CN116064808B

CN116064808B - Melanoma biomarker and application thereof

Info

Publication number: CN116064808B
Application number: CN202211351111.8A
Authority: CN
Inventors: 吴晓凤; 汪丽; 孙慧; 游凡
Original assignee: Hangzhou Tianfan Biotechnology Co ltd
Current assignee: Hangzhou Tianfan Biotechnology Co ltd
Priority date: 2022-10-31
Filing date: 2022-10-31
Publication date: 2025-05-27
Anticipated expiration: 2042-10-31
Also published as: CN116064808A

Abstract

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

Description

Melanoma biomarker and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a melanoma biomarker and application thereof.

Background

Cutaneous melanoma is the 19 th cancer type worldwide in 2020, accounting for 1.7% of all cancer cases, accounting for 0.6% of all cancer death cases. Although the early melanoma has high cure rate, the melanoma has high malignancy and is easy to metastasize, so that the early melanoma has important significance.

At present, chemotherapy and biological treatment are main treatment means for advanced melanoma, wherein traditional chemotherapy drugs of the melanoma mainly comprise azomethine (DTIC), vinblastine, nitrourea, PCZ and platinum drugs (such as cisplatin DDP, carboplatin and oxaliplatin), the DTIC is the first-choice drug for treating the melanoma, the single drug effective rate of the DTIC is about 20%, and the immune treatment drugs of the melanoma mainly comprise interleukin-2 (IL-2), interferon alpha (IFN-alpha) and the like.

In order to improve the curative effect, a biochemical treatment (biochemotherapy) method is developed at present, namely, the biological treatment and the chemotherapy are combined for use, the effective rate and the remission rate of the method are higher than those of the single chemotherapy or the single biological treatment, and the method is one of the best methods for treating melanoma at present.

At present, the DTIC+DDP+IL2+IFN-alpha combined drug is the biological chemotherapy scheme with the widest application for treating the domestic melanoma, the effective rate can reach 20% -30%, but the overall treatment effect for the advanced melanoma is poor, and the median survival time is only about half a year. On the one hand, the melanoma cells have poor sensitivity to the chemotherapeutic drugs, and on the other hand, the melanoma cells are easy to generate drug resistance to the chemotherapeutic drugs, so that the overall curative effect is reduced and the prognosis is poor.

Therefore, there is a need to explore new methods for overcoming melanoma resistance to chemotherapeutic agents.

Disclosure of Invention

The invention aims to provide a melanoma biomarker and application thereof, and provides a new thought and approach for early detection and treatment of melanoma.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

A melanoma biomarker, which is a miR-324-3p or G3BP2 gene.

The invention discovers for the first time that compared with the expression level of miR-324-3P in human normal skin melanocytes and normal serum, the expression level of miR-324-3P in human melanoma cells, tissues and serum of melanoma patients is obviously reduced (P < 0.001), and meanwhile, compared with the expression level of G3BP2 gene in human normal skin melanocytes and normal serum, the expression level of G3BP2 gene in human melanoma cells, tissues and serum of melanoma patients is obviously up-regulated (P < 0.05), and meanwhile, experiments show that miR-324-3P can be combined with G3BP2 gene so as to achieve the purpose of silencing or inhibiting G3BP2 gene expression. The results show that miR-324-3p and G3BP2 genes can be used as melanoma biomarkers, are used for assisting in early diagnosis or prognosis prediction of melanoma, and provide a new approach for early diagnosis or prognosis prediction of melanoma.

Based on the above, the invention also provides application of the melanoma biomarker in preparation of a melanoma early diagnosis or prognosis prediction reagent.

The invention also provides a reagent for early diagnosis or screening of melanoma, a kit for early diagnosis or screening of melanoma containing the reagent for early diagnosis or screening of melanoma, a reagent for prognosis prediction of melanoma, and a kit for prognosis prediction of melanoma containing the reagent for prognosis prediction of melanoma.

The melanoma early diagnosis or screening reagent or the melanoma prognosis prediction reagent can be a specific amplification primer or probe of miR-324-3p or a coding gene thereof, or a specific amplification primer or probe of G3BP2 gene or RNA expressed by the G3BP2 gene, or an antibody specifically combined with a protein expressed by the G3BP2 gene.

