The aim of our study was to identify miRNAs that were obviously differentially expressed in OC tissue compared with normal tissue and to improve ovarian cancer patients’ overall survival by exploring the mechanisms regulating particular pathways. We selected MIR502 as our main miRNA after screening miRNAs through a strict selection process. Our survival analysis showed that MIR502 conferred a protective phenotype to OC patients, with a higher expression of MIR502 predicting a longer overall survival. MIR502 is located in the third intron of the CLCN5 gene, and it shows a strong positive correlation with CLCN5 in ovarian cancer. We predicted NRF1 as a transcription factor regulating CLCN5, and ChIP-seq data of various tumour cells verified the binding peak between NRF1 and CLCN5. We demonstrated that NRF1, as a transcription factor regulating CLCN5, regulated the expression of MIR502 indirectly, which clarified the upstream regulatory mechanism of MIR502.
To explore the downstream regulatory mechanism of MIR502 in ovarian cancer, we further predicted and analysed genes correlated with MIR502. We identified a set of biological functions and related signalling pathways that MIR502 might regulate in ovarian cancer. Furthermore, the GSEA annotation analysis results showed that MIR502 negatively regulated anti-apoptosis and pro-proliferation genes, such as CCND1, FGF1, MYC, and GLI2, in the Hippo signalling pathway. All of these results demonstrated that the expression of MIR502 was down-regulated in OC, which increased the expression levels of the oncogenes CCND1, FGF1, MYC and GLI2, which have important functions in anti-apoptosis and promote the development of OC. The PPI network also suggested that CCND1 and MYCN were both target genes regulated by MIR502, and they were at the centre position of interaction with other proteins.
CCND1, also known as cyclin D1, is a member of the cell cycle family of proteins[26]. CCND1 regulates cell cycle progression by promoting the cell cycle transition from G1 to S phase[27-29]. The abnormal expression of CCND1 promotes cell proliferation by regulating the cell cycle[30]. Previous researchers have demonstrated that CCND1, identified as a proto-oncogene, has an essential role in the development of many kinds of tumours, including lung adenocarcinoma, glioma and renal cell cancer[31-33]. In addition, some studies have shown that overexpression of CCND1 promotes tumour cell invasion and metastasis in breast cancer gastric cancer, leading to a poor prognosis[34, 35]. Compared with that in normal tissues, the expression of CCND1 is obviously higher in bladder cancer tissues, reproductive system tumours, gastric cancer tissues and lung cancer tissues, and it is correlated with the pathological type and clinical stage of the tumour[36-38]. CCND1 expression is closely related to cell proliferation ability and apoptosis in epithelial ovarian cancer cells. A study of epithelial ovarian cancer observed that overexpression of CCND1 leads to stronger cell growth ability and less apoptosis[39]. In our study, MIR502 was down-regulated in ovarian cancer, and the expression of CCND1 was negatively correlated with MIR502, which means CCND1 is overexpressed in OC. In addition, the PPI network showed that CCND1 plays a core function in interacting with other proteins, which further verified the important role of CCND1 in regulating the progression of OC. The development of OC may be slowed down by up-regulating MIR502, which decreases the expression of CCND1 and restrains the cell cycle.
The MYC family of proto-oncogenes is comprised of c-MYC, MYCN and MYCL[40]. c-MYC as an oncogene in numerous cancer cells plays an important role in a myriad of biological processes, including cell growth, cell cycle progression and proliferation[41, 42] by cooperating with YAP and activating a large number of target genes[43]. In fact, the amplification of c-MYC has been reported in ovarian cancer[44]. Previous studies showed that higher levels of c-MYC expression led to a faster recurrence and worse overall survival rate of patients with high grade serous ovarian cancer and was related to cisplatin resistance of ovarian cancer cells. Silencing of c-MYC inhibited the growth of cisplatin-resistant ovarian cancer. Thus, c-MYC targeted therapy is a potential treatment for ovarian cancer patients with high expression of c-MYC, including those who are resistant to cisplatin. This means that c-MYC may act as a new biomarker and therapy target for the chemotherapy response. Another member of the MYC family, MYCN, controls the basic process of embryonic development. MYCN signalling disorders leads to a variety of tumours, including neuroblastoma, medulloblastoma, rhabdomyosarcoma, Wilms tumour, prostate cancer and lung cancer. In neuroblastoma, a genetic aberration of MYCN amplification is related to a poor prognosis and failure of therapy. MYCN targeted therapy has been proposed as a new strategy for cancer treatment, and many effort has been made to develop direct and indirect MYCN inhibitors with potential clinical applications[45].
FGF1 belongs to the fibroblast growth factors (FGFs) family, whose function is regulating many cellular processes, including cell proliferation, differentiation and survival as an oncogene [46-48]. FGF1 is associated with tumour development, as it is upregulated in various cancers, including breast cancer, gliomas and ovarian cancer. The expression of FGF-1 has a strong relationship with a poor prognosis and chemoresistance of tumours[49-52]. FGF1 has been considered as a potential prognostic marker for OC[53]. Compared with other family members, FGF1 genetic variation has the most significant correlation with an increased risk of ovarian cancer[54]. In addition, FGF1 expression is also an important determinant of survival and response to platinum chemotherapy. Therefore, the regulation of FGF1 by different mechanisms may play an important role in the development of ovarian cancer[55]. Our study suggested that MIR502 had a counter-regulatory expression effect on FGF1, and a low level of MIR502 expression increases FGF1 expression in ovarian cancer, which may lead to OC development and platinum chemotherapy resistance.
GLI family zinc finger proteins mediate Sonic hedgehog (Shh) signalling, and they exist in embryonic tumour cells as effective oncogenes. The proteins encoded by GLI2 belong to the C2H2-type zinc finger protein subclass of the GLI family. Researchers have found that the expression of GLI2 is regulated by Yap/TAZ, which activates the downstream regulatory factors of Shh signalling and promotes proliferation[56]. A large body of evidence has implicated GLI2 as a key regulator link in the cell cycle. Nagao et al. reported that silencing the expression of GLI2 made the cell cycle stop in G1 phase, which prevented the growth of osteosarcoma[57]. Similar mechanisms have been reported in human vascular smooth muscle cells[58] and myofibroblasts[59]. The same thing was observed in cervical cancer, that overexpression of GLI2 increased proliferation. All of the research has demonstrated that GLI2 promoted cell proliferation and exerted a tumour-promoting role in cancer. In our study, GLI2 as a downstream target of the Hippo signalling pathway was highly expressed due to the negative regulation by MIR502, resulting in an acceleration of the pathological process of ovarian cancer. GLI2 may be targeted as a novel therapeutic strategy in the future.
In summary, we have discovered that MIR502 expression in ovarian cancer is lower than that in normal tissue, which means that MIR502 acts as a significant tumour suppressor in ovarian cancer. MIR502 expression level was also correlated with ovarian cancer overall survival outcomes. Additionally, our analysis showed that the expression of MIR502 was regulated by NRF1 and further induced apoptosis and inhibiting proliferation by regulating genes downstream of the Hippo signalling pathway, including CCND1, FGF1, MYC and GLI2. In our study, we propose novel mechanisms between MIR502 and ovarian cancer that have not been elucidated previously. The immediate application of our findings is that MIR502 can be used as a prognostic tool in ovarian cancer. A better result is that our research on MIR502 in ovarian cancer will promote more extensive research on the molecular mechanisms of MIR502 and provide a reference for improving the clinical treatment of ovarian cancer.