Long Non-coding RNA CCAT2 Promotes Hepatocellular Carcinoma Proliferation And Metastasis Through Up-regulaton Of NDRG1

Background:Hepatocellular carcinoma(HCC)is the most common pathological type of liver cancer,accounting for 85-90 %.Meanwhile,the incidence and mortality of HCC keep high levels in the world.Due to occult onset,rapid progression and high degree of malignancy,the survival prognosis of patients with advanced HCC is still poor even though the diagnosis and treatment of HCC are progressing.It is urgent to improve the diagnosis and treatment of HCC by revealing its pathogenesis.Studies showed that the abnormal expressions of many long non-coding RNAs(lncRNAs)played important roles in HCC.LncRNA CCAT2 has been reported to play a crucial role in many cancers.However,its role in HCC remains unclear.Objective:1.To investigate the biological functions of lncRNA CCAT2 in HCC progress.2.To explore the molecule mechanism of CCAT2 in HCC progress.Methods:Part one: Detection the expressions of CCAT2 in HCC cells and tissues1.RT-qPCR was performed to detect the expressions of CCAT2 in four HCC cells(SMMC7721,Huh7,HepG2,SK-Hep1)and the normal hepatocytes(L02).2.Then CCAT2 RNA levels were detected in 20 HCC tissues and corresponding tissues.Part two: Analysis for the biological functions of CCAT2 in HCC1.The overexpression plasmid of CCAT2 was constructed and shRNA targeting CCAT2 was designed and tested.Then,a series of biological function assays such as MTS,colony formation assay,transwell assay(with / without matrigel),wound healing assay and flow cytometry were used to testify the effects of CCAT2 in SMMC7721 and SK-Hep1 cells.2.CCAT2 overexpression and shRNA targeting CCAT2 stable cell lines were constructed.The effects of CCAT2 on tumorigenesis and metastasis of HCC in vivo were verified by subcutaneous tumorigenesis and in situ liver metastasis in nude mice.Part three: Screening target genes for CCAT2 and verifying the biological functions of the target gene1.RT-qPCR were used to detect the influence of CCAT2 on related genes,and RT-qPCR,WB were used to test the expressions of the target gene regulated by CCAT2.2.RT-qPCR,WB and immunohistochemistry(IHC)were performed to test the expression levels of the target gene NDRG1 in HCC cells and 20 tissue samples.Moreover,the correlation between CCAT2 and NDRG1 expressions in HCC cases were analyzed.3.NDRG1 full-length overexpression plasmid was constructed.A series of biological function experiments were carried out to verify the role of NDRG1 in SMMC7721 and SK-Hep1 cells.The biological function assays of CCAT2 co-transfected with NDRG1 were tested to explore how CCAT2 could play a role in HCC cells through NDRG1.4.To verify the regulatory effect of CCAT2 on NDRG1 expression in vivo.Part four: Molecular mechanisms of CCAT2 regulating the target gene NDRG11.The promoter sequence of NDRG1 was found through NCBI database,and the full-length double luciferase reporter gene plasmid was constructed.The effect of CCAT2 on the activity of NDRG1 promoter was detected by double luciferase reporter gene assay.2.Double luciferase reporter gene plasmids with truncated promoter regions of NDRG1 were constructed.Double luciferase reporter gene assays were used to detect the effects of CCAT2 on the activity of each region of NDRG1 promoter,and to determine the active range.Results:Part one: The expressions of CCAT2 in HCC cells and tissues1.The expressions of CCAT2 were significantly increased in HCC cells compared with those in L02 cells.2.The expressions of CCAT2 were significantly increased in 20 HCC tissue samples compared with those in corresponding paracancerous tissues.Part two: Analysis for the biological functions of CCAT2 in HCC1.CCAT2 promoted proliferation,migration and invasion abilities of SMMC7721,SK-Hep1 cells.2.Overexpression of CCAT2 promoted tumorigenesis,liver in situ metastasis and lung metastasis of HCC cells in vivo,while interference had the opposite result.Part three: Screening target genes for CCAT2 and verifying the biological functions of the target gene1.Overexpression of CCAT2 up-regulated the expression of NDRG1,while interference was the opposite.NDRG1 was the downstream target gene of CCAT2.2.The NDRG1 levels were up-regulated in HCC cells and 20 HCC tissues.There was a positive corelation between the RNA levels of CCAT2 and NDRG1 in HCC sampes.3.Overexpression of NDRG1 could promote the proliferation,migration and invasion of SMMC7721 and SK-Hep1 cell lines.4.In vivo experiments,the expressions of NDRG1 in tumor tissues of mice overexpressing CCAT2 group were increased,while the interference was the opposite.Part four: Molecular mechanisms of CCAT2 regulating the target gene NDRG11.CCAT2 enhanced the transcriptional activity of NDRG1 promoter.2.The effective regon of CCAT2 on NDRG1 promoter was-1000 to-500 bp.Conclusion:LncRNA CCAT2 promoted the proliferation,migration and invasion of HCC cells by enhancing the transcriptional activity of NDRG1 promoter and then increasing the expression of NDRG1.