| 【Objective】Hepatocellular carcinoma(HCC)is one of the most common malignant tumor of digestive system with high incidence and mortality that threaten human health.Research shows that the occurrence and development of HCC is related to a variety of risk factors,of which chronic hepatitis B virus(HBV)infection is an important cause.Clinical data showed that about 54% of the global HCC can be attributed to HBV infection,and HBV positive HCC and HBV negative HCC is significantly different in the pathogenesis and prognosis,but the molecular mechanism is still unclear.At present,many studies have revealed that the long non-coding RNA(lnc RNA)can be widely involved in the progression of human cancers,including HCC.In order to explore the role of lnc RNA in HBV related HCC,we analysis the abnormal expression of lnc RNA in HBV negative and positive HCC tissue,then selected a high expressed lnc RNA named n335586.The present study aims to investigate the regulation mechanism of abnormal expression of n335586 in HCC cells,and explore its effect on the malignant behavior of cancer cells,so as to clarify the role of n335586 in the development of HCC,in order to provide new molecular and theoretical basis for the diagnosis and treatment of HCC.【Methods】First,RT-q PCR was used to detect the expression of n335586 in HBV negative and positive HCC tissues and hepatoma cell lines.Then we transiently transfected Hep G2 cells with p HBV1.3 plasmid and HBV encoded protein to examine the expression level of n335586.Colony formation,MTT,Transwell migration and invasion assays and Western Blot assays were taken to test the influence of n335586 on malignant behavior and EMT process.In order to analyze the mechanism of n335586,we firstly using RT-q PCR and Blot assay to explore the effect of n335586 on its host gene CKMT1 A,and then cell function experiments and Western Blot assays were were carried out to analyze the role of CKMT1 A in HCC.Then bioinformatics were done to predict the common potential binding candidate mi RNA of n335586 and CKMTA,meanwhile RT-q PCR,EGFP fluorescent report and Western Blot assays were applied to confirm that mi R-924 can bind both n335586 and CKMT1 A.In addition we investigated whether n335586 regulated the expression of CKMT1 A dependent on mi R-924.Finally rescue experiments were performed to confirm that mi R-924 mediated the function of n335586 on hepatoma cells.【Results】RT-q PCR experimental results showed that compared with HBV negative group,n335586 expression was significantly up-regulated in HBV positive HCC tissues.In addition,the expression level of n335586 in HBV positive cells Hep G2.2.15 were prevalently higher than that in HBV negative cell lines Hep G2.At the same time n335586 levels were markedly increased after transfected with HBV replication plasmid p HBV1.3 in Huh7 cells than that of the control group.These data further confirmed our deep sequencing results.When transfected in Hep G2 and Huh7 cells with HBx,n335586 expression levels increased,suggesting that increased expression of n335586 were induced by X protein which is encoded by HBV itself.A series of assays showed that n335586 can function as an oncogene,by promoting the invasion and migration of HCC cells and the EMT process.RT-q PCR and Western Blot assays indicated that n335586 can regulate and promote the expression of its host gene CKMT1 A.Moreover the function of cell biology experiment showed that CKMT1 A could also promote the migration and invasion of hepatocellular carcinoma cells and EMT.Bioinformatics predicted that mi R-924 has potential binding sites on both n335586 and CKMT1 A,and EGFP fluorescent report assay and RT-q PCR experiments proved that mi R-924 could compete with n335586 and CKMT1 A.Subsequently we using RT-q PCR,EGFP fluorescent reporter assay and Western Blot experiments further confirmed that n335586 regulate the expression of CKMT1 A in a mi R-924 dependent manner.Finally rescue experiments were performed to prove that mi R-924 mediates the biological function of n335586 in hepatoma cells.【Conclusions】Our study found that n335586 were ectopic expressed in HBV positive hepatocellular carcinoma tissues,which was induced by HBx protein.Studies of cell function showed that n335586 acts as an oncogene in HCC by combined with mi R-924 and CKMT1 A. |
