Transcriptional Regulation And Functional Mechanism Of Long Non-coding RNA DRHC In Hepatocellularcarcinoma

BackgroundHepatocellular carcinoma(HCC)is one of the most prevalent cancer in alimentary system.HCC progresses rapidly without typical symptom.Due to its high recurrence and metastatic rates,the prognosis is very dismal.In recent years,although the comprehensive treatment strategy based on surgery has made great progress,the 5-year overall survival rate is still less than 30%.To develop effective and specific targets for intervention and improve the prognosis,the mechanism underlying HCC development need to be elucidated.Long non-coding RNA(lncRNA)is a class of non-coding RNA with a length of more than 200 nucleotides without protein coding capacity in general.LncRNA,previously thought to be the "noise" of gene transcription,is a by-product of the RNA polymerase ? transcription.Recent studies found that lncRNA participated in cell proliferation,cell cycle,cell differentiation,apoptosis and epithelial-mesenchymal transition(EMT)regulation.Currently,only a small number of lncRNAs such as lnCRNA HULG,IncRNA HOTTIP,lncRNA DANCR have been investigated.The roles and mechanisms of the majority of the lncRNAs remain unknown in HCC.AimsThis study aims to find differently expressed lncRNAs in the HCC tissues and paired adjacent normal tissues using IncRNA array.By exploring the expression pattern,transcriptional regulation and mechanisms of the differentially expressed IncRNA,we hope to identify new effective therapeutic targets for HCC.Methods1.We performed IncRNA array analysis in 5 HCC tissues and paired adjacent normal tissues(without vascular invasion,single tumor nodule,diameter less than 5cm,without downstaging treatment like TACE).2.The differentially expressed lncRNAs were validated in another cohort of HCC patients(n=112)receiving partial hepatectomy by qRT-PCR.The significantly downregulated IncRNA CTC-505O3(lncRNA downregulated in HCC,lncRNA DRHC)was analyzed with clinical parameters.To reveal its potential of acting as a diagnostic and prognostic serum marker,the expression level of IncRNA DRHC was also detected in serum.3.We studied the effect of histone modification,DNA methylation on transcription of IncRNA DRHC using specific inhibitors;Based on JASPAR database,the transcription factors of IncRNA DRHC were predicted and further validated by luciferase reporter assay.4.IncRNA DRHC was overexpressed in HCC cell lines Huh-7and SK-Hep-1 by transfection of plasmid or lentivirus.The effects of lncRNA DRHC on HCC cell lines were evaluated by cell counting kit-8(CCK-8)experiment,cell proliferation detection,cell apoptosis detection,cell migration assay,cell invasion assay,xenograft tumor model.5.We used mRNA array to reveal the pathways related to lncRNA DRHC and validated by Western Blot;Specific inhibitor was used to perform the rescue experiment.6.Proteins bind to lncRNA DRHC were revealed by RNA pulldown and mass spectrum.The potential proteins were validated by Western Blot.We then knocked down the transcription factor modulated by the protein bound to lncRNA DRHC and observed the changes in related pathway.We also predicted the miRNA potentially sponged by lncRNA DRHC and validated by qRT-PCR.Results1.Compared with adjacent normal tissues,97 lncRNAs were upregulated and 100 lncRNAs were downregulated.LncRNA DRHC was verified to be downregulated in a validation cohort.The expression level of lncRNA DRHC was associated with tumor diameter,while not correlated with tumor number,differentiation or AFP level in the serum.Patients with higher lncRNA DRHC expression level in HCC showed improved prognosis.It was undetectable in the serum.2.Histone deacetylase inhibitor promoted lncRNA DRHC expression at transcriptional level;Histone methyltransferase PRMT5 and EZH2,DNA methylation did not participate in transcriptional regulation of lncRNA DRHC;Dual luciferase reporter assay showed that cells transfected with EGR1,FOS or GATA3 did not increase luciferase reporter expression.3.LncRNA DRHC inhibited proliferation,migration,invasion and EMT of HCC cell lines in vitro.It also inhibited tumorigenicity in vivo.KEGG analysis based on mRNA array indicated MAPK signaling was most affected.As shown by Western Blot,lncRNA DRHC inhibited the phosphorylation level of MEK1/2 and ERK1/2.The difference in proliferation,migration,invasion and EMT resulted from IncRNA DRHC overexpression was attenuated by MEK1/2 specific inhibitor Trametinib.4.By RNA pulldown assay and mass spectrum,a list of proteins potentially bound to lncRNA DRHC were identified.Among them,MYBBP1A was validated by Western Blot.Knockdown of c-Myb,which was modulated by MYBBP1A,diminished the difference in ERK1/2 phosphorylation.By miRDB,31 miRNAs were predicted to be sponged by IncRNA DRHC.However,none of them showed higher concentration in the precipitation in the RNA pulldown assay using the sense probe.Conclusions1.IncRNA DRHC is downregulated in HCC compared with the adjacent normal tissues.Its expression level is correlated with tumor diameter and the prognosis of the patients receiving partial hepatectomy,so it may serve as a prognostic marker for those patients;2.Downregulation of IncRNA DRHC in HCC is partially resulted from increase in histone deacetylation;3.IncRNA DRHC bind with MYBBP1A,which regulates MEK/ERK signaling through modulating transcription factor c-Myb,and finally inhibites proliferation,migration,invasion and EMT of HCC cells.