A melanoma biomarker and its application

Abstract

The present invention provides a melanoma biomarker and its application. The melanoma biomarker is miR-324-3p or the G3BP2 gene. The present invention discovers for the first time that miR-324-3p is significantly highly expressed in melanoma cells, serum or tissues, while the G3BP2 gene is significantly lowly expressed in melanoma cells, serum or tissues; therefore, it can be used as a melanoma biomarker for assisting in the early diagnosis or prognosis prediction of melanoma; the present invention also discovers that a miR-324-3p function enhancer or a G3BP2 gene silencer can improve the drug resistance of melanoma cells to chemotherapeutic drugs and enhance the chemosensitivity of melanoma cells to chemotherapeutic drugs, thereby achieving the purpose of improving the efficacy of chemotherapeutic drugs against melanoma, providing new ideas and approaches for the treatment of melanoma.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a melanoma biomarker and its application. Background Art

[0002] Cutaneous melanoma is the 19th most common cancer type globally in 2020, accounting for 1.7% of all cancer cases and 0.6% of all cancer deaths. Although the cure rate of early-stage melanoma is high, melanoma is highly malignant and prone to metastasis; therefore, the early occurrence of melanoma is of great significance.

[0003] Currently, for advanced melanoma, chemotherapy and biotherapy are the main treatment methods. Among them, the traditional chemotherapy drugs for melanoma mainly include dacarbazine (DTIC), vinca alkaloids, nitrosoureas, PCZ, and platinum drugs (such as cisplatin DDP, carboplatin, oxaliplatin). DTIC is the first-choice drug for treating melanoma, and its single-agent effective rate is about 20%; the immunotherapy drugs for melanoma mainly include interleukin-2 (IL-2), interferon α (IFN-α), etc.

[0004] To improve the curative effect, a biochemotherapy method has been developed currently, that is, biotherapy and chemotherapy are used in combination. Its effective rate and remission rate are both higher than those of simple chemotherapy or simple biotherapy, and it is one of the best methods for treating melanoma currently.

[0005] Currently, the combined use of DTIC + DDP + IL2 + IFN-α is the most widely used biochemotherapy regimen for melanoma treatment in China. Its effective rate can reach 20% - 30%, but the overall treatment effect for advanced melanoma is poor, and the median survival period is only about half a year. The reasons are, on the one hand, that melanoma cells themselves are less sensitive to chemotherapy drugs, and on the other hand, melanoma cells are prone to develop drug resistance to chemotherapy drugs, resulting in a reduced overall curative effect and poor prognosis.

[0006] Therefore, it is necessary to explore some new methods to overcome the drug resistance of melanoma to chemotherapy drugs. Summary of the Invention

[0007] The invention object of the present invention is to provide a melanoma biomarker and its application, so as to provide new ideas and approaches for the early detection and treatment of melanoma.

[0008] To achieve the above invention object, the technical solution of the present invention is as follows:

[0009] A melanoma biomarker, which is miR-324-3p or the G3BP2 gene.

[0010] The present invention discovers for the first time that, compared with the expression levels of miR-324-3p in normal human skin melanocytes and normal serum, the expression levels of miR-324-3p in human melanoma cells, tissues and the serum of melanoma patients are significantly down-regulated (P<0.001); meanwhile, compared with the expression levels of the G3BP2 gene in normal human skin melanocytes and normal serum, the expression levels of the G3BP2 gene in human melanoma cells, tissues and the serum of melanoma patients are significantly up-regulated (P<0.05); at the same time, the experiment shows that miR-324-3p can bind to the G3BP2 gene to achieve the purpose of silencing or inhibiting the expression of the G3BP2 gene. The above results indicate that both miR-324-3p and the G3BP2 gene can be used as melanoma biomarkers for assisting in the early diagnosis or prognosis prediction of melanoma, providing a new approach for the early diagnosis or prognosis prediction of melanoma.

[0011] Based on this, the present invention also provides the application of the above-mentioned melanoma biomarker in the preparation of early diagnosis or prognosis prediction reagents for melanoma.

[0012] The present invention also provides an early diagnosis or screening reagent for melanoma, an early diagnosis or screening kit for melanoma containing the early diagnosis or screening reagent for melanoma; and, a prognosis prediction reagent for melanoma, a prognosis prediction kit for melanoma containing the prognosis prediction reagent for melanoma.

[0013] Among them, the early diagnosis or screening reagent for melanoma or the prognosis prediction reagent for melanoma can both be specific amplification primers or probes for miR-324-3p or its coding gene; or, specific amplification primers or probes for the G3BP2 gene or the RNA expressed by it; or, an antibody that specifically binds to the protein expressed by the G3BP2 gene.