The expression level of miR-324-3p or G3BP2 in the detected object is detected by using the reagent, so that the melanoma disease condition or the melanoma prognosis condition can be known.

Preferably, in the early diagnosis or screening reagent for melanoma or the prognosis pre-test for melanoma, the specific amplification primer of the coding gene of miR-324-3p comprises:

An upstream primer 5'-CTGGGTCGTATCCAGTGCAA-3';

and a downstream primer 5'-GTCGTATCCAGTGCGTGTCG-3'.

The specific amplification primer of the G3BP2 gene comprises:

an upstream primer 5'-GAGCTGCAAGAGAGCGAGAA-3';

And a downstream primer 5'-GACCTCGATCATTGCGCCTA-3'.

The invention also provides application of the miR-324-3p function enhancer in preparation of melanoma treatment medicines, wherein the miR-324-3p function enhancer comprises a miR-324-3p mimic or a plasmid containing the miR-324-3p mimic, and the miR-324-3p mimic has the following nucleotide sequence:

5’-CCCACUGCCCCAGGUGCUGCUGG-3’。

meanwhile, the invention also provides application of the G3BP2 gene silencing agent in preparation of melanoma treatment drugs.

The invention also researches and discovers that miR-324-3pmimic can be transfected in oxaliplatin drug-resistant melanoma cells to obviously up-regulate the expression level of miR-324-3p, and can also obviously down-regulate the expression level of G3BP2, so that the miR-324-3p function enhancer or G3BP2 gene silencer can improve the drug resistance of the melanoma cells to a chemotherapeutic drug and enhance the chemosensitivity of the melanoma cells to the chemotherapeutic drug, thereby achieving the purpose of improving the curative effect of the chemotherapeutic drug on melanoma and providing a new thought and approach for the treatment of melanoma.

Wherein, the G3BP2 gene silencing agent can be miR-324-3p function enhancer or other medicines capable of silencing the G3BP2 gene.

Compared with the prior art, the invention has the beneficial effects that:

(1) The invention discovers for the first time that compared with the expression level of miR-324-3P in human normal skin melanocytes and normal serum, the expression level of miR-324-3P in human melanoma cells, tissues and serum of melanoma patients is obviously reduced (P < 0.001), and meanwhile, compared with the expression level of G3BP2 gene in human normal skin melanocytes and normal serum, the expression level of G3BP2 gene in human melanoma cells, tissues and serum of melanoma patients is obviously up-regulated (P < 0.05), and meanwhile, experiments show that miR-324-3P can be combined with G3BP2 gene so as to achieve the purpose of silencing or inhibiting G3BP2 gene expression. The results show that miR-324-3p and G3BP2 genes can be used as melanoma biomarkers, are used for assisting in early diagnosis or prognosis prediction of melanoma, and provide a new approach for early diagnosis or prognosis prediction of melanoma.

(2) The invention also researches and discovers that miR-324-3p mimic can be transfected in oxaliplatin drug-resistant melanoma cells to obviously up-regulate the expression level of miR-324-3p, and can also obviously down-regulate the expression level of G3BP2, so that the miR-324-3p function enhancer or G3BP2 gene silencer can improve the drug resistance of the melanoma cells to a chemotherapeutic drug and enhance the chemosensitivity of the melanoma cells to the chemotherapeutic drug, thereby achieving the purpose of improving the curative effect of the chemotherapeutic drug on melanoma and providing a new thought and approach for the treatment of melanoma.

