| Objective:Ovarian Cancer(OC)is the leading cause of death in gynecologic malignancies.In2020,there were 207252 deaths from ovarian cancer worldwide,and 37519 deaths in our country,accounting for 18.10%of global deaths.OC has an insidious onset,non-specific symptoms and signs,and the lack of effective early diagnostic approaches,70%of the patients were diagnosed at advanced stages.The 5-year survival rate of ovarian cancer is still less than 50%,despite the existence of surgical treatment,chemotherapy,targeted therapy,immunotherapy.Chemotherapy resistance of tumor cells is the main cause of poor prognosis and high mortality in patients with ovarian cancer.Platinum-based chemotherapy is the first-line chemotherapy for ovarian cancer.Therefore,it is of great significance to further explore the mechanism of platinum resistance in OC to find new therapeutic targets to improve the prognosis of patients with OC.The m~6A modification in RNA Epigenetics(N6-methyladenosine,m~6A)can not only affect various aspects of m RNA metabolism,including splicing,translation,and stability,involving in many biological processes,such as cell development,maintenance of stem characteristics,and mitosis control,but also play an important role in tumorigenesis,disease progression and drug resistance of tumor cells.In this study,Methylated RNA Immunoprecipitation with Next Generation Sequencing(Me RIP-Seq)and bioinformatics analysis were used to screen the differential molecule MEF2C between platinum-resistant and platinum-sensitive ovarian cancer cells.Cell,tissue and in vivo experiments were conducted to verify that MEF2C could inhibit the proliferation of OC cells,induce cell cycle arrest,promote apoptosis of OC cells,and inhibit the cisplatin resistance of OC through transcriptional regulation of the expression of DNA damage regulated autophagy modulator 1(DRAM1).The aim of this study is to provide a new target for the treatment of cisplatin resistance in ovarian cancer from the Epigenetics of m~6A,which has important scientific significance and clinical value.Methods:1.IC50 of A2780,SKOV3,A2780-DDP and SKOV3-DDP was determined by MTT Assay.The m~6A dot blot assay was used to detect the difference in overall m~6A modification levels between cisplatin-resistant and cisplatin-sensitive ovarian cancer cell lines.Me RIP-Seq was used to screen m~6A-modified genes between cisplatin-resistant ovarian cancer cell line and cisplatin sensitive cell line.TCGA data,GTEx data and GSE14407 data set were used to screen the differentially expressed genes between ovarian cancer and normal ovarian tissues.GSE15372 data set,GSE47856 data set and GSE45553 data set were used to screen differentially expressed genes between platinum-resistant and platinum-sensitive ovarian cancer cell lines.MEF2C was selected as the research target.q RT-PCR and Western blot were used to verify the difference of MEF2C expression in OC cisplatin-resistant cells and tissues,as well as OC cisplatin-sensitive cells and tissues.The expression of MEF2C was detected by immunohistochemistry in 101 cases of ovarian cancer.2.Bioinformatics analysis of MEF2C expression in pan-cancer and single-gene difference analysis of MEF2C in ovarian cancer:Based on TCGA database,the MEF2C expression in ovarian cancer was ranked from low to high,using 50%as the cut-off value,the differential expression molecules between high and low expression groups were analyzed.The cluster profiler package[version 3.14.3]was used for GSEA(Gene Set Enrichment Analysis)Analysis.The reference gene set is h.all.v7.2.symbols.gmt[Hallmarks],further speculating on possible functions or pathways involved in MEF2C.3.The silencing plasmid was transfected with MEF2C in A2780,SKOV3,the over-expression plasmid of MEF2C was transfected in A2780-DDP and SKOV3-DDP.The transfection efficiency was verified by q RT-PCR and Western blot.MTT assay,colony formation assay,Flow cytometry and IC50 assay were used to detect the proliferation of ovarian cancer cells,colony formation ability,apoptosis level and cisplatin resistance of ovarian cancer cells.4.Database mining of DRAM1,a downstream target gene as a transcription factor,was performed to validate the differential expression in OC cisplatin-resistant cells and tissues and OC cisplatin-sensitive cells and tissues using q RT-PCR and Western blot.DRAM1 expression was detected by immunohistochemistry in 101 cases of ovarian cancer.The silencing plasmid of MEF2C was transfected into A2780 and SKOV3,and the overexpression plasmid of MEF2C was transfected into A2780-DDP and SKOV3-DDP,q RT-PCR and Western blot were used to validate the effect of intervention with MEF2C on m RNA and protein expression of DRAM1.5.The m~6A reader of MEF2C was predicted to be YTHDF2 based on the difference in m~6A modification abundance and m RNA expression amount of MEF2C between platinum-resistant and