[0014] By using the above reagents to detect the expression levels of miR-324-3p or G3BP2 in the test subject, the melanoma disease status or melanoma prognosis of the test subject can be understood.

[0015] Preferably, in the above-mentioned early diagnosis or screening reagent for melanoma or prognosis prediction reagent for melanoma, the specific amplification primers for the coding gene of miR-324-3p include:

[0016] Forward primer: 5’-CTGGGTCGTATCCAGTGCAA-3’;

[0017] Reverse primer: 5’-GTCGTATCCAGTGCGTGTCG-3’.

[0018] The specific amplification primers for the G3BP2 gene include:

[0019] Forward primer: 5’-GAGCTGCAAGAGAGCGAGAA-3’;

[0020] Reverse primer: 5’-GACCTCGATCATTGCGCCTA-3’.

[0021] The present invention also provides the use of a miR-324-3p function enhancer in the preparation of a therapeutic drug for melanoma. Among them, the miR-324-3p function enhancer includes a miR-324-3p mimic or a plasmid containing the miR-324-3p mimic. The miR-324-3p mimic has the following nucleotide sequence:

[0022] 5’-CCCACUGCCCCAGGUGCUGCUGG-3’.

[0023] Meanwhile, the present invention also provides the use of a G3BP2 gene silencer in the preparation of a therapeutic drug for melanoma.

[0024] The present invention also studies and finds that transfection of miR-324-3p mimic in oxaliplatin-resistant melanoma cells can significantly up-regulate the expression level of miR-324-3p and simultaneously significantly down-regulate the expression level of G3BP2, indicating that the miR-324-3p function enhancer or the G3BP2 gene silencer can improve the drug resistance of melanoma cells to chemotherapeutic drugs and enhance the chemosensitivity of melanoma cells to chemotherapeutic drugs, so as to achieve the purpose of improving the efficacy of chemotherapeutic drugs against melanoma, providing new ideas and approaches for the treatment of melanoma.

[0025] Among them, the G3BP2 gene silencer can be a miR-324-3p function enhancer or other drugs capable of silencing the G3BP2 gene.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] (1) It was first discovered in the present invention that, compared with the expression levels of miR-324-3p in normal human skin melanocytes and normal serum, the expression levels of miR-324-3p in human melanoma cells, tissues and the serum of melanoma patients were significantly down-regulated (P<0.001); meanwhile, compared with the expression levels of the G3BP2 gene in normal human skin melanocytes and normal serum, the expression levels of the G3BP2 gene in human melanoma cells, tissues and the serum of melanoma patients were significantly up-regulated (P<0.05); at the same time, the experiment showed that miR-324-3p could bind to the G3BP2 gene to achieve the purpose of silencing or inhibiting the expression of the G3BP2 gene. The above results indicate that both miR-324-3p and the G3BP2 gene can be used as melanoma biomarkers for assisting in the early diagnosis or prognosis prediction of melanoma, providing a new approach for the early diagnosis or prognosis prediction of melanoma.

[0028] (2) The present invention also found through research that transfection of miR-324-3p mimic into oxaliplatin-resistant melanoma cells could significantly up-regulate the expression level of miR-324-3p and simultaneously significantly down-regulate the expression level of G3BP2, indicating that miR-324-3p function enhancer or G3BP2 gene silencer can improve the drug resistance of melanoma cells to chemotherapeutic drugs and enhance the chemosensitivity of melanoma cells to chemotherapeutic drugs, so as to achieve the purpose of improving the efficacy of chemotherapeutic drugs against melanoma, providing new ideas and approaches for the treatment of melanoma. Brief Description of the Drawings

[0029] Figure 1 Comparison of the relative expression levels of miR-324-3p in the serum of melanoma patients and the serum of healthy donors;

[0030] Among them, Cancer represents the serum of melanoma patients, Normal represents the serum of healthy donors, Relative miR-324-3p expression level represents the relative expression level of miR-324-3p, *** indicates that compared with the normal group, the P value <0.001, the same below;

[0031] Figure 2 Comparison of the relative expression levels of G3BP2 in melanoma tissues and normal tissues;

[0032] Among them, Tumor represents melanoma tissue, Normal represents normal tissue, num(T)=461 indicates that the number of samples of melanoma tissue is 461, num(N)=558 indicates that the number of samples of normal tissue is 558, G3BP2 expression-log 2(TPM + 1) represents the logarithm of the G3BP2 gene expression level; * indicates that compared with the Normal group, the P value < 0.05, the same below;