Drawings

FIG. 1 is a comparison of the relative expression levels of miR-324-3p in serum from melanoma patients and in serum from healthy donors;

Wherein Cancer represents serum of melanoma patients, normal represents serum of healthy donors, RELATIVE MIR-324-3P expression level represents relative expression level of miR-324-3P, P value is less than 0.001 compared with Normal group, and the following is the same;

FIG. 2 is a comparison of the relative expression levels of G3BP2 in melanoma tissue and normal tissue;

Wherein Tumor represents melanoma tissue, normal represents Normal tissue, num (T) =461 represents the number of samples of melanoma tissue as 461, num (N) =558 represents the number of samples of Normal tissue as 558,G3BP2 expression-log ₂ (TPM+1) represents the logarithmic value of the G3BP2 gene expression level;

FIG. 3 is a comparison of the relative expression levels of miR-324-3p in human melanoma A375 and MNT-1 cells and human normal skin melanocyte PIG 1;

Wherein, P is less than 0.001 as compared with PIG1, and the same applies;

FIG. 4 is a comparison of the relative expression levels of the G3BP2 gene in human melanoma A375 and MNT-1 cells and human normal skin melanocyte PIG 1;

wherein RELATIVE G BP2 mRNA expression level represents the relative expression level of the G3BP2 gene, as follows;

FIG. 5 is a nucleotide sequence segment at a miR-324-3p binding site in a wild-type G3BP2 gene and a mutant G3BP2 gene designed in the embodiment;

Wherein, position 1-31of G3BP2 3'UTR WT represents 1-31 site nucleotide of wild type G3BP2 gene, position 1-31of G3BP2 3'UTR WUT represents 1-31 site nucleotide of mutant type G3BP2 gene;

FIG. 6 is a graph showing the relative activity of firefly luciferase in melanoma cell A375 when co-transfected with MiR-324-3p mimic or MiR-324-3p mimic control and pmirGLO-G3BP2-WT reporter plasmid or pmirGLO-G3BP2-MUT reporter plasmid;

Wherein MC represents MiR-324-3p mimic control,M represents MiR-324-3p mimic,G3BP2 WT represents wild type G3BP2 gene, G3BP2 MUT represents mutant type G3BP2 gene, relative luciferase activity represents relative luciferase activity, # # represents P value <0.001 compared with M-G3BP2 WT group, and the same applies;

FIG. 7 shows the relative activity of firefly luciferase in melanoma cells MNT-1 co-transfected with MiR-324-3p mimic or MiR-324-3p mimic control and pmirGLO-G3BP2-WT reporter plasmid or pmirGLO-G3BP2-MUT reporter plasmid;

FIG. 8 is a comparison of the relative cell viability of melanoma cells and oxaliplatin resistant melanoma cells at different oxaliplatin treatment concentrations;

Wherein A375/OXR represents drug-resistant melanoma cells A375, MNT-1/OXR represents drug-resistant melanoma cells MNT-1,Relative cell viability (%) represents relative cell viability (percent),. Times.represents P value <0.001 compared to group A375,. Times.A represents P value <0.001 compared to group MNT-1,. Times.A represents P value <0.01 compared to group MNT-1, and the same applies below;

FIG. 9 is an IC50 value of different melanoma cells under oxaliplatin treatment;

Wherein IC50 value of Oxaliplatin (μM) represents the IC50 value (micromoles per liter) of oxaliplatin;

FIG. 10 is a comparison of the relative expression levels of miR-324-3p in different melanoma cells;

FIG. 11 is a comparison of the relative expression levels of G3BP2 in different melanoma cells;

FIG. 12 is an effect of transfection of miR-324-3p mimic or miR-324-3p mimic control on the relative expression level of miR-324-3p in drug resistant melanoma cells A375/OXR;

Wherein, +++ representation and A375 in contrast to the group of the/OXR, P value <0.001, the same applies;

FIG. 13 is the effect of transfection of miR-324-3p mimic or miR-324-3p mimic control on the relative expression level of miR-324-3p in drug resistant melanoma cells MNT-1/OXR;

Wherein, ++ means and MNT-in contrast to the 1/OXR group, P value <0.001, the same applies;

FIG. 14 is the effect of transfection of 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;

wherein RELATIVE G BP2 protein expression level represents the relative expression level of the G3BP2 protein, and P value <0.01 compared to the a375/OXR group;

FIG. 15 is the effect of transfection 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;

Wherein, P is less than 0.01 compared with MNT-1/OXR group;

FIG. 16 is the effect of transfection miR-324-3p mimic or miR-324-3p mimic control on cell viability of drug resistant melanoma cells A375/OXR;

