The Experimental Study Of Long Non-Coding RNA MYLK-AS1 Upregulating CDC25A To Promote The Progression Of Hepatocellular Carcinoma By Sponging MiR-141-3p

Background: Hepatocellular carcinoma(HCC)is one of the most prevalent type of malignancy worldwide.A number of recent studies have demonstrated that aberrantly expressed long non-codingRNAs(lncRNAs)play an important role in HCC and these lncRNAs may be used as biomarkers for predicting patient survival.However,the molecular function of lncRNAs in HCC progression remain largely unclear.Aim: To screen out the specific lncRNAs which was related to the prognosis of hepatocellular carcinoma,and further study the oncogenic role of MYLK-AS1 in HCC.Methods: TheRNA-seq data and patients` clinical records with LIHC were download from TCGA.R packages and cytoscape software were used for data processing and ceRNA Network constructing.Correlation between the DELs and overall survival(OS)was analyzed using Kaplan-Meier survival analysis.Quantitative real-time polymerase chain reaction and in situ hybridization were used to detect the expression of MYLK-AS1 in HCC tissues.The role of MYLK-AS1 was further evaluated in vitro in gain-of-function and loss-of-function studies by colony formation assay,cell cycle analysis,quantification of apoptosis,RNA fluorescence in situ hybridization and luciferase reporter.Western bloting was used to detect the main protein expression of cell cycle.Results: 1.We discovered 1992 differential expressed mRNAs,1082 differential expressed lncRNAs and 126 differential expressed miRNAs,then we constructed lncRNA-miRNA-mRNA network for HCC.Then Kaplan-Meier survival analysis depicted that 13 lncRNAs were closely related with overall survival of HCC patients.Among the 13 lncRNAs,we choose four candidate gene for further characterization in three hepatocarcinoma cell lines(Huh7,Hep G2,Hep3B)and non-cancerous human hepatocyte cell line(LO2).q RT-PCR quantification revealed that one of these genes,MYLK-AS1 expression was up-regulated in all three HCC cell lines relative to the expression level detected in normal LO2 cell.2.q RT-PCR results showed that MYLK-AS1 expression level was higher in 44 liver cancer tissues than in adjacent non-cancerous tissues,and the level of MYLK-AS1 expression was positively correlated with TNM stage and tumor differentiation degree.Then the results of in situ hybridization of 72 HCC tissues showed that MYLK-AS1 was expressed in both HCC tissues and adjacent non-cancerous tissues,but the staining score of MYLK-AS1 in HCC tissues was higher than that in adjacent non-cancerous tissues and the expression level of MYLK-AS1 was not only correlated with TNM stage and tumor differentiation degree,but also correlated with microvascular invasion and patient prognosis.Multivariate COX regression analysis showed that MYLK-AS1 expression and tumor volume were independent risk factors for prognosis of patients with HCC.3.Fluorescence in situ hybridization(FISH)results showed that MYLK-AS1 was mainly expressed in the cytoplasm of HCC cells Huh7 and HCC tissue samples.4.CCK-8 proliferation,clonal formation and tumorigenesis experiments in mice showed that MYLK-AS1 promoted the proliferation of hepatoma cells.Flow cytometry and apoptosis assay showed that knockdown of MYLK-AS1 expression in Huh7 cells could significantly induce apoptosis and arrest cell cycle of HCC cells.While overexpression of MYLK-AS1 in Hep G2 cells promoted the proliferation of liver cancer cells,accelerated the cell cycle progression and inhibited the cell apoptosis,and MYLK-AS1 promoted the cell cycle progression by activating the Rb/E2 F signaling pathway.5.The expression of miR-141-3p was lower in HCC cells and HCC tissues.MYLK-AS1 and miR-141-3p had a mutual regulation effect,and the dual luciferase reporting assay confirmed the binding of MYLK-AS1 and miR-141-3p.6.CDC25 A is the target gene of miR-141-3p,and CDC25 A expression was increased in HCC tissues.CDC25 A expression level is positively correlated with MYLK-AS1,and miR-141-3p can negatively regulates CDC25 A expression in HCC cells.Cell functional recovery assay showed that miR-141-3p could weaken the regulation of MYLK-AS1 on CDC25 A expression and the effect on cell cycle.Conclusion: We identified an oncogene MYLK-AS1 in HCC,which was with high expression level in HCC and related to tumor TNM stage,tumor differentiation and the prognosis of HCC.MYLK-AS1 expression and the size of tumor were independent risk factors of HCC.MYLK-AS1 plays a role in the occurrence and development of tumors by promoting the proliferation,cell cycle progression and inhibiting apoptosis of HCC cells.MYLK-AS1 could upregulated CDC25 A expression by sponging miR-141-3p.MYLK-AS1 could affect the process of cell cycle through the Rb/E2 F signaling pathway.The findings of this study provide new ideas and research basis for the specific mechanism of the occurrence and development of liver cancer.