[0033] Figure 3 Comparison of the relative expression levels of miR - 324 - 3p in human melanoma A375 and MNT - 1 cells and human normal skin melanocyte PIG1;

[0034] Among them, ^^^ indicates that compared with PIG1, the P value < 0.001, the same below;

[0035] Figure 4 Comparison of the relative expression levels of the G3BP2 gene in human melanoma A375 and MNT - 1 cells and human normal skin melanocyte PIG1;

[0036] Among them, Relative G3BP2 mRNA expression level represents the relative expression level of the G3BP2 gene, the same below;

[0037] Figure 5 Nucleotide sequence segments at the miR - 324 - 3p binding site in the wild - type G3BP2 gene and mutant G3BP2 gene designed in this example;

[0038] Among them, Position 1 - 31of G3BP2 3’UTR WT represents the nucleotides at positions 1 - 31 of the wild - type G3BP2 gene, and Position 1 - 31of G3BP2 3’UTR WUT represents the nucleotides at positions 1 - 31 of the mutant G3BP2 gene;

[0039] Figure 6 Relative luciferase activity of firefly luciferase in melanoma cell A375 under co - transfection of MiR - 324 - 3p mimic or MiR - 324 - 3p mimic control with pmirGLO - G3BP2 - WT reporter plasmid or pmirGLO - G3BP2 - MUT reporter plasmid;

[0040] Among them, MC represents MiR - 324 - 3p mimic control, M represents MiR - 324 - 3p mimic, G3BP2 WT represents the wild - type G3BP2 gene, G3BP2 MUT represents the mutant G3BP2 gene, Relative luciferase activity represents relative luciferase activity, indicates that compared with the M - G3BP2 WT group, the P value < 0.001, the same below;

[0041] Figure 7The relative activity of firefly luciferase in melanoma cell MNT-1 under co-transfection of MiR-324-3p mimic or MiR-324-3p mimic control with pmirGLO-G3BP2-WT reporter plasmid or pmirGLO-G3BP2-MUT reporter plasmid;

[0042] Figure 8 Comparison of the relative cell viability of melanoma cells and oxaliplatin-resistant melanoma cells at different oxaliplatin treatment concentrations;

[0043] Among them, A375 / OXR represents drug-resistant melanoma cell A375, MNT-1 / OXR represents drug-resistant melanoma cell MNT-1, Relative cell viability(%) represents relative cell viability (percentage), *** indicates that compared with the A375 group, the P value < 0.001; ^^^ indicates that compared with the MNT-1 group, the P value < 0.001; ^^ indicates that compared with the MNT-1 group, the P value < 0.01; the same below;

[0044] Figure 9 The IC50 values of different melanoma cells under oxaliplatin treatment;

[0045] Among them, IC50 value of Oxaliplatin(μM) represents the IC50 value of oxaliplatin (micromoles per liter); the same below;

[0046] Figure 10 Comparison of the relative expression levels of miR-324-3p in different melanoma cells;

[0047] Figure 11 Comparison of the relative expression levels of G3BP2 in different melanoma cells;

[0048] Figure 12 The effect of transfection with miR-324-3p mimic or miR-324-3p mimic control on the relative expression level of miR-324-3p in drug-resistant melanoma cell A375 / OXR;

[0049] Among them, +++ indicates that compared with the A375 / OXR group, the P value < 0.001, the same below;

[0050] Figure 13 The effect of transfection with miR-324-3p mimic or miR-324-3p mimic control on the relative expression level of miR-324-3p in drug-resistant melanoma cell MNT-1 / OXR;

[0051] Among them, +++ indicates that the P value < 0.001 compared with the MNT-1 / OXR group, and the same applies hereinafter;

[0052] Figure 14 Effect of transfection with miR-324-3p mimic or miR-324-3p mimic control on the relative expression level of G3BP2 protein in drug-resistant melanoma cells A375 / OXR;

[0053] Among them, Relative G3BP2 protein expression level indicates the relative expression level of G3BP2 protein, ** indicates that the P value < 0.01 compared with the A375 / OXR group; the same applies hereinafter;

[0054] Figure 15 Effect of transfection with miR-324-3p mimic or miR-324-3p mimic control on the relative expression level of G3BP2 protein in drug-resistant melanoma cells MNT-1 / OXR;

[0055] Among them, ^^ indicates that the P value < 0.01 compared with the MNT-1 / OXR group; the same applies hereinafter;