Wherein P value <0.001 compared to group a 375; +++ indicates that compared to the a375/OXR group, P value <0.001; ++ means that the P value is <0.01 compared to the A375/OXR group;

FIG. 17 is the effect of transfection of miR-324-3p mimic or miR-324-3p mimic control on oxaliplatin IC ₅₀ values of drug resistant melanoma cells A375/OXR;

FIG. 18 is the effect of transfection miR-324-3p mimic or miR-324-3p mimic control on cell viability of drug resistant melanoma cells MNT-1/OXR;

Wherein, the value of P <0.001 compared with MNT-1, the value of P <0.01 compared with MNT-1, ++ is <0.001 compared with MNT-1/OXR, ++ is <0.01 compared with MNT-1/OXR, and the same applies;

FIG. 19 is the effect of transfection of miR-324-3p mimic or miR-324-3p mimic control on the oxaliplatin IC ₅₀ values of drug resistant melanoma cells MNT-1/OXR;

FIG. 20 is the effect of transfection miR-324-3p mimic or miR-324-3p mimic control on the migration capacity of drug resistant melanoma cells A375/OXR;

FIG. 21 is the effect of transfection miR-324-3p mimic or miR-324-3p mimic control on the migration capacity of drug resistant melanoma cells MNT-1/OXR;

FIG. 22 is the effect of transfection miR-324-3p mimic or miR-324-3p mimic control on the relative mobility of drug resistant melanoma cells A375/OXR;

wherein Relative migration rates (%) represents the relative mobility (percent);

FIG. 23 is the effect of transfection miR-324-3p mimic or miR-324-3p mimic control on the relative mobility of drug resistant melanoma cells MNT-1/OXR;

FIG. 24 is the effect of transfection miR-324-3p mimic or miR-324-3p mimic control on drug resistant melanoma cell proliferation capacity;

FIG. 25 is the effect of transfection miR-324-3p mimic or miR-324-3p mimic control on proliferation rate of drug resistant melanoma cells A375/OXR;

wherein EdU/DAPI (%) indicates cell proliferation rate (percent), P <0.01 compared to group A375, + indicates P <0.05 compared to group A375/OXR;

FIG. 26 is the effect of transfection miR-324-3p mimic or miR-324-3p mimic control on proliferation rate of drug resistant melanoma cells MNT-1/OXR;

Wherein, the value of P is <0.001 compared with MNT-1 group, and++ is <0.01 compared with MNT-1/OXR group;

FIG. 27 is the effect of transfection miR-324-3p mimic or miR-324-3p mimic control on the invasive capacity of drug resistant melanoma cells;

FIG. 28 is the effect of transfection miR-324-3p mimic or miR-324-3p mimic control on the relative invasion rate of drug resistant melanoma cells A375/OXR;

Wherein Relative invasion rate (%) represents the relative attack rate (percent), the same as follows;

FIG. 29 is the effect of transfection miR-324-3p mimic or miR-324-3p mimic control on the relative invasion rate of drug resistant melanoma cells MNT-1/OXR;

Wherein Relative invasion rate (%) represents the relative attack rate (percent), and P represents a P value <0.01 compared to MNT-1.

Detailed Description

The technical scheme of the invention is further described in detail below with reference to the attached drawings and the detailed mode.

In the examples of the present invention, all sets of data were analyzed by GRAPHPAD PRISM 8.0.0 (GraphPad Software Inc, USA). Results are presented as mean ± Standard Deviation (SD) from multiple independent experiments. The significance of the differences between paired groups was analyzed by independent sample t-test. Significance of differences between the groups was analyzed using one-way analysis of variance (ANOVA). Multiple comparison tests of Tukey were used for post hoc testing. P <0.05 is considered statistically significant.

EXAMPLE 1 expression of miR-324-3p and G3BP2 genes in melanoma

1. RT-qPCR (reverse transcription-quantitative polymerase chain reaction) detection of expression level of miR-324-3p and G3BP2 genes in cells and tissues

1. Clinical specimen collection

Serum was collected from melanoma patients (n=25) and healthy donors (n=25) approved by the ethical committee (number: MSER 201806050), each participant signed all written informed consent.

