Human La Protein Inhibitor H11 Regulates HBV Gene Expression Profile Changes And Its Bioinformatics Studies

Part 1 Alteration of micro RNA profiles by a novel inhibitor of human La protein in HBV-transformed human hepatoma cellsAims: La protein functions as a RNA molecular chaperone to stabilize HBV RNA structure and promote HBV replication.HBSC11(H11)is a pyrazolo and pyridine compound with the chemical name of methyl pyrazolo [1,5-a] pyridine-2-carboxylate.It was previously identified as a novel inhibitor of human La protein with antihepatitis B virus(HBV)activity.However,the underlying mechanism(s)of HBV inhibition by H11 remains unclear.This study aimed to examine the regulation of miRNA by H11 in HBV-transformed human hepatoma Hep G2.2.15 cells.Methods: HBV gene stably transformed Hep G2.2.15 cells were treated with or without H11.Total RNA was extracted and miRNA array analyses were performed with Agilent human miRNA microarray.Differentially expressed miRNAs were validated by q PCR.Target genes of the differentially expressed miRNAs were predicted and investigated by bioinformatics techniques,including GO and KEGG pathway analyses.Results: The expression of three miRNAs(miR-3912-5p,miR-6793-5p,and miR-7159-5p)was significantly upregulated in Hep G2.2.15 cells treated with H11 as identified by miRNA array analysis and confirmed by qRT-PCR.A total of 3184 target genes were obtained via miRNA target prediction.Bioinformatics study revealed that these target genes of the three miRNAs are mainly involved in the regulation of nucleic acid-templated transcription,negative regulation of gene expression,nucleic acid binding transcription factor activity,as well as regulation of phosphorylation..Further KEGG analysis suggested that the target genes are enriched in certain regulatory pathways of HBV infection and-associated diseases progression,such as TGF-?,wnt,and p53 signaling pathways.Conclusion: Our study demonstrates the involvement ofmiR-3912-5p,miR-6793-5p,and miR-7159-5p and the potential modulation of specific pathways(TGF-?,Wnt,and p53 signaling)in H11-mediated inhibition of HBV replication.This study provides insight into the molecular mechanism of the action of H11 against HBV infection.Part 2:LncRNA expression profiles in HBV-transformed human hepatocellular carcinoma cells treated with a novel inhibitor of human La proteinAims:We previously identified a novel inhibitor of La protein,H11,inhibited HBV replication by inhibiting the interaction between La protein with HBV RNA.However,whether other cellular factors are involved in this process is unclear.The present study aimed to investigate the underlying mechanism of H11 mediated HBV inhibition.Methods: A lnc RNA microarray was performed in Hep G2.2.15 cells with or without H11 treatment.The differentially expression profile of lnc RNAs and m RNAs were identified and further analyzed using GO and pathway analysis.An lnc RNA-m RNA co-expression network was constructed to investigate the associations of differentially expressed lnc RNAs with m RNAs.There were 96 nodes in the network,including 51 lnc RNA nodes and 45 m RNA nodes.Of the 585 lnc RNA-lnc RNA,m RNA-m RNA and lnc RNA-m RNA pairs identified by expression,322 pairs correlated with each other positively and 263 pairs correlated with each other negatively.Results: The microarray data showed 61 lnc RNAs were up-regulated and 74 lnc RNAs were down-regulated,as well as 43 m RNAs were up-regulated and 44 m RNAs were down-regulated in H11 treated group which were consistent with the result of qRT-PCR Validation.Using GO analysis,the differentially expressed lnc RNAs were found to be involved in posttranscriptional gene silencing by RNA and regulation of viral genome replication biological process as well as Jak-STAT signaling pathway and apoptosis pathways.The differentially expressed m RNAs enriched in negative regulation of wnt signaling pathway and negative regulation of growth GO terms.Pathways analysis indicated they were related with regulation of nuclear beta catenin signaling and target gene transcription,direct p53 effectors,PPAR signaling pathway and peroxisome pathways.Conclusion: qRT-PCR results confirmed the results of microarray data and indicated that H11 inhibited HBV replication through regulating wnt/?-catenin and PPAR signaling pathwayPart 3:Expression of the La in Hepatocellular Carcinoma Cells and TissueAims: The La protein has been found to be a RNA binding protein which was related to different aspects of RNA metabolism and plays an important role in human tumor diseases.However,the precise role of La in hepatocellular carcinoma(HCC)has not been fully elucidated.The aim of this study was to investigate the expression level of La proteins in hepatocellular carcinoma cells and tissues.Methods: La expression was examined in 8 HCC cell lines using real-time PCR and western blot analysis(WB).La expression was also studied in 30 paired and 16 paired HCC lesions and the adjacent non-cancerous tissue samples.The expression of La in two independent HCC datasets of the oncomine database was also analyzed.Results: The results showed that La was differentially expressed in the various HCC cell lines at both the m RNA and protein level.Among them,the Hep G2,Hep G2.2.15,Huh7 and SMMC7721 cell lines expressed high levels of La,but LM3 and MHCC-97 L cells had low La expression levels.La m RNA was down-regulated in 70%(21/30)of CRC tissues compared with the matched adjacent normal tissues(P < 0.01),which was consistent with the result from the oncomine database.La protein expression was significantly higher in tumor tissues compared with that in the paired adjacent normal tissues.Conclusion: These results confirmed that La expression increased both at the transcriptional and translational levels in HCC cell and tissues.La protein may represent a promising therapeutic target for hepatocellular carcinoma treatment.