The aim of our study was to distinguish miRNAs that were obviously different expression in OC comparing with normal tissue, and to improve ovarian cancer patients’ clinical survival outcome by exploring the mechanism attending in particular pathway. We selected MIR502 as our interested miRNA after screening miRNAs through strict selection process. Our survival analysis showed that MIR502 conferred a protective phenotype to OC patients in which higher expression of MIR502 predicted longer overall survival. MIR502 located in the third intron of the CLCN5 gene, and it showed strong positive correlation with CLCN5 in ovarian cancer, we predicted NRF1 as a transcription factor of CLCN5, and ChIP-seq data of various tumor cells verified the binding peak between NRF1 and CLCN5. We demonstrated NRF1 as transcription factor of CLCN5 regulated the expression of MIR502 indirectly, which expounded the upstream regulatory mechanism of MIR502 deeply.
In order to explore downstream regulatory mechanism of MIR502 in the process of ovarian cancer, we further predicted and analyzed genes correlated with MIR502. We identified a set of biological functions and related signaling pathways which MIR502 might regulate in ovarian cancer. Furthermore, GSEA annotation analysis results showed that MIR502 regulated anti-apoptosis and pro-proliferation genes such as CCND1, FGF1, MYC, GLI2 negatively in Hippo signaling pathway. All the results demonstrated strongly that the expression of MIR502 was down regulated in OC, which increased the expression level of oncogene CCND1, FGF1, MYC and GLI2, they served an important function of anti-apoptosis and promote development of OC. PPI network also suggested that CCND1 and MYCN were both target genes regulated by MIR502, and they were at the center position interaction with other proteins.
CCND1,also known as cyclin D1, is a member of cell cycle family protein . CCND1 regulates cell cycle progression by promoting the cell cycle transition from the G1 to S phase [27–29]. The abnormal expression of CCND1 promotes cell proliferation by regulating cell cycle.. Previous researches have demonstrated that CCND1, identified as a proto-oncogene, served as an essential role in the development of many kinds of tumors, including lung adenocarcinoma, glioma and renal cell cancer[31–33]. In addition, some studies have shown that overexpression of CCND1 promoted tumor cell invasion and metastasis in breast cancer gastric cancer, leading to a poor prognosis[34, 35]. Compared with normal tissues, the expression of CCND1 were obviously higher in bladder cancer, reproductive system tumors, gastric cancer and lung cancer, which were correlated with the pathological type and clinical stage of the tumor[36–38] .CCND1 expression was closely related with 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 fewer apoptosis. In our study, MIR502 is down regulated in ovarian cancer, and CCND1 was negatively correlated with MIR502, which means the expression of CCND1 is overexpressed in OC. In addition, PPI network showed that CCND1 played a core function in interacting with other proteins, further verified the important role of CCND1 in regulating progress of OC. The development of OC may be slowed down by up regulating MIR502, which decreases expression of CCND1 and restrains cell cycle.
The MYC family proto-oncogenes is comprised of c-MYC, MYCN and MYCL .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 great number of target genes. In fact, amplification of c- MYC has been reported in ovarian cancer.And previous studies showed that higher levels of c-MYC expression led faster recurrence and worse overall survival rate of patients with high grade serous ovarian cancer, and was related to cisplatin resistance in ovarian cancer cells. Silencing of c-MYC inhibited cell 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. It means that c-MYC may act as a new biomarker and therapy target of chemotherapy response. Another member of the MYC family, MYCN, controls the basic process of embryonic development. MYCN signaling disorder leads to a variety of tumors, including neuroblastoma, medulloblastoma, rhabdomyosarcoma, Wilms tumor, prostate cancer and lung cancer. In neuroblastoma, genetic aberration of MYCN amplification is the most related to poor prognosis and failure of therapy. MYCN targeted therapy has been proposed as a new strategy for cancer treatment, and a lot of efforts have been made to develop direct and indirect MYCN inhibitors with potential clinical applications.
FGF1 belongs to 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 tumor development, as it is upregulated in various cancers, including breast cancer, gliomas and ovarian cancer. The expression of FGF-1 has strong relationship with severity of prognosis and chemoresistance of tumor[49–52]. FGF1 has been considered as a potential prognostic marker for OC. Compared with other family members, FGF1 genetic variation has the most significant correlation with the increased risk of ovarian cancer. 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.Our study suggested that MIR502 played counter-regulation expression effect on FGF1, MIR502 low level of expression increases FGF1expression in ovarian cancer, which may lead OC development and platinum chemotherapy resistance.
GLI family zinc finger proteins mediate Sonic hedgehog (Shh) signaling and they exist in embryonic tumor 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 was regulated by Yap/TAZ, which activated the downstream regulatory factors of Shh signaling and promote proliferation. A large number of evidences implicate that GLI2 regulates the key link of 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 . Similar mechanisms have been reported in human vascular smooth muscle cells and myofibroblasts. Same cases occurred in cervical cancer that overexpression of GLI2 increased proliferation. All researches demonstrated that GLI2 promoted cell proliferation and exerted a tumor-promoting role in cancer. In our study, GLI2 as a downstream molecular of Hippo signaling pathway, highly expressed due to the negatively regulated by MIR502, resulting in accelerating the pathological process of ovarian cancer. And GLI2 may be targeted as a novel therapeutic strategy in the future.
In summary, we have discovered that MIR502 expression in ovarian cancer was lower than that in normal tissue, which means MIR502 acting as a significant tumor-suppressor in ovarian cancer and through K-M analysis, MIR502 expression is connected to ovarian cancer patient overall survival outcomes. Additionally, analysis showed that the expression of MIR502 was regulated by NRF1 and further exerted apoptosis and inhibiting proliferation by regulating genes downstream of the Hippo signaling 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 prognostic tools in ovarian cancer. The better result is that our research on MIR502 in ovarian cancer will promote the more extensive research on the molecular mechanism of MIR502 and provide reference for improving the clinical treatment of ovarian cancer.