| Aims:Hepatocellular carcinoma(HCC)has a high morbidity and mortality both in our country and all over the whole world.It is of great importance to determine biomarkers for accurate diagnosis and precise treatment of HCC.p53 is a critical tumor suppressor in hepatocellular carcinoma,and associates with a variety of cellular processes,including cell cycle arrest,apoptosis and cellular senescence.Recently,long noncoding RNAs(lncRNAs)have been reported to be involved with series of malignant biological behaviors.Some of them are directly or indirectly regulated by p53 and contribute to the p53 signaling pathway.Both lincRNA p21 and PANDA are regulated by p53,and transcribed from CDKN1A gene locus.However,the biological roles and molecular mechanisms of these two important p53 associated IncRNAs in HCC are still poorly understood and remain to be further explored.Methods;Firstly,we used the method of qRT-PCR to detect lincRNA-p21 and PANDA in p53 dependent DNA damage model and sorafenib in vitro treatment model and in two cohorts of hepatocellular carcinoma patients following liver resection and liver transplantation.Secondly,we employed siRNA to knock down lincRNA-p21 in liver cancer cells and used CCK-8,flow cytometry and transwell assay to analyze lincRNA-p21's effect on cell proliferation,migration and invasion.Moreover,we used immunofluorescence and western blots to analyze the underlying molecular mechanisms.Thirdly,we employed lentivirus to overexpress PANDA in liver cancer cells and used CCK-8,EdU,xenograft,cell cycle,cellular senescence,western blots and immunohistochemistry to illustrate PANDA's influence on proliferation and senescence of liver cancer.Moreover,by qRT-PCR,we explored PANDA downstream target genes including IL8,Through transfection of IL8 plasmid,we performed the rescue experiments.Last,we performed the RNA pull down assay and the promoter dual-fluorescence reporter assay to explore the molecular mechanism how PANDA regulate IL8.The correlation between PANDA and IL8 in clinical samples were also determined.Results:Firstly,we found that lincRNA-p21 and PANDA were significantly upregulated in p53 dependent DNA damage model,but significantly lower in the liver tumor tissues compared with adjacent peri-tumor tissues.After treated with sorafenib,lincRNA-p21 was induced.Secondly,knockdown of lincRNA-p21 promoted proliferation,cell cycle transition,migration and invasion of BEL-7402.Importantly,lincRNA-p21 regulated cytoskeleton plasticity thus affected epithelial-mesenchymal transition(EMT).Moreover,reduction of lincRNA-p21 evoