by qRT-PCR is shown in SEQ ID No. 10.
9. A method for assessing the risk of melanoma metastasis without the purpose of diagnosis, which comprises detecting the expression level of miR-182-5p in melanoma using the diagnostic product according to claim 8, and determining that the subject is at high risk of melanoma metastasis if the expression level of miR-182-5p in melanoma is significantly increased.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170275703A1 (en) * | 2014-08-25 | 2017-09-28 | The Council Of The Queensland Institute Of Medical Research | Treatment and detection of melanoma |
| CN107326026A (en) * | 2017-07-12 | 2017-11-07 | 河南师范大学 | MiR 182 analogies, mortifier and its recombinant expression carrier and application |
| CN112426538A (en) * | 2011-08-04 | 2021-03-02 | 耶达研究及发展有限公司 | micro-RNAs and compositions comprising micro-RNAs |
| CN113230267A (en) * | 2021-03-22 | 2021-08-10 | 四川大学华西医院 | Application of microRNA sequence in preparation of malignant melanoma gene therapy drug |
-
2021
- 2021-11-22 CN CN202111385245.7A patent/CN113897435A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112426538A (en) * | 2011-08-04 | 2021-03-02 | 耶达研究及发展有限公司 | micro-RNAs and compositions comprising micro-RNAs |
| US20170275703A1 (en) * | 2014-08-25 | 2017-09-28 | The Council Of The Queensland Institute Of Medical Research | Treatment and detection of melanoma |
| CN107326026A (en) * | 2017-07-12 | 2017-11-07 | 河南师范大学 | MiR 182 analogies, mortifier and its recombinant expression carrier and application |
| CN113230267A (en) * | 2021-03-22 | 2021-08-10 | 四川大学华西医院 | Application of microRNA sequence in preparation of malignant melanoma gene therapy drug |
Non-Patent Citations (2)
| Title |
|---|
| 李旭文等: "miR-182 增强黑色素瘤细胞增殖和侵袭的作用研究", 中华全科医学 * |
| 陈诚等: "ｍｉＲ－１８２受ＴＥＡＤ１转录调控参与黑素瘤细胞转移", 中国临床医学 * |
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