Roles And Regulation Mechanisms Of Long Non-coding RNA:00441 In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the common malignant tumors in the world which is characterized by aggressiveness,invasiveness,especially intrahepatically,and frequent recurrence after resection.According to recent statistics,there are more than 780000 new patients which were diagnosed as HCC and 750000 patients died from this disease.In China,HCC patients account for about half of all global HCC patients,so HCC is considered as a severe threat for people's health and life.At present,tumor resection remains the most effective measures to cure HCC.But owing to the high growth speed and patient at early stage without apparent symptom,when the patients were diagnosed as HCC,they tended to miss the best remedy period.Therefore,exploring the specific mechanisms of HCC initiation and progression are very important for HCC diagnosed at early stage and improving prognosis.With the development of High-throughput sequencing,more and more non-coding RNA which were considered as junk gene before,and their biological function have been fully elaborated.Long non-coding RNA,as one of non-coding RNA,is a kind of molecule that have no ability to code and whose transcript length is more than 200nt.LncRNA has been demonstrated to involve in some important life process including genomic imprinting,chromatin modification,transcription activation and protein function regulation.Besides,lncRNAs also play an important role in tumor initiation and progression.In human genome,nearly 11%genes are arranged as bidirectional gene pair in the pattern of head to head,divergent manner and controlled by bidirectional promoters including IncRNAs and protein-coding genes.Retinoblastoma 1(RB1)gene is the firstly discovered cancer suppressor gene and its cancer suppression functions were correlated with cell cycle,cell differentiation,senility and apoptosis.Based on a large amount of researches before,this study focused on lnc00441 which share a promoter with RB1,and explored whether lnc00441 could affect HCC progression via regulating RB1 expression and the specific mechanism.We deeply studied the functions and mechanism of lnc00441 in the process of HCC progression and gene modification via regulating RBI expression in HCC cell lines level,in vivo and clinic HCC tissues level,through adopting some methods such as high-throughput sequencing,bioinformation analysis and classical molecular biology experiment.We found that:1.Long non-coding RNA 00441 is highly expressed in HCC tissues and has a close expression relationship with RBI in space in HCC lines.Firstly,compared to their corresponding adjacent tissues and 16 normal liver tissues using qRT-PCR,we found that the expression level of lnc00441 is highly expressed in HCC tissues,but RBI expression level were inverse.Pearson correlation analysis showed a negative correlation ship between RB1 and lnc00441 expression.Besides,In situ Hybridization(ISH)assay revealed that the expression of lnc00441 and RB1 in space have a close relationship in HCC lines.Furthermore,clinic data about the tissues showed that the patients with high lnc00441 expression were more easily to suffer larger tumor.2.Inc00441can strengthen the proliferation ability of HCCAfter treated with lentivirus expressing lnc00441 or inducing low expression of lnc00441,HCC lines can grow faster compared to control group by inducing G1?S transition in cell cycle.Meanwhile,in vivo,the HCC lines expressing high lnc00441 had some results in nude mouse model.3.The specific mechanisms of high expression of lnc00441 in HCC and regulating the expression of RB1.Based on the combination of bioinformatics analysis and papers before,we have known that lnc00441 and RB1 share a promoter but show inverse expression tendency.So,we performed an RNA immunoprecipitation(RIP)assay and found that lnc00441 can contribute to high methylation of CpG islands in the promoter region of RBI gene via recruiting DNMT3A,which can be suppressed by methyltransferase inhibitor.Meanwhile,we also found that high rate of H3K27 acetylation in the promoter region of lnc00441 from HCC expressing high level of lnc00441 and it can be up-regulated by transcription factor:TCF-4.In conclusion,we studied the roles and molecular mechanisms of functional long non-coding RNA00441 in HCC initiation and development.Our researches provide new views and landscapes to understand the complex signaling network in HCC,and potential new strategies for the early prognosis and therapy of HCC.