The Molecular Mechanism Of MiR-92a-3p Targeting Wilms Tumor Over-expressed Genes To Regulate The Migration And Invasion Of Human Cholangiocarcinoma Cells

Cholangiocarcinoma(CCA)is an aggressive bile duct epithelial adenocarcinoma,and is one of the most common primary hepatobiliary malignancies.The incidence of CCA is second only to hepatocellular carcinoma.So far,surgical resection is still the most effective treatment for CCA.Although many advances have been made in surgery and chemotherapy,the prognosis of CCA patients remains poor.Due to the insufficient understanding of the molecular mechanism of the occurrence and development of CCA,the development and application of CCA-related new drugs are hindered to a certain extent.Therefore,there is an urgent need to find new target molecules to improve the survival rate of CCA patients.In this study,we conducted a bioinformatics analysis on the published public data set of cholangiocarcinoma,and finally screened out Nephroblastoma overexpressed gene(NOV)that were significantly up-regulated at both the m RNA and protein levels and analyzed its function in depth,further design experiments to verify its role in the proliferation,migration and invasion of cholangiocarcinoma cells and the study of molecular mechanisms.NOV(also known as CCN3)is one of the 6 members of the CCN family.Based on the important role played by the CCN family in the biological process of tumors,we conducted a bioinformatics analysis of 6 members of its family in 10 pan-cancer datasets.The results showed that NOV has differential expression diversity in different tumor patients.It was significantly up-regulated in cholangiocarcinoma,ovarian cancer,and pancreatic cancer,and significantly down-regulated in the other 7 tumors.In cholangiocarcinoma,NOV is the most up-regulated molecule among the 6 members of the CCN family.NOV has been shown to regulate various pathophysiological processes in various human cancers,but its expression and biological functions in cholangiocarcinoma are still unknown.The purpose of this study is to explore the regulation of NOV on the proliferation,migration and invasion of cholangiocarcinoma cells.First,we verified whether NOV in CCA cells HCCC9810(hereinafter referred to as 9810)and RBE were consistent with the bioinformatics prediction(high expression of NOV in CCA)by fluorescence quantitative PCR(q-PCR)and Western Blot experiments.Then we connect the NOV gene to the pc DNA3.0 vector to effectively overexpress it in 9810 and RBE cells,and divide the experiment into a negative control group(pc DNA3.0)and an overexpression group(NOV);use RNA interference(RNAi)technology transfects small interfering RNA(siRNA)targeting NOV into 9810 and RBE cells to effectively silence the expression of NOV.The experiment is divided into negative control group(si-NC)and silencer Group(si-NOV-1,si-NOV-2).The following experiments were carried out: In 9810 and RBE cells,the m RNA and protein expression of NOV in the cells were detected by q-PCR and Western Blot respectively;observe the effect of NOV on the proliferation of 9810 and RBE cells through CCK-8 cell proliferation experiments and cell colony formation experiments;use wound healing assay and Transwell chambers to conduct migration experiments to detect the effect of NOV on the migration of 9810 and RBE cells;use Transwell cell microporous membrane to evenly coat matrigel on the cell invasion experiment to observe the effect of NOV on the invasion ability of 9810 and RBE cells;Western Blot technology was used to detect the expression levels of epithelial marker protein E-cadherin and interstitial marker proteins Vimentin,N-cadherin,Snail and MMP9 in cells;use Western Blot technology to explore downstream regulatory molecules of NOV;use bioinformatics to predict and Dual luciferase reporter assay to explore the mechanism of micro RNA regulating NOV upregulation in CCA cells.The results showed that the expression of NOV in 9810 and RBE was significantly higher than that in human normal bile duct epithelial cells HIBEC(P <0.001).Overexpression of NOV significantly promoted the migration and invasion of 9810 and RBE cells,while silencing NOV significantly inhibited the migration and invasion of 9810 and RBE cells.However,NOV had no significant effect on the proliferation of 9810 and RBE cells(P> 0.05).In addition,the expression of epithelial cell marker E-cadherin was significantly down-regulated in 9810 and RBE cells overexpressing NOV,while the expression of mesenchymal cell markers Vimentin,N-cadherin,Snail,and MMP9 was significantly up-regulated;on the contrary,9810 and RBE that silence NOV The expression of epithelial cell marker E-cadherin was significantly up-regulated in cells,while the expression of mesenchymal cell markers Vimentin,N-cadherin,Snail,and MMP9 was significantly down-regulated,indicating that NOV can promote the epithelial-mesenchymal transition of cholangiocarcinoma cells 9810 and RBE(Epithelial-Mesenchymal Transition,EMT)process.In terms of mechanism,We first detected the downstream regulatory molecules of NOV,NOV can positively regulate the Notch1 signaling pathway;Next,we found that miR-92a-3p,which has the opposite expression pattern of NOV in CCA cells,can directly bind to the 3'UTR region of NOV to negatively regulate NOV expression.The miR-92a-3p mimic and the NOV overexpression plasmid were co-transformed into 9810 cells can effectively reverse the cell migration and invasion effects caused by NOV overexpression.Similarly,the addition of miR-92a-3p mimics can also effectively reverse NOV.The process of epithelial-mesenchymal transition caused by overexpression.In summary,miR-92a-3p is involved in NOV-mediated migration and metastasis of CCA cells.Therefore,NOV can be used as a potential target for pre-CCA diagnosis and treatment.This study provides experimental data and molecular theoretical basis for the development of CCA anti-tumor drugs targeting NOV,and enriches the understanding of the molecular mechanism of CCA.