[0056] Figure 16 Effect of transfection with miR-324-3p mimic or miR-324-3p mimic control on the cell viability of drug-resistant melanoma cells A375 / OXR;

[0057] Among them, *** indicates that the P value < 0.001 compared with the A375 group, +++ indicates that the P value < 0.001 compared with the A375 / OXR group, ++ indicates that the P value < 0.01 compared with the A375 / OXR group; the same applies hereinafter;

[0058] Figure 17 Effect of transfection with miR-324-3p mimic or miR-324-3p mimic control on the oxaliplatin IC 50 value of drug-resistant melanoma cells A375 / OXR;

[0059] Figure 18 Effect of transfection with miR-324-3p mimic or miR-324-3p mimic control on the cell viability of drug-resistant melanoma cells MNT-1 / OXR;

[0060] Among them, ^^^ indicates that the P value < 0.001 compared with the MNT-1 group; ^^ indicates that the P value < 0.01 compared with the MNT-1 group; +++ indicates that the P value < 0.001 compared with the MNT-1 / OXR group; ++ indicates that the P value < 0.01 compared with the MNT-1 / OXR group; the same below;

[0061] Figure 19 Effect of transfection with miR-324-3p mimic or miR-324-3p mimic control on the oxaliplatin IC 50 value of drug-resistant melanoma cells MNT-1 / OXR;

[0062] Figure 20 Effect of transfection with miR-324-3p mimic or miR-324-3p mimic control on the migration ability of drug-resistant melanoma cells A375 / OXR;

[0063] Figure 21 Effect of transfection with miR-324-3p mimic or miR-324-3p mimic control on the migration ability of drug-resistant melanoma cells MNT-1 / OXR;

[0064] Figure 22 Effect of transfection with miR-324-3p mimic or miR-324-3p mimic control on the relative migration rate of drug-resistant melanoma cells A375 / OXR;

[0065] Among them, Relative migration rates(%) represents the relative migration rate (percentage); the same below;

[0066] Figure 23 Effect of transfection with miR-324-3p mimic or miR-324-3p mimic control on the relative migration rate of drug-resistant melanoma cells MNT-1 / OXR;

[0067] Figure 24 Effect of transfection with miR-324-3p mimic or miR-324-3p mimic control on the proliferation ability of drug-resistant melanoma cells;

[0068] Figure 25 Effect of transfection with miR-324-3p mimic or miR-324-3p mimic control on the proliferation rate of drug-resistant melanoma cells A375 / OXR;

[0069] Among them, EdU / DAPI (%) represents the cell proliferation rate (percentage). ** indicates that compared with the A375 group, the P value < 0.01; + indicates that compared with the A375 / OXR group, the P value < 0.05; the same below;

[0070] Figure 26 Effects of transfection with miR-324-3p mimic or miR-324-3p mimic control on the proliferation rate of drug-resistant melanoma cells MNT-1 / OXR;

[0071] Among them, ^^^ indicates that compared with the MNT-1 group, the P value < 0.001; ++ indicates that compared with the MNT-1 / OXR group, the P value < 0.01; the same below;

[0072] Figure 27 Effects of transfection with miR-324-3p mimic or miR-324-3p mimic control on the invasion ability of drug-resistant melanoma cells;

[0073] Figure 28 Effects of transfection with miR-324-3p mimic or miR-324-3p mimic control on the relative invasion rate of drug-resistant melanoma cells A375 / OXR;

[0074] Among them, Relative invasion rate (%) represents the relative invasion rate (percentage), the same below;

[0075] Figure 29 Effects of transfection with miR-324-3p mimic or miR-324-3p mimic control on the relative invasion rate of drug-resistant melanoma cells MNT-1 / OXR;

[0076] Among them, Relative invasion rate (%) represents the relative invasion rate (percentage), ^^ indicates that compared with MNT-1, the P value < 0.01. Detailed implementation manners

[0077] The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific manners.

[0078] In the embodiments of the present invention, all group data were analyzed by Graphpad Prism 8.0 (GraphPad Software Inc, USA). The results were presented in the form of mean ± standard deviation (SD) from multiple independent experiments. The significance of differences between paired groups was analyzed by independent samples t-test. One-way analysis of variance (ANOVA) was used to analyze the significance of differences among multiple groups. Tukey's multiple comparison test was used for post hoc test. P < 0.05 was considered statistically significant.