2. Cell culture

Human melanoma A375 (CRL-1619) and MNT-1 (CRL-3450) cell lines were purchased from the American type culture Collection (USA), all were cultured in 37℃in a 5% CO ₂ incubator (3110,Thermo Fisher Scientific,USA) using DMEM medium (30-2002, ATCC) to which 10% or 20% fetal bovine serum (FBS, 10099,Gibco,USA), 100U/mL penicillin and 100. Mu.g/mL streptomycin (15140122, gibco) were added, as required.

Human normal skin melanocyte PIG1 cell line (BNCC 340321) was purchased from the institute of biotechnology for North Innova (Beijing, china) and cultured in relevant complete medium (BNCC 342329, north Nah).

3. RT-qPCR to detect the expression levels of miR-324-3p and G3BP2 genes in cells and tissues Total RNA in cells/serum was isolated using a TRIzol Reagent (15596026, invitrogen) and the isolated mRNA was reverse transcribed into cDNA using a reverse transcription Reagent (AH 401-01, transGen, beijin, china), miRNA in cells/serum was isolated using a miPure cell/tissue miRNA kit (RC 201, vazyme, china) and the isolated miRNA was reverse transcribed into cDNA using a reverse transcription Reagent (MR 101-01, vazyme), and then the expression levels of miR-324-3p (5'-CCCACUGCCCCAGGUGCUGCUGG-3') and G3BP2 genes (NC 000004.12) in the two cDNAs were detected using a CFX Opus real-time PCR system (CFX 96, bio-rad, CA, USA) and SYBR Green real-time PCR premix (AQ 301-01, transGen) respectively, with GAPDH or U6 as the internal delta Ct method. The primers used for RT-qPCR are shown in Table 1.

TABLE 1

GEPIA2 (http:// gepia2.Cancer-pku. Cn/# analysis) was used to predict the expression of G3BP2 in skin melanoma (SKCM) tissue (n=461) and normal tissue (n=558).

The detection results are shown in fig. 1,2, 3 and 4.

As can be seen from fig. 1, the relative expression level of miR-324-3P in the serum of melanoma patients was significantly down-regulated (P < 0.001) compared to the relative expression level of miR-324-3P in the serum of healthy donors;

As can be seen from fig. 2, the relative expression level of G3BP2 in melanoma tissue was significantly down-regulated compared to the relative expression level of G3BP2 in normal tissue;

As can be seen from fig. 3, the relative expression levels of miR-324-3P in human melanoma a375 and MNT-1 cells were significantly down-regulated (P < 0.001) compared to the relative expression levels of miR-324-3P in human normal skin melanocyte PIG 1;

As can be seen from fig. 4, the relative expression level of mRNA of G3BP2 in human melanoma a375 and MNT-1 cells was significantly down-regulated (P < 0.001) compared to the relative expression level of mRNA of G3BP2 in human normal skin melanocyte PIG 1.

The results show that miR-324-3p and G3BP2 genes are obviously differentially expressed in melanoma cells/tissues and normal cells/tissues, and the miR-324-3p and G3BP2 genes can be used as biomarkers of melanoma and used for assisting early diagnosis or prognosis prediction of the melanoma.

2. Investigation of interaction between G3BP2 and miR-324-3p

1. Material preparation

MiR-324-3p mimic (hereinafter referred to as M, the nucleotide sequence of which is shown as SEQ ID No. 9: 5'-CCCACUGCCCCAGGUGCUGCUGG-3') and MiR-324-3p mimic control (hereinafter referred to as MC) are all available from Thermo FISHER SCIENTIFIC (USA; 4464066);

The possible binding sites for G3BP2 and miR-324-3p were predicted using TARGETSACN (http:// www.targetscan.org/vert_72 /), then the wild-type G3BP2 (G3 BP 2-WT) sequence was commissioned GENEPHARMA (Shanghai, china) to synthesize the mutant G3BP2 (G3 BP 2-MUT) sequence with altered miR-324-3p binding sites (as shown in FIG. 5), and G3BP2-WT and G3BP2-MUT were inserted into pmirGLO dual-luciferase miRNA target expression vectors (E1330, promega, madison, WI, USA) respectively, to obtain pmirGLO-G3BP2-WT reporter plasmids, pmirGLO-G3BP2-MUT reporter plasmids, respectively.

