As one of the three major gynecological malignancies, ovarian cancer is a highly destructive and heterogeneous gynecological malignancy. Even with the development of many surgical techniques and chemotherapy, the overall five-year survival rate is still as low as 47%. Besides, OC is the fourth leading cause of cancer-related deaths in China. It is worth noting that the vast majority of ovarian cancer patients are in advanced stage when they are diagnosed, and the 5-year survival rate is less than 30%. These unsatisfactory data are mainly due to poor early diagnosis rate of OC[40, 41] and poor prognosis. At present, the internationally recognized treatment for ovarian cancer is cytoreductive surgery, followed by platinum based chemotherapy. However, primary and secondary drug resistance may occur during chemotherapy of ovarian cancer, which makes it fail to achieve the desired effect. Recently,it was discussed that the unknown pathogenesis of OC is the main challenge to develop new diagnostic markers and therapeutic targets. This prompted us to further explore the molecular mechanism of ovarian cancer progression, in order to find valuable biomarkers and new treatment methods.
As one of the most common mRNA modifications, m6A is closely related to the development of human beings.RNA m6A methylation is a post transcriptional modification of adenine at position 6. This modification has been found in most eukaryotic mRNA, tRNA, rRNA and other noncoding RNAs. Its important regulatory functions include the regulation of gene expression, biological development and cancer development.Especially the proliferation, apoptosis and metastasis of cancer. In breast cancer (BC), METTL3 (“Reader” protein of m6A modification) promotes HBXIP expression by increasing m6A modification, thus promoting BC proliferation. Another study on glioblastoma stem cells (GSC) showed that the m6A modification of METTL3 and METTL14 in GSC inhibited the expression of ADAM19, EPHA3 and other oncogenes, inhibited the growth and self-renewal of GSC, thus inhibiting tumorigenesis. In addition, it was reported that M6A modification also plays an important role in endometrial carcinoma(EC). High expression of METTL3 promotes the development of endometrioid epithelial ovarian cancer (EEOC) by regulating abnormal methylation of m6A RNA, indicating malignant tumor and low survival rate of patients. However, the study of m6A modification in ovarian cancer has only started in recent years. The fat mass and obesity associated protein (FTO) is a kind of m6A demethylase ("Eraser" protein of m6A modification). It can inhibit the self-renewal of ovarian tumor and cancer stem cells (CSC) and inhibit tumorigenesis in vivo, this process is completed by inhibiting cAMP signal transduction. Overexpression of TLR4 activates the NF - κ B pathway to upregulate the expression of ALKBH5 and increase the level of m6A, which promotes the occurrence of ovarian cancer. This discovery provides clues for the invention of new targeted therapy methods in OC. YTHDF1 (“Reader” proteins of m6A modification) can promote the occurrence and metastasis of ovarian cancer by binding with E6A modified EIF3C mRNA, and the up regulation of YTHDF1 is associated with poor prognosis of ovarian cancer patients. Another study showed that METTL3 plays a carcinogenic role partly through AkT signaling pathway in the process of esophageal cancer, which indicates that METTL3 can be used as a potential therapeutic target for esophageal cancer treatment. Thus, the above results demonstrated that m6A modification often depends on the related protein signaling pathway to abnormal expression of targeted genes, to participating in the proliferation, apoptosis and metastasis of cancer.Herein,t he purpose of our study is to identify the differentially expressed genes(DEGs) modified by m6A in OC and investigate the protein pathway that the modification depends on.
In this study, two mRNA microarray data sets were analyzed to obtain DEG between OC and non-cancerous tissues. A total of 152 DEG were identified in two data sets, including 75 down-regulated genes and 77 up-regulated genes. GO and KEGG enrichment analysis was carried out to explore the interaction between DEGs. The over-expressed genes were mostly enriched in cell cycle, mitotic spin, activation of protein kinase activity, response to hydroxyia and oocyte meiosis, while the low-expressed genes were mostly enriched in signal transduction and protein domestication activity. It has been reported that the imbalance of cell cycle process and mitotic cell cycle play an important role in the occurrence or development of tumor[52, 53, 54]. Furthermore, A large number of reports have suggested that complement activation is another way to promote tumor. In addition, hypoxia is the common result of rapid growth of solid tumors and abnormal vascular structure, which has been recognized as one of the most important characteristics of tumor microenvironment (hallmark). More importantly, Hypoxia also induces the invasion of ovarian cancer to increase, and to chemoradiotherapy,including not sensitive or even resistant[57, 58, 59, 60]. Consistent with these results, our results are consistent with all these theories.
Subsequently,we also screened 15 m6A-modified DEGs through the m6AVar database. The candidate genes were analyzed byKaplan-Meier Plotter database,GEPIA and cBioPortal online platform, two DEGs were selected for detailed analysis: SCN7A and GATM. In different data sets, SCN7A and GATM were significantly low expressed in OC, and then the impact of the expression levels of the two genes on the prognosis of patients was studied. The results confirmed that the expression levels of SCN7A and GATM had a significant impact on the overall survival time of patients.Besides, the expression levels of SCN7A and GATM in OC of different TNM stages were different, and the expression of SCN7A and GATM in stage II and stage III was higher than that in stage IV. Mounting studies have shown that during the transcriptional regulation of BCL2 family and IAP family genes by ras-pi3k-akt-nf - κ B pathway, SCN7A is regulated and dramatically down regulated, leading to tumor proliferation and inhibiting tumor apoptosis. SCN7A as a BM related gene carrying frequent brain metastasis (BM) specific mutations. In colorectal cancer (CRC), SCN7A mutation leads to down-regulation of gene expression and promotes BM of CRC. It can be seen that our study on SCN7A is consistent with the above conclusion. GATM is a proximal tubular enzyme involved in creatine biosynthesis, In renal cell carcinoma (RCC), the expression of GATM RNA chimeras is increased, compared with benign adjacent kidneys, this up regulation of RNA chimeras may regulate the cellular mechanism and may affect the survival of patients. All these revealed that the role of SCN7A and GATM in OC needs further research and exploration.
The analysis of the methylation of the two genes was completed through the m6AVar database. The results included 11 GATM studies (2 mouse and 9 human ) and 94 SCN7A studies (6 mouse and 88 human). The modification of SCN7A by m6A is mainly concentrated on chromosome 2, but the protein pathway dependent on m6A modification has not been recorded. On the other hand, the m6A modification of GATM mainly concentrated on chromosome 2, 15 and 16. The related binding proteins were EIF4A3 and FUS. EIF4A3 mainly occurred in the downstream of 3'AG and FUS occurred in the upstream of 5'GT. The present study aimed that CircPVRL3 can activate FUS, LIN28A, PTB and EIF4A3 binding proteins, promote the methylation of m6A, thus promoting the occurrence of gastric cancer (GC). Linc00667 up-regulated in non-small cell lung cancer (NSCLC) can promote the production of vascular endothelial growth factor A (VEGFA) by binding with EIF4A3, thus promoting the proliferation, migration and angiogenesis of NSCLC cells.It has been previously shown that LncRNA EMX2OS binds directly to FUS protein, and overexpression of both can inhibit the proliferation, migration and invasion of prostate cancer cells and activate the GMP-PKG pathway. In the case, combined with the above information, we can assume that m6A can promote OC cell development by reducing GATM gene expression through EIF4A3 or FUS. Of course, this hypothesis needs to be further verified in future research.