[0079] Example 1 Expression of miR-324-3p and G3BP2 genes in melanoma

[0080] I. Detection of miR-324-3p and G3BP2 gene expression levels in cells and tissues by RT-qPCR

[0081] 1. Collection of clinical specimens

[0082] With the approval of the ethics committee (No.: MSER201806050), sera from melanoma patients (n = 25) and healthy donors (n = 25) were collected, and each participant signed all written informed consent forms.

[0083] 2. Cell culture

[0084] Human melanoma A375 (CRL-1619) and MNT-1 (CRL-3450) cell lines were purchased from the American Type Culture Collection (USA). All cells were cultured in an incubator (3110, Thermo Fisher Scientific, USA) at 37 °C with 5% CO 2 using DMEM medium (30-2002, ATCC). As needed, 10% or 20% fetal bovine serum (FBS, 10099, Gibco, USA), 100 U / mL penicillin, and 100 μg / mL streptomycin (15140122, Gibco) were added to the medium.

[0085] The human normal skin melanocyte PIG1 cell line (BNCC340321) was purchased from BeiNa ChuangLian Biotechnology Research Institute (Beijing, China) and cultured in the relevant complete medium (BNCC342329, BeiNa).

[0086] 3. Detection of the expression levels of miR-324-3p and G3BP2 genes in cells and tissues by RT-qPCR Total RNA in cells / serum was isolated using TRIzol Reagent (15596026, Invitrogen), and the isolated mRNA was reverse-transcribed into cDNA using a reverse transcription reagent (AH401-01, TransGen, Beijing, China); miRNA in cells / serum was isolated using the miPure Cell / Tissue miRNA Kit (RC201, Vazyme, China), and the isolated miRNA was reverse-transcribed into cDNA using a reverse transcription reagent (MR101-01, Vazyme); then, the expression levels of miR-324-3p (5'-CCCACUGCCCCAGGUGCUGCUGG-3', as shown in SEQ ID No. 9) and G3BP2 gene (NC_000004.12) in the two kinds of cDNA were detected using a CFX Opus real-time PCR system (CFX96, Bio-rad, CA, USA) and SYBR Green real-time PCR premix (AQ301-01, TransGen), respectively. GAPDH or U6 was used as an internal reference, and the results were calculated using the 2-ΔΔCt method. The primers used in RT-qPCR are shown in Table 1.

[0087] Table 1

[0088]

[0089]

[0090] GEPIA2 (http: / / gepia2.cancer-pku.cn / #analysis) was used to predict the expression of G3BP2 in skin cutaneous melanoma (SKCM) tissues (n = 461) and normal tissues (n = 558).

[0091] The detection results are as Figure 1 、 Figure 2 、 Figure 3 and Figure 4 shown.

[0092] It can be seen from Figure 1 that the relative expression level of miR-324-3p in the serum of melanoma patients was significantly downregulated compared with that in the serum of healthy donors (P < 0.001);

[0093] It can be seen from Figure 2 that the relative expression level of G3BP2 in melanoma tissues was significantly downregulated compared with that in normal tissues;

[0094] From Figure 3 It can be seen that compared with the relative expression level of miR-324-3p in normal human skin melanocytes PIG1, the relative expression levels of miR-324-3p in human melanoma A375 and MNT-1 cells were significantly down-regulated (P < 0.001).

[0095] From Figure 4 It can be seen that compared with the relative mRNA expression level of G3BP2 in normal human skin melanocytes PIG1, the relative mRNA expression levels of G3BP2 in human melanoma A375 and MNT-1 cells were significantly down-regulated (P < 0.001).

[0096] The above results indicate that there are significant differential expressions of miR-324-3p and G3BP2 genes in melanoma cells / tissues and normal cells / tissues, suggesting that both miR-324-3p and G3BP2 genes can be used as biomarkers for melanoma to assist in the early diagnosis or prognosis prediction of melanoma.

[0097] II. Research on the interaction between G3BP2 and miR-324-3p

[0098] 1. Material preparation

[0099] MiR-324-3p mimic (hereinafter referred to as M, nucleotide sequence as shown in SEQ ID No. 9: 5’-CCCACUGCCCCAGGUGCUGCUGG-3’), MiR-324-3p mimic control (hereinafter referred to as MC) were both purchased from ThermoFisher Scientific (USA; 4464066);

[0100] TargetScan (http: / / www.targetscan.org / vert_72 / ) was used to predict the possible binding sites between G3BP2 and miR-324-3p, and then GenePharma (Shanghai, China) was commissioned to synthesize the wild-type G3BP2 (G3BP2-WT) sequence and the mutant G3BP2 (G3BP2-MUT) sequence with altered miR-324-3p binding sites (as Figure 5 shown), and G3BP2-WT and G3BP2-MUT were respectively inserted into the pmirGLO dual-luciferase miRNA target expression vector (E1330, Promega, madison, WI, USA) to obtain the pmirGLO-G3BP2-WT reporter plasmid and the pmirGLO-G3BP2-MUT reporter plasmid respectively.