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

Oxaliplatin (HY-17371, purity not less than 98.0%) is diluted into aqueous solution, preserved at-20 ℃, A375 or MNT-1 cells are sequentially exposed to increasing doses (0, 2, 4, 6, 8 and 10 mu M) of oxaliplatin to establish an oxaliplatin-resistant A375 or MNT-1 cell line (construction method, see literature: establishment of human colon cancer oxaliplatin-resistant cells HCT116/L-OHP and initial detection of drug resistance mechanism thereof), and after successful construction, the drug-resistant cells are preserved in a culture medium containing 55 mu g/mL of oxaliplatin to maintain drug resistance.

3. Dual luciferase reporter assay

According to the provider's procedure, oxaliplatin-resistant melanoma cells A375 and MNT-1 were cultured overnight in advance, and each cell was further divided into two groups, M and MC, the M and MC being further divided into two groups, the G3BP2 wild-type group and the G3BP2 mutant group, wherein the M-G3BP2 wild-type group co-transfects M and pmirGLO-G3BP2-WT reporter plasmids into oxaliplatin-resistant melanoma cells using Lipofectamine 2000 reagent, the M-G3BP2 mutant group co-transfects M and pmirGLO-G3BP2-MUT reporter plasmids into oxaliplatin-resistant melanoma cells, and the MC-G3BP2 wild-type group co-transfects MC and pmirGLO-G3BP2-WT reporter plasmids into oxaliplatin-resistant melanoma cells using Lipofectamine 2000 reagent, and the MC-G3BP2 mutant group and pmirGLO-G3BP2-MUT reporter plasmids into oxaliplatin-resistant melanoma cells.

Firefly luciferase activity and Renilla luciferase activity of each group of cells were measured using a GloMax microplate fluorometer (E6521, promega) with a double syringe, and Renilla luciferase activity was used as an internal reference. The detection results are shown in fig. 6 and 7.

As can be seen from FIGS. 6 and 7, the firefly luciferase activity of either the A375 cells or MNT-1 cells of the M-G3BP2 wild group was significantly reduced (P < 0.001) compared to that of the MC-G3BP2 wild group, whereas the firefly luciferase activities of either the A375 cells or MNT-1 cells of the M-G3BP2 mutant group and the MC-G3BP2 mutant group were substantially equivalent, indicating that MiR-324-3P and G3BP2 have interactions in the cells, miR-324-3P being able to affect the expression of the G3BP2 gene by binding to G3BP 2.

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

1. MTT assay

MTT (ST 1537, beyotime, china) experiments were used to evaluate cell viability of melanoma cells or oxaliplatin resistant melanoma cells.

Three different cells were seeded into 96-well plates, each set containing 3X 10 ³ cells, three were placed in parallel, 0, 2,4, 6, 8 and 10. Mu.M oxaliplatin were added to each well, after 24 hours of treatment, 10. Mu.L MTT at a concentration of 5mg/mL was added to each well of the plate, the plate was then cultured at 37℃for another 4 hours, the supernatant of each well was then discarded, 100. Mu.L of dimethyl sulfoxide (A600163-0250, bio, china) was added to each well, and finally, the value of optical density at 570nm was examined by an enzyme-labeling instrument (Sunrise, tecan, austraia) to evaluate the corresponding cell viability. The results of the cell viability assay are shown in FIG. 8, and the results of the IC ₅₀ value assay are shown in FIG. 9.

As can be seen from FIG. 8, the cell viability of both A375 and MNT-1 cell lines decreased gradually with increasing oxaliplatin treatment concentration, while the cell viability of both A375/OXR and MNT-1/OXR cells decreased with increasing oxaliplatin treatment concentration, but remained higher and significantly different (P < 0.001) compared to the A375 and MNT-1 cell lines, indicating that both A375/OXR and MNT-1/OXR cells acquired oxaliplatin resistance.

