Study On The Mechanism Of LncRNA RNU12 Inhibiting The Apoptosis Of Human Hepatocellular Carcinoma Cells Induced By Recombinant Human TRAIL Protein

ObjectiveTo study the changes in the long non-coding RNA(lncRNAs)expression profiles between sub-lethal heat treatment hepatocellular carcinoma(HCC)cells and normal liver cells,and carry out bioinformatics analysis preliminarily.We investigate the molecular mechanism of inhibiting the apoptosis of human hepatocellular carcinoma cells induced by recombinant human TRAIL protein at 46 ~ 50 ? in "Dual effect" phenomenon of temperature.MethodsTo establish a sub-lethal heat treatment model of human hepatocellular carcinoma cell line SMMC-7721,in order to simulate the microenvironment of the transition zone of insufficient radiofrequency ablation of HCC.Using the latest Agilent Human lncRNA+m RNA Array V4.0 chip to detect lnc RNA/mRNA expression level.The original data were standardized.GO and KEGG analysis of differentially expressed m RNAs were carried out.And the co-expression network of differentially expressed lnc RNA/m RNA was constructed.20 lncRNAs(FC(abs)?2 and p<0.05)were selected for further validated by using q RT-PCR in three HCC cell lines(SMMC-7721?MHCC97-H and Hep G2).The significant differentially expressed ENST00000602478.1(lnc RNA RNU12)at 50 ? was finally picked as the focus of this study.The effects of recombinant human TRAIL protein on the apoptosis of MHCC97-H cells were detected by using Annexin-V/PI fluorescent dyes under the conditions of four different temperatures(37 ??43 ??46 ??50 ?).The expression of lncRNA RNU12 in MHCC97-H cells was inhibited by lentivirus interference technique,and the expression of mRNA and protein of HSP72 were detected by qRT-PCR and Western blot before and after transfection with lentiviral vector.Annexin-V/PI staining was used to detect the effect of recombinant human TRAIL protein on the apoptosis rate of MHCC97-H cells before and after transfection.Finally,fluorescence in situ hybridization was used to detect the intracellular localization of lnc RNA RNU12.ResultsCapital Bio lncRNA+m RNA microarray chip contains a total of 41000 lnc RNAs and 34000 m RNAs.1475 lncRNAs and 1015 m RNAs were found up-regulated,similarly,775 lncRNAs and 3374 m RNAs were found down-regulated(FC(abs)?2 and p<0.05).ENST00000602478.1(lnc RNA RNU12)remained stable up regulation in three HCC cell lines(SMMC-7721 ? MHCC97-H and Hep G2)in sub-lethal heat treatment model.ENST00000602478.1(lncRNA RNU12)were increased by 37.00 times,24.60 times,84.31 times,respectively.Among the differentially expressed m RNA,2314 were involved in the biological process,2252 involved in molecular function,and 2443 involved in the cell component.Pathway analysis showed that mRNA regulated by differential expression of 20 genes in the pathway,the highest enrichment is oxidative phosphorylation,a total of 44 genes were involved.43 ? promoted the apoptosis of HCC cells induced by recombinant human TRAIL protein,which were inhibited at a temperature of 46 ?~ 50 ?.The expression level of HSP72 m RNA in the four groups were significantly decreased after transfection of lentiviral vector(P<0.01).Similarly,the expression level of HSP72 protein were significantly reduced at 37 ??46 ??50 ? groups(P<0.05).Although there was no significant difference in 43 ? group,the specific number was still lower in this group.Silencing lncRNA RNU12 can significantly increase the apoptosis rate of HCC cells induced by TRAIL protein at 46 ?.RNA-FISH showed that lnc RNA RNU12 was expressed in the nucleus and cytoplasm of MHCC97-H at 37 ?,while the expression was significantly increased in the nucleus at 50 ?.Conclusion(1)LncRNA+m RNA microarray chip was used for the first time to reveal the differential expression of lncRNAs in sub-lethal heat treated HCC cells.(2)The expression of lnc RNA RNU12 was significantly increased after the stimulation of sub-lethal heat treatment,which may be involved in the development of HCC.(3)"Dual effect" phenomenon of temperature also exists in HCC cells.(4)LncRNA RNU12 can upregulate HSP72 expression at the transcriptional level at 46~50 ?.(5)Increased expression of HSP72 could inhibit the apoptosis of HCC cells induced by recombinant human TRAIL protein.