The Role Of LncRNA UCA1 Upregulated By HBx In Hepatocarcinogenesis

Although it is widely accepted that Hepatitis B virus(HBV)-encoded X protein(HBx)is one of the most important proteins which associated with HBV-induced oncogenesis.In order to discover more new effective therapeutic targets for hepatocellular carcinoma(HCC),the underlying molecular signaling and epigenetic mechanisms still need to be fully elucidated.Recent studies have shown the diversity of biological functions of lncRNA,but the mechanism about lncRNA in HCC is still unclear.The purpose of this study is to investigate the effects of HBx on lncRNA expression profiles,to explore the biological functions and potential mechanisms in hepatocarcinogenesis of lncRNA induced by HBx,particularly the epigenetic mechanisms of HBx involved in the malignancy of HCC.The lncRNA differential expression profiles obtained earlier which induced by HBx are validated by qPCR(Real-time Fluorescent Quantitative Reverse Transcriptase-polymerase Chain Reaction)method firstly.The remarkable upregulated IncRNA UCA1 is selected as the candidate for further study.The regulatory effects of HBx on UCA1 expression were evaluated in HBx transfection and HBx RNAi cell lines using qPCR.The expression levels of HBx and UCA1 in HCC clinical specimens are detected by qPCR and the correlation of HBx and UCA1 was further analyzed.The functions of UCA1 are measured using CCK-8 cell proliferation assay,flow cytometry analysis,colony formation assay and xenograft mouse tumorigenicity assay.Western blot method analyze the effect of UCA1 on the expression of apoptosis-related protein cleaved caspase-9,caspase-3 and caspase-8.The impact of UCA1 on the expression of its target gene p27 mRNA and protein levels was analyzed by qPCR and western blot.Further detect the expression of p27 in HCC clinical specimens and analyze of the correlation between p27 and UCA1.The RIP(RNA Immunoprecipitation)assay is applied to detect the physical association of UCA1 and EZH2,and ChIP(Chromatin Immunoprecipitation Assay)assay applied to determine the specific region of p27 binding with EZH2,which is the subunit of PRC2.qPCR are performed to detect the alteration of the binding of EZH2 and H3K27me3 on p27 promoter after UCA1 knockdown.qPCR assay are employed to explore the expression of UCA1 in normal liver,hepatitis tissues,tumor and paratumor tissues.We analyze the expression pattern of UCA1 and further explore whether UCA1 could participate in "inflammation-carcinoma" transformation.Then,miR-122 was predicted and chosen as a potential target of UCA1.qPCR assay was used to examine the effects of UCAl expression knock-down on miR-122 expression.And the expression of miR-122 in normal liver and hepatitis tissues was detected for analyzing the correlation between miR-122 expression and inflammation.The effect of UCA1 on pri-miR-122 expression was explored by UCA1 specific knock-down.The correlation between the expression of miR-122 and UCA1,pri-miR-122 and UCA1 were analyzed followed with the measurement of miR-122 and pri-miR-122 expression in tumor and paratumor tissues of HCC by qPCR.Furthermore,the binding capacity of miR-122 and CCL23'UTR was predicted by using bioinformatics analysis,and the effects of miR-122 on CCL2 expression was discovered via using miR-122 mimics and inhibitors transfection.To investigate the effects of the inflammatory microenvironment on UCA1 expression,LO2 cells,the immortalized human hepatocyte line,were treated with TGF-?,because the TGF-?signaling usually be activated under inflammatory microenvironment.Following results were obtained through the present study:1)The expression of lncRNA UCAl was significantly upregulated by HBx,and depend on HBx dose.2)There was a positive correlation between UCA1 and HBx expression levels in HCC clinical specimens.3)UCA1 promoted cell proliferation,inhibited cell apoptosis and accelerated cell cycle progression in vitro,and UCA1 also increased tumorigenicity in vivo.4)Enforced UCA1 expression inhibited p27 expression in HCC cells,and the expression level of p27 was negatively correlated with UCA1 level in HCC clinical specimens.5)UCA1 affected the binding of EZH2 to p27 promoter region,which subsequently regulated H3K27 triple-methylation of p27 promoter region.The results showed that UCA1 decreased p27 expression at least partly via recruiting EZH2 to p27 promoter region and leading to epigenetic silenced UCA1.6)Loss or low expression level of UCA1 was detected in normal liver tissue,but high in hepatitis and paratumor tissues,and the expression of UCA1 was significantly reduced in tumor tissues compared to paratumor tissues.miR-122 expression was significantly lower in two hepatitis tissues than in normal liver tissues,and there was a negative correlation of UCA1 and miR-122 expression among 21 pairs of paratumor and tumor tissues in HCC.UCA1 knock-down significantly increased the expression of miR-122.7)The expression of pri-miR-122 was significantly reduced with endogenous UCA1 knock-down,and there was a positive correlation between UCA1 and pri-miR-122 expression in clinical cases.It suggested that UCA1 inliibited the level of mature miR-122 might by binding to pri-miR-122.8)Down-regulating of miR-122 level increased proinflammatory factor CCL2 expression.And inflammatory cytokine TGF-? could significantly stimulate the expression of UCA1 in hepatocyte L02 cells.In summary,the present study showed the expression of IncRNA UCA1 was regulated by HBx and involved in HCC progression.This is the first time to report the role of HBx-UCA1/EZH2-p27Kip1 axis in HCC carcinogenesis.Furthermore,these data implied that UCA1 might contribute to "inflammation-carcinoma" transformation.