[0101] 2. Construction of oxaliplatin-resistant A375 or MNT-1 cell lines

[0102] Oxaliplatin (HY-17371, purity ≥98.0%) was diluted into an aqueous solution and stored at -20°C; A375 or MNT-1 cells were successively exposed to increasing doses (0, 2, 4, 6, 8, and 10 μM) of oxaliplatin to establish oxaliplatin-resistant A375 or MNT-1 cell lines (for the construction method, see the literature: Establishment and preliminary exploration of the drug resistance mechanism of oxaliplatin-resistant human colon cancer cell line HCT116 / L-OHP). After successful construction, the drug-resistant cells were stored in a medium containing 5 μg / mL of oxaliplatin to maintain drug resistance.

[0103] 3. Dual-luciferase reporter gene assay

[0104] According to the provider's protocol, oxaliplatin-resistant melanoma cells A375 and MNT-1 were cultured overnight in advance. The next day, each type of cell was divided into two groups: group M and group MC. The cells in group M and group MC were further divided into two groups: the G3BP2 wild-type group and the G3BP2 mutant group; among them, the M-G3BP2 wild-type group transfected M and the pmirGLO-G3BP2-WT reporter plasmid into oxaliplatin-resistant melanoma cells using Lipofectamine 2000 reagent, and the M-G3BP2 mutant group transfected M and the pmirGLO-G3BP2-MUT reporter plasmid into oxaliplatin-resistant melanoma cells; the MC-G3BP2 wild-type group transfected MC and the pmirGLO-G3BP2-WT reporter plasmid into oxaliplatin-resistant melanoma cells using Lipofectamine 2000 reagent, and the MC-G3BP2 mutant group transfected MC and the pmirGLO-G3BP2-MUT reporter plasmid into oxaliplatin-resistant melanoma cells.

[0105] The firefly luciferase activity and Renilla luciferase activity of each group of cells were detected using a GloMax 96 microplate luminometer (E6521, Promega) with a dual syringe, and the Renilla luciferase activity was used as an internal reference. The detection results are shown in Figure 6 and Figure 7 .

[0106] by Figure 6 and Figure 7It can be seen that compared with the MC-G3BP2 wild group, the firefly luciferase activity of A375 cells or MNT-1 cells in the M-G3BP2 wild group was significantly decreased (P<0.001); while the firefly luciferase activity of A375 cells or MNT-1 cells in the M-G3BP2 mutant group and the MC-G3BP2 mutant group was basically equivalent; indicating that miR-324-3p and G3BP2 interact in cells, and miR-324-3p can affect the expression of the G3BP2 gene by binding to G3BP2.

[0107] III. Analysis of the expression levels of miR-324-3p and G3BP2 in oxaliplatin-resistant A375 and MNT-1 cell lines

[0108] 1. MTT assay

[0109] The MTT (ST1537, Beyotime, China) assay was used to evaluate the cell viability of melanoma cells or oxaliplatin-resistant melanoma cells.

[0110] 3×10 3 different cells were seeded into 96-well plates, with three parallels set for each group; 0, 2, 4, 6, 8, and 10 μM oxaliplatin were added to each well respectively. After 24 h of treatment, 10 μL of MTT at a concentration of 5 mg / mL was added to each well of the plate; then the plate was incubated at 37 °C for another 4 hours, and then the supernatant of each well was discarded. 100 μL of dimethyl sulfoxide (A600163-0250, Sangon Biotech, China) was added to each well; finally, the value of the optical density at 570 nm was detected by a microplate reader (Sunrise, Tecan, Austria) to evaluate the corresponding cell viability. The detection results of cell viability are as Figure 8 shown, and the determination results of the IC 50 value are as Figure 9 shown.

[0111] It can be Figure 8 seen that with the continuous increase of the oxaliplatin treatment concentration, the cell viability of both A375 and MNT-1 cell lines gradually decreased; while the cell viability of A375 / OXR and MNT-1 / OXR cells also decreased with the increase of the oxaliplatin treatment concentration, but was still higher than that of A375 and MNT-1 cell lines and showed a significant difference (P<0.001), indicating that A375 / OXR and MNT-1 / OXR cells acquired oxaliplatin resistance.

