Biological Functions Of Metastasis-related LncRNA-8439 In Hepatocellular Carcinoma

Backgrounds and AimsHepatocellular carcinoma(HCC)is the most common pathological type in hepatic malignancies.It progresses rapidly,has a higher degree of malignancy,has a poor prognosis,and has a higher mortality rate.The main cause of poor prognosis of HCC is metastasis and recurrence.In all cancer types including liver cancer,mortality caused by tumor metastasis is 90%.Therefore,exploring mechanism of liver cancer metastasis is helpful for both prevention and treatment strategies.Related studies have shown that some long non-coding RNAs(lncRNAs)regulate key molecules in playing an important role in tumor metastasis.But so far,there are still a large number of unknown lncRNAs that might involve in tumor metastasis,and their roles are not clear.Pluripotency factors are core molecules which maintain the pluripotency and self-renewal of cells.Among them,the most important ones are OCT4,NANOG and SOX2.Studies have shown that they are involved in tumor metastasis regulated by up-stream molecules.The aim of this study was to use hepatocellular carcinoma as an entry point to find long-chain non-coding RNAs associated with liver cancer metastasis and to explore the relationship between them and pluripotency factors,and their influence on liver cancer cells.This study could provide an insight in mechanism of liver cancer metastasis on an experimental basis and provide possible targets for the intervention and treatment of liver cancer metastasis.MethodsFirst,we searched for lncRNA with significant differential expression in primary and metastatic liver cancer tissues by lncRNA sequencing,and then screened differentially-expressed lncRNAs related to pluripotency factors nanog,oct4,and sox2 by bioinformatics analysis.Next,real-time quantitative PCR(RT-PCR)was used to detect the expression of differentially-expressed lncRNAs in human cancer cell line Hep3 B,Huh7 cells and their corresponding tumor suspension spheres.The differentially-expressed lncRNA levels were tested by real-time quantitative PCR(qPCR)in human hepatocellular carcinoma cell lines,Hep3 B and Huh7,and their corresponding suspended spheres.Through bioinformatics analysis,we predicted that lncRNA-8439 might bind to NANOG.The localization of lncRNA-8439 in Hep3 B and Huh7 cells was subsequently observed by fluorescence in situ hybridization(FISH).The efficiency of overexpression and knockdown was detected by transfection overexpression and knockdown of lncRNA-8439.Subsequently,after knocking down lncRNA-8439,the expression of NANOG in the two hepatocarcinoma cells was detected by RT-PCR and Western blotting,and the growth of the suspension sphere was observed.After overexpression of lncRNA-8439,the expression of NANOG in the two hepatocellular carcinoma cells was detected by RT-PCR and Western blotting,and the growth of the suspension sphere was observed.ResultsLncRNA-Sequencing revealed a total of 85 significant differentially-expressed lncRNAs.Compared with the primary liver cancer tissue,there were 62 highly-expressed lncRNAs in the metastatic liver cancer tissues;23 of the lncRNAs were significantly lowly expressed in the metastatic liver cancer tissues.Based on the above screen,9 differentially-expressed lncRNAs associated with pluripotent factors NANOG,OCT4,and SOX2 were found through bioinformatic methods;Subsequently,the expression levels of the above 9 lncRNAs in hepatoma carcinoma cells Huh7 and Hep3 B and their corresponding tumor suspension spheres were detected.The results showed that only expression of lncRNA-8439 was consistently reduced or increased in the two hepatoma carcinoma tumor suspension spheres,which was statistically different from that in the corresponding cell lines,and the expression difference was the most significant(P<0.01).FISH revealed that lncRNA-8439 was mainly distributed in the nucleus;and it was found that lncRNA-8439 might bind to NANOG 3' end;Using interfering small RNA(siRNA)and reverse complement sequences(RCS),we successfully down-regulated and up-regulated lncRNA-8439 in the two hepatoma carcinoma cell lines for subsequent studies.And then,interference and overexpression experiments revealed that after knocking down lncRNA-8439,the RNA and protein expression levels of NANOG were significantly reduced in the Hep3 B and Huh7 cells(all P<0.01),and the number of suspended spheres was reduced.On the contrary,after overexpressing lncRNA-8439,the RNA and protein expression levels of NANOG were significantly increased in the two kinds of hepatoma carcinoma cells(all P<0.01),and the number of suspended spheres was also increased.ConclusionsThe results showed that lncRNA-8439 was significantly differentially expressed in the primary and metastatic tissues of hepatocellular carcinoma.LncRNA-8439 could affects self-renewal ability of hepatoma carcinoma cells through regulating expression of pluripotency factor NANOG,which might play an important role of invasion and metastasis of hepatocellular carcinoma.