As can be seen from figure 9, oxaliplatin IC ₅₀ values for a375/OXR and MNT-1/OXR cells increased to higher levels with the acquisition of oxaliplatin resistance and were significantly different (P < 0.001) compared to a375 and MNT-1 cell lines.

2. RT-qPCR (reverse transcription-quantitative polymerase chain reaction) detection of expression level of miR-324-3p and G3BP2 genes in cells

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

As can be seen from FIG. 10, the relative expression level of miR-324-3P in oxaliplatin resistant A375/OXR, MNT-1/OXR cells was much lower than that of the parent cells A375, MNT-1, with a significant difference (P < 0.001);

As can be seen from FIG. 11, the relative expression level of the G3BP2 gene was much higher in oxaliplatin resistant A375/OXR, MNT-1/OXR cells than in their parent cells A375, MNT-1, with a significant difference (P < 0.001).

3. Research on interaction between G3BP2 and miR-324-3p

To clearly illustrate the role of miR-324-3p and G3BP2 in melanoma, this example transfected miR-324-3p mimic, miR-324-3p mimic control into melanoma cells, respectively, and then examined 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 at different oxaliplatin treatment concentrations, and the IC ₅₀ values, the results of which are shown in fig. 12 to 19.

Wherein the protein expression level of G3BP2 was determined by Western Blotting (WB), the assay method comprising extracting total protein by Radioimmunoassay (RIPA) lysis buffer (P0013B, beyotime) supplemented with protease and phosphatase inhibitors (P1051, beyotime) and detecting protein concentration using BCA protein assay kit (P0010, beyotime), diluting all protein samples used in one experiment to the same concentration, then subjecting the same amount of protein to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, P0012A, beyotime), transferring to polyvinylidene fluoride (PVDF) membrane (IPSN 07852, millipore, USA), blocking the PVDF membrane with 5% skimmed milk for 1 hour, incubating the membrane with G3BP2 (ab 86135,54kDa, ab 1:2000, abcam) and GAPDH (ab 9485,37kDa, dilution 1:2500, abcam) primary antibodies at 4℃overnight, then subjecting to a final wash of 0.1M and finally subjecting the membrane to a two-dimensional signal enhancement by a specific dye-buffer (Biotec) at 4℃and a specific dye-buffer density of two-buffer (Biotec 1: 205718, biob) to a chemiluminescent signal enhancement by a PCR system (Biotec-1, biob) at a specific dye-4 ℃.

As can be seen from FIGS. 12 and 13, transfection of miR-324-3P mimic control had substantially no effect on the expression levels of miR-324-3P in the A375/OXR and MNT-1/OXR cell lines, whereas transfection of miR-324-3P mimic significantly promoted expression of miR-324-3P in the A375/OXR and MNT-1/OXR cell lines (P < 0.001).

As can be seen from FIGS. 14 and 15, transfection of miR-324-3P mimic control had no effect on the relative expression levels of the G3BP2 protein in the A375/OXR and MNT-1/OXR cell lines, whereas transfection of miR-324-3P mimic significantly reduced the relative expression levels of the G3BP2 protein in the A375/OXR and MNT-1/OXR cell lines (P < 0.05).

As can be seen from FIGS. 16-19, transfection of miR-324-3P mimic control had no significant effect on the cell viability of the A375/OXR and MNT-1/OXR cell lines, whereas transfection of miR-324-3P mimic significantly reduced the cell viability of the A375/OXR and MNT-1/OXR cell lines, and also significantly reduced the IC ₅₀ values (P < 0.001) of the A375/OXR and MNT-1/OXR cell lines under oxaliplatin. EXAMPLE 2 correlation of miR-324-3p with the migration, proliferation and invasion Capacity of melanoma cells

To assess the effect of miR-324-3p on melanoma migration, proliferation and invasion, the following experiments were performed in this example.