[0112] It can be Figure 9 seen that with the acquisition of oxaliplatin resistance, the oxaliplatin IC 50The value rises to a higher level and shows a significant difference compared with A375 and MNT-1 cell lines (P < 0.001).

[0113] 2. Detection of the expression levels of miR-324-3p and G3BP2 genes in cells by RT-qPCR

[0114] Using the same RT-qPCR method as above, the relative expression levels of miR-324-3p and G3BP2 genes in A375, MNT-1, A375 / OXR, and MNT-1 / OXR cells were detected, and the detection results are shown in Figure 10 and Figure 11 .

[0115] As can be seen from Figure 10 in oxaliplatin-resistant A375 / OXR and MNT-1 / OXR cells, the relative expression level of miR-324-3p is much lower than that of their parental cells A375 and MNT-1, and there is a significant difference (P < 0.001);

[0116] As can be seen from Figure 11 in oxaliplatin-resistant A375 / OXR and MNT-1 / OXR cells, the relative expression level of G3BP2 gene is much higher than that of their parental cells A375 and MNT-1, and there is a significant difference (P < 0.001).

[0117] 3. Study on the interaction between G3BP2 and miR-324-3p

[0118] To clearly clarify the roles of miR-324-3p and G3BP2 in melanoma, in this example, miR-324-3p mimic and miR-324-3p mimic control were transfected into melanoma cells respectively, and then the expression level of miR-324-3p in melanoma cells, the expression level of G3BP2 protein in melanoma cells, the cell viability of melanoma cells under different oxaliplatin treatment concentrations, and the IC 50 value were detected, and the detection results are as Figures 12 to 19 shown.

[0119] Among them, the expression level of G3BP2 protein was determined by Western blot (WB). The determination method included: extracting total protein with radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime) supplemented with protease and phosphatase inhibitors (P1051, Beyotime), and detecting the protein concentration using a BCA protein assay kit (P0010, Beyotime); diluting all protein samples used in one experiment to the same concentration, and then performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, P0012A, Beyotime) on equal amounts of protein, transferring it to a polyvinylidene difluoride (PVDF) membrane (IPSN07852, Millipore, USA), and blocking the PVDF membrane with 5% skim milk for 1 hour; incubating the membrane with primary antibodies against G3BP2 (ab86135, 54 kDa, dilution 1:2000, Abcam) and GAPDH (ab9485, 37 kDa, dilution 1:2500, Abcam) overnight at 4°C, and then washing it 3 times with 0.1 M TBST; finally, incubating the membrane with an HRP-conjugated secondary antibody (ab205718, dilution 1:5000, Abcam) at 25°C for 2 hours; testing the specific protein signal using an enhanced chemiluminescence Western blot substrate (4AW011-200, 4A Biotech, China) and a ChemiDoc MP imaging system (Bio-rad), and quantifying the integrated optical density of the protein bands by ImageJ software.

[0120] It can be seen from Figure 12 and Figure 13 that transfection with miR-324-3p mimic control had basically no effect on the expression level of miR-324-3p in the A375 / OXR and MNT-1 / OXR cell lines, while transfection with miR-324-3p mimic significantly promoted the expression of miR-324-3p in the A375 / OXR and MNT-1 / OXR cell lines (P < 0.001).

[0121] It can be seen from Figure 14 and Figure 15 that transfection with miR-324-3p mimic control had no effect on the relative expression level of G3BP2 protein in the A375 / OXR and MNT-1 / OXR cell lines, while transfection with miR-324-3p mimic significantly decreased the relative expression level of G3BP2 protein in the A375 / OXR and MNT-1 / OXR cell lines (P < 0.05).

[0122] It can be seen from Figures 16 to 19It can be seen that transfection of miR-324-3p mimic control has no obvious effect on the cell viability of A375 / OXR and MNT-1 / OXR cell lines, while transfection of miR-324-3p mimic significantly reduces the cell viability of A375 / OXR and MNT-1 / OXR cell lines. At the same time, it also significantly reduces the IC 50 value (P < 0.001) of A375 / OXR and MNT-1 / OXR cell lines under oxaliplatin. Example 2 Correlation between miR-324-3p and migration, proliferation and invasion abilities of melanoma cells

[0123] To evaluate the effects of miR-324-3p on melanoma migration, proliferation and invasion, the following experiments were conducted in this example.

