SNHG6 Maintains The Genomic Hypomethylation Of HCC Through The MiR-1297-mediated Feedback Loops

Part 1.Effects of SNHG6 on the methylation level and profile in hepatocellular carcinomaObjective: In this section,we intended to elucidate the influence of long non-coding RNA(lnc RNA)-SNHG6 on the methylation level in hepatocellular carcinoma(HCC)tissue and hepatoma cells,and to detect the methylation profile impacted by alternative expressions of SNHG6.In addition,we initially investigated the mechanism of this methylation variation.Methods: In HCC tissues,paired adjacent tissues and hepatoma cell lines,reverse transcription-fluorescence quantitative polymerase chain reaction(RT-qPCR),enzyme-linked immunosorbent assay(ELISA),and ultra-performance liquid chromatography(UPLC)were used to detect the expression of SNHG6,5-methylcytosine(5mC)and S-adenosylmethionine(SAMe)respectively,so that we could analyze the association among them by utilizing bivariate correlation analysis.Subsequently,SNHG6 was overexpressed or knocked down in HCC cell lines and the level of 5mC was detected with ELISA.Then we applied UPLC to measure the changes in the concentration of SAMe and S-adenosylhomocysteine(SAH),followed by observing the aberrant methylation profile in hepatoma cell lines by Illumina Methylation 850 K sequencing.Results: SNHG6 expression was elevated in HCC tissues,while 5mC and SAMe showed lower levels.In HCC tissues,adjacent tissues,and hepatoma cell lines,we recognized that SNHG6 was negatively correlated with 5mC and SAMe,whereas 5mC was positively correlated with SAMe.Moreover,it was suggested that SNHG6 negatively regulated 5mC levels and SAMe concentrations,as well as the SAMe/SAH ratio.Methylation sequencing showed that changes of SNHG6 level altered the genomic methylation profile along with methylation modifications of multiple sites.Furthermore,SNHG6 seemed to regulate the promoter region methylation of multiple HCC-related genes disclosed by multiple hypothesis tests.Conclusion: long non-coding RNA-SNHG6 represses methylation levels of hepatoma genome and changes its methylation profile through downregulating SAMe concentrations.Part 2.Molecular mechanism of SNHG6 down-regulating intracellular S-adenosylmethionine concentrationObjective: To identify the molecular mechanisms underlying the regulation of SNHG6 on SAMe concentrations in HCC via series experiments in vitro.Methods: We overexpressed or/and knocked down SNHG6 in hepatoma cell lines and detected the expression of methionine adenosyltransferase(MAT),the key enzyme regulating SAMe,by utilizing qPCR and western blotting.Then we resorted to bioinformatics predictions and noticed that miR-1297 and Fused in Sarcoma(FUS)protein may be the intermediate regulatory molecules.Therefore,fluorescence in situ hybridization(FISH)was applied to observe the subcellular distribution of SNHG6 and miR-1297,along with empty-vector RNA-RNA co-precipitation analysis(MS2-RIP)operated to verify the direct binding of miR-1297 to SNHG6.Rescue experiments were implemented to confirm the adsorption of SNHG6 on miR-1297 in parallel.The downregulation of MAT2A mRNA and FUS mRNA by miR-1297 was explored by dual luciferase reporter gene system and RNA decay assay.Lastly,we carried out immunohistochemistry and cytochemical staining(IHC/ICC)to illustrate the subcellular localization of FUS,RNA immunoprecipitation analysis(RIP)to verify the direct binding of FUS to MAT1A mRNA,qPCR and western blotting to detect the expression of MAT1A in the condition of overexpressing or knocking down FUS in hepatoma cells,and FISH to visually observe the subcellular distribution of MAT1A mRNA influenced by FUS overexpression.Results: According to the qPCR and western blotting results mentioned above,MAT2A mRNA and protein levels were upregulated by SNHG6.Meanwhile,SNHG6 could also downregulate MAT1A protein expression,but had no significant effect on its mRNA level.In terms of the regulation of SNHG6 on MAT2A,luciferase reporter system and RNA decay assay revealed that miR-1297 bound to the 3’ untranslated region(UTR)of MAT2A and inhibited mRNA levels of which to accelerate its degradation.MS2-RIP and rescue experiments showed that SNHG6 released the inhibition of MAT2A 3’ UTR by adsorbing miR-1297 and consequently upregulated MAT2A expression.Regarding the regulation of MAT1A,similarly to MAT2A,SNHG6 could absorb miR-1297 to promote the expression of nuclear protein FUS which directly bound to MAT1A mRNA according to the results of RIP.FISH visualization showed that some MAT1A mRNAs were trapped in the nucleus with FUS overexpression,which meant SNHG6 regulated MAT1A protein expression through FUS-mediated nuclear-cytoplasmic shuttling.Conclusion: SNHG6 upregulates MAT2A and downregulates MAT1A expression through two pathways mediated by miR-1297,thereby suppressing intracellular SAMe concentration.miR-1297 adsorption.Part 3.SNHG6 activates two SAMe-dependent positive feedback pathways throughObjective: To confirm whether both the two regulatory pathways mentioned above play important roles,and if expressions of the key molecules,namely FUS and MAT2A,were SAMe-dependent.Meantime,we planned to verify that the regulatory pathways activated by SNHG6,which maintained low SAMe concentrations worked as 2 positive feedback loops in order to cause genomic hypomethylation.Methods: In hepatoma cells,after MAT1A overexpression or/and MAT2A knockdown,intracellular 5mC levels were measured with ELISA.Subsequently,we treated hepatoma cells with SAMe or SAH,and detected the expressions of SNHG6,miR-1297,FUS,MAT1A,and MAT2A utilizing qPCR and western blotting.Furthermore,methylated DNA immunoprecipitation(MeDIP)was applied to evaluate the methylation of MAT2A and FUS promoter,which could be determined by SAMe and SAH concentrations.Results: Overexpression of MAT1A or/and knocking down MAT2A in hepatoma cells both increased the 5mC content with a synergistic effect.SAMe or SAH treatments revealed that the mRNA levels of MAT2A and FUS were regulated by the SAMe/SAH ratio,while the expressions of other genes were not significantly influenced.Lastly,it was disclosed by MeDIP that SAMe facilitated the methylation of MAT2A and FUS promoter,which meant they were SAMe-dependent expressions.MAT2A and FUS would be further improved when the level of intracellular SAMe was decreased,thereby forming two positive feedback loops in these regulations.Conclusion: MAT2A and FUS,two key molecules in pathways determining SAMe concentration were also regulated by SAMe in return.This may indicate two positive feedback pathways activated by SNHG6 absorbing miR-1297 existing in order to induce the genomic hypomethylation.