platinum-sensitive cells of ovarian cancer.The expression of YTHDF2 in ovarian cancer and its relationship with the clinicopathological parameters of patients with ovarian cancer was analyzed in bioinformatics,the expression of YTHDF2 was detected in platinum-resistant and platinum-sensitive ovarian cancer tissues,and YTHDF2 expression was detected in A2780,SKOV3,A2780-DDP and SKOV3-DDP cells by q RT-PCR and Western blot,respectively.The expression of YTHDF2 in 101 cases of ovarian cancer was detected by immunohistochemistry.6.The overexpression plasmid of YTHDF2 was transfected into A2780 and SKOV3,and the silencing plasmid of YTHDF2 was transfected into A2780-DDP and SKOV3-DDP,the transfection efficiency was verified by q RT-PCR and Western blot.The overexpression plasmids of YTHDF2,the overexpression plasmids of MEF2C,the overexpression plasmids of YTHDF2 and MEF2C were transfected into A2780 and SKOV3,respectively.The apoptosis level of ovarian cancer cells was examined by Flow cytometry,q RT-PCR and Western blot were used to validate the effect of intervention with YTHDF2 and MEF2C on m RNA and protein expression of MEF2C and DRAM1.At the same time,the effect of YTHDF2 intervention on the m RNA stability of MEF2C was verified by RNA stability experiment.7.Co-immunoprecipitation of methylated RNA combined with high-throughput sequencing,SRAMP website and cat RAPID website were used to predict m~6A sites on MEF2C.RNA pulldown experiments were used to verify the direct binding between YTHDF2 and MEF2C.8.Fifty healthy BALB/C athymic nude mice were injected with 5×10~6 A2780 cells,and divided into five groups.The five groups were transfected with oe-NC,oe-MEF2C,oe-NC,oe-YTHDF2 and oe-MEF2C+oe-YTHDF2 plasmids,respectively.When the mean tumor size reached 10×10×10 mm,each group of nude mice was randomly divided into two groups and treated with cisplatin(5 mg/kg)or normal saline(NS)every week.Tumor width(W)and length(L)were measured weekly.Tumor Volume(V)was calculated as V=(W2×L)/2.After receiving 6 cycles of cisplatin or saline treatment,the nude mice were killed.Then,the tumor volume and weight were recorded,and the expression of YTHDF2 and MEF2C were verified by q RT-PCR,Western blot,and immunohistochemistry.9.The SPSS23.0 software and Graph Pad Prism 8 software were used to statistically analyze all data,which were presented as the mean±standard deviation(x±SD)of three independent experiments.Independent-sample t-test or one-way ANOVA was used to compare the differences among the groups.P<0.05 was considered statistically significant.Results:1.Me RIP-Seq and bioinformatics analysis were used to screen the differential molecular.1.1 The m~6A modification level of the platinum-resistant OC cell lines was higher than that of the platinum-sensitive OC cell lines.1.2 Me RIP-Seq in platinum-resistant and platinum-sensitive ovarian cancer cell lines indicated that m~6A was located in the CDS region(56.12%),36.43%were in the 3’UTR and 7.45%were in the 5’UTR.Based on the condition of fold change≥1.5,p-value<0.05,a total of 2459 genes with m~6A modification differences were obtained,of which358 genes with increased m~6A modification levels and 2101 genes with decreased m~6A modification levels were obtained for A2780-DDP compared with A2780 cell line.1.3 TCGA data,GTEx data and GSE14407 data were used to screen the differentially expressed genes between OC and normal ovarian tissues.GSE15372 data set,GSE47856 data set,GSE45553 data set were used to screen differentially expressed genes between platinum-resistant and platinum-sensitive ovarian cancer cell lines.The above results were used to obtain MEF2C as the target molecule combined with Me RIP-Seq results.1.4 The results of q RT-PCR,Western blot and immunohistochemistry showed that the expression of MEF2C in platinum-resistant ovarian cancer tissues was lower than that in platinum-sensitive ovarian cancer tissues,the expression of MEF2C in A2780-DDP and SKOV3-DDP was lower than that in A2780 and SKOV3.2.Study on the biological characteristics of platinum resistance regulated by MEF2C in ovarian cancer.2.1 Expression of MEF2C in TCGA pan-cancer:the expression of MEF2C was significantly down-regulated in 10 cancer types and up-regulated in 4 cancer types.Pan-cancer analysis based on TCGA data and GTEx data showed that MEF2C expression was significantly down-regulated in 16 cancer types and up-regulated in 10 cancer types.2.2 Single gene differential analysis of MEF2C in TCGA database:there were 1896differentially expressed genes,including 964 up-regulated genes and 932 down-regulated genes.The results of GSEA analysis suggested that the gene sets Hallmark P53 Pathway and Hallmark Apoptosis were enriched in MEF2C high expression groups.2.3 MEF2C inhibits cisplatin resistance in ovarian cancer cells:Knockdown of MEF2C can enhance the proliferative and clonogenic capacity of A2780 