1. Cell migration ability test

Cell mobility was measured using a wound healing assay by inoculating 400 μl/well cell suspension (containing 3×10 ⁵ cells) into a 6-well plate, scraping with a 200 μl pipette tip to form a good monolayer of cells, then rinsing the floating cells with phosphate buffered saline, then adding FBS-free medium to each well for further testing, collecting 0h and 24h cell images at 100 x magnification by an inverted microscope (CKX 41, olympus, japan), calculating scratch area by Image J software (Wayne Rasband, USA), and subtracting 24h scratch area in the control group as 100% mobility, and the test results are shown in fig. 20-23.

As can be seen from FIGS. 20-23, A375/OXR and MNT-1/OXR cells with oxaliplatin resistance had a higher relative mobility (P < 0.001) than either A375 or MNT-1 cells, but transfected miR-324-3P mimic control had substantially no effect on the relative mobility of A375/OXR and MNT-1/OXR cells, whereas transfected miR-324-3P mimic significantly reduced the relative mobility of A375/OXR and MNT-1/OXR cells (P < 0.001).

2. Cell proliferation potency test

The proliferation capacity of melanoma cells was determined by EdU staining, by inoculating melanoma cells into 100. Mu.L of complete medium in 96-well plates at 1X 10 ⁴ cells per well, incubating the cells with EdU reagent (20. Mu.M, ab219801, abcam, UK) for 4 hours, fixing the cells using the provided fixing solution and permeabilizing them with the provided permeabilizing buffer, staining the cells with the provided iFluor azide dye, counterstaining the nuclei with DAPI (C1005, beyotime), and finally photographing with a fluorescence microscope (Zeiss Axio Vert. A1, germany) at a magnification of 200 times at 491nm excitation wavelength and 520nm emission wavelength, and the photographic and statistical results are shown in FIGS. 24, 25 and 26.

As can be seen from FIGS. 24, 25 and 26, the proliferation capacity of A375/OXR and MNT-1/OXR cells was greater with oxaliplatin-resistant than that of A375 or MNT-1 cells, but the proliferation performance of transfected miR-324-3p mimic control on A375/OXR and MNT-1/OXR cells was substantially unaffected, whereas transfected miR-324-3p mimic significantly reduced the proliferation rate of A375/OXR and MNT-1/OXR cells.

3. Cell invasion Capacity test

The ability of melanoma cells to invade was tested using a Transwell test method using a conventional 24 well Transwell system (3464,8.0 μm well, corning), each filter covered with Max Gel extracellular matrix (E0282, sigma-Aldrich) to test cell invasion;

each well of the Transwell system comprised an upper chamber and a lower chamber, 100 μl of cell suspension (1×10 ⁵ cells) was inoculated into the upper chamber, and each lower chamber was filled with 600 μl of complete medium supplemented with 20% FBS, after 24 hours, fixed with 4% paraformaldehyde (P0099, beyotime, china) at 25 ℃ for 30 minutes, then stained with 0.1% crystal violet solution (G1063, solarbio) for 20 minutes, then washed with PBS for excess staining, gently removing the cells remaining on the upper side of the filter with a cotton swab, then photographed using an inverted microscope at 250 magnification, photographed and counted as shown in fig. 27, 28 and 29.

As can be seen from FIGS. 27, 28 and 29, A375/OXR and MNT-1/OXR cells with oxaliplatin resistance had higher relative mobilities than either A375 or MNT-1 cells, but transfected miR-324-3p mimic control had substantially no effect on the migration properties of A375/OXR and MNT-1/OXR cells, whereas transfected miR-324-3p mimic significantly reduced the relative mobilities of A375/OXR and MNT-1/OXR cells.

The results show that the transfection of miR-324-3p mimic can obviously reduce migration, invasion and proliferation performance of oxaliplatin-resistant melanoma cells under oxaliplatin treatment.

Claims

Application of miR-324-3p function enhancer in preparation of oxaliplatin-resistant melanoma chemosensitivity enhancing reagent for oxaliplatin, characterized in that miR-324-3p function enhancer comprises miR-324-3p mimics, and the nucleotide sequence of the miR-324-3p mimics is as follows:

5’-CCCACUGCCCCAGGUGCUGCUGG-3’。