[0124] 1. Cell migration ability test

[0125] The wound healing assay was used to detect the cell migration rate. The test method: Inoculate 400 μL / well of cell suspension (containing 3×10 5 cells) into a 6-well plate, and use a 200 μL pipette tip to scratch to form a good monolayer of cells, and then rinse the floating cells with phosphate-buffered saline; then add medium without FBS to each well for further testing; Collect cell images at 0 h and 24 h with an inverted microscope (CKX41, Olympus, Japan) at a magnification of 100 times; Calculate the scratch area through Image J software (Wayne Rasband, USA), and set the scratch area at 0 h minus the scratch area at 24 h in the control group as 100% migration rate; The test results are as Figures 20 to 23 shown.

[0126] As Figures 20 to 23 can be seen, compared with A375 or MNT-1 cells, A375 / OXR and MNT-1 / OXR cells with oxaliplatin resistance have a higher relative migration rate (P < 0.001); However, transfection of miR-324-3p mimic control has basically no effect on the relative migration rate of A375 / OXR and MNT-1 / OXR cells, while transfection of miR-324-3p mimic significantly reduces the relative migration rate of A375 / OXR and MNT-1 / OXR cells (P < 0.001).

[0127] 2. Cell proliferation ability test

[0128] The EdU staining method was used to determine the proliferation ability of melanoma cells. The detection method: Inoculate melanoma cells into 100 μL of complete medium in a 96-well plate, and the inoculation amount is 1×10 per well 4The cells were incubated with EdU reagent (20 μM, ab219801, Abcam, UK) for 4 h, fixed with the provided fixation solution and permeabilized with the provided permeabilization buffer, stained with the provided iFluor 488 azide dye, and counterstained with DAPI (C1005, Beyotime); finally, the images were taken with a fluorescence microscope (Zeiss Axio Vert.A1, Germany) at 491 nm excitation wavelength and 520 nm emission wavelength at 200-fold magnification. The images and statistical results are shown in Figure 24 , Figure 25 and Figure 26 .

[0129] Depend on Figure 24 , Figure 25 and Figure 26 It can be seen that compared with A375 or MNT-1 cells, A375 / OXR and MNT-1 / OXR cells with oxaliplatin resistance have stronger proliferation ability; however, transfection of miR-324-3p mimic control has little effect on the proliferation performance of A375 / OXR and MNT-1 / OXR cells, while transfection of miR-324-3p mimic significantly reduces the proliferation rate of A375 / OXR and MNT-1 / OXR cells.

[0130] 3. Cell invasion ability test

[0131] Transwell was used to detect the invasion ability of melanoma cells. The test method was as follows: a commonly used 24-well Transwell system (3464, 8.0 μm pores, Corning) was used, and each filter was covered with Max Gel extracellular matrix (E0282, Sigma-Aldrich) to detect cell invasion;

[0132] Each well of the Transwell system consists of an upper chamber and a lower chamber. 100 μL of cell suspension (1×10 5 10 cells) were inoculated into the upper chamber, while each lower chamber was filled with 600 μL of complete medium supplemented with 20% FBS; after 24 hours, 4% paraformaldehyde (P0099, Beyotime, China) was used to fix at 25°C for 30 minutes, and then 0.1% crystal violet solution (G1063, Solarbio) was used to stain for 20 minutes; then the excess stain was washed with PBS, and the cells remaining on the upper side of the filter were gently removed with a cotton swab, and then an inverted microscope was used to photograph at a magnification of 250 times. The photographing and statistical results are shown in Figure 27 , Figure 28 and Figure 29 .

[0133] Depend onFigure 27 , Figure 28 and Figure 29 It can be seen that compared with A375 or MNT-1 cells, A375 / OXR and MNT-1 / OXR cells with oxaliplatin resistance have a higher relative migration rate. However, transfection with miR-324-3p mimic control has basically no effect on the migration performance of A375 / OXR and MNT-1 / OXR cells, while transfection with miR-324-3p mimic significantly reduces the relative migration rate of A375 / OXR and MNT-1 / OXR cells.

[0134] The above results indicate that transfection with miR-324-3p mimic can significantly reduce the migration, invasion and proliferation performance of oxaliplatin-resistant melanoma cells under oxaliplatin treatment.

Claims

1. Use of miR-324-3p enhancer in preparing a reagent for enhancing the chemosensitivity of oxaliplatin-resistant melanoma to oxaliplatin, characterized in that, the miR-324-3p enhancer includes miR-324-3p mimics, and the nucleotide sequence of the miR-324-3p mimics is as follows: 5’-CCCACUGCCCCAGGUGCUGCUGG-3’.

Images