and SKOV3,attenuate apoptosis capacity,and promote cisplatin resistance in ovarian cancer cells.Overexpression of MEF2C can attenuate the proliferative and clonogenic capacity of A2780-DDP and SKOV3-DDP,enhance the apoptotic capacity,and inhibit cisplatin resistance in ovarian cancer cells.2.4 MEF2C suppresses cisplatin resistance in ovarian cancer cells through transcriptional regulation of DRAM1 expression:mining MEF2C as a downstream target gene of transcription factors by exploring Ch IP-Atlas databases;exploring the HGSOC-Platinum database to mine the platinum resistance-related genes with clear literature reports,and analyzing the TCGA database to screen the genes with a Spearman correlation coefficient greater than 0.5 with MEF2C expression in ovarian cancer data,P53 pathway-related genes were downloaded,and the four gene sets were intersected to get DRAM1.DRAM1 expression in OC was significantly lower than that in normal ovarian,and DRAM1 expression gradually decreased with the increase of stage in OC,the expression of MEF2C and DRAM1 was highly correlated in OC with a Spearman correlation coefficient of 0.542.The results of q RT-PCR,Western blot and immunohistochemistry showed that DRAM1 expression was lower in platinum-resistant tissues than in platinum-sensitive tissues.The m RNA and protein expression of DRAM1in A2780-DDP and SKOV3-DDP was lower than that in A2780 and SKOV3.Knockdown of MEF2C in A2780 and SKOV3 cells significantly reduced the m RNA and protein expression of DRAM1.Overexpression of MEF2C in A2780-DDP cells and SKOV3-DDP cells significantly increased the m RNA and protein expression of DRAM1.3.YTHDF2 affects the stability of MEF2C by directly binding to the m~6A modification site of MEF2C,which leads to platinum resistance in ovarian cancer.3.1 YTHDF2 was an independent risk factor for poor prognosis of ovarian cancer:Bioinformatics analysis showed that YTHDF2 m RNA expression in OC was higher than that in normal tissues,YTHDF2 protein expression in OC was higher than that in normal tissues,and increased with the increase of clinical pathological stages of OC.Based on TCGA data,multivariate Cox regression analysis indicated that YTHDF2 was an independent risk factor for poor prognosis in OC,and survival analysis results indicated that in drug-resistant patients with OC,the OS and DSS of YTHDF2 high expression group were significantly lower than that of YTHDF2 low expression group.3.2 YTHDF2 inhibits the effect of MEF2C on Cisplatin resistance in OC:q RT-PCR,Western blot and immunohistochemistry showed that YTHDF2 expression was higher in platinum-resistant tissues than in platinum-sensitive tissues.The m RNA and protein expression of YTHDF2 in A2780-DDP and SKOV3-DDP was higher than that in A2780and SKOV3.After overexpression of YTHDF2,the m RNA and protein expression of MEF2C and DRAM1 were significantly reduced in A2780 and SKOV3 cells,and after knockdown of YTHDF2,the m RNA and protein expression of MEF2C and DRAM1were significantly elevated in A2780-DDP and SKOV3-DDP cells.The oe-NC,oe-MEF2C,oe-YTHDF2,oe-MEF2C and oe-YTHDF2 plasmids were transfected into cisplatin-treated A2780 and SKOV3 cells,respectively.The results of q RT-PCR and Western blot showed that YTHDF2 could inhibit m RNA and protein expression of MEF2C.The results of RNA stability experiments showed that the degradation of MEF2C was accelerated with the increase of YTHDF2 expression.YTHDF2 could decrease the m RNA stability and expression of MEF2C.The m~6A binding sites of YTHDF2 to MEF2C were predicted by SRAMP and cat RAPID websites,and RNA pull-down experiments were performed.The m~6A probe of MEF2C can drag out more YTHDF2 protein.YTHDF2 could reduce the stability and expression of MEF2C by recognizing and directly binding the m~6A modified site of MEF2C,and inhibit the MEF2C-mediated cisplatin resistance in OC.3.3 The effect of YTHDF2 on cisplatin resistance in OC was validated in nude mouse xenograft tumor experiments in vivo:overexpression of MEF2C decreased the volume and tumor weight of the xenograft,while overexpression of YTHDF2 increased the volume and tumor weight of the xenograft,overexpression of YTHDF2 could decrease the expression of MEF2C and inhibit the changes of tumor volume and weight induced by overexpression of MEF2C.Conclusions:1.The abundance of m~6A modification of MEF2C was increased in cisplatin-resistant ovarian cancer cells while the m RNA expression was decreased.MEF2C inhibited the proliferation of OC,promoted the apoptosis of OC,and inhibited the platinum resistance of OC by transcriptional regulation of DRAM1.2.YTHDF2 could inhibit the effects of MEF2C on cisplatin resistance of ovarian cancer cells.3.YTHDF2 could directly bind MEF2C and reduce the m RNA stability and expression of MEF2C in an m~6A-dependent manner,thereby mediating cisplatin resistance in OC. |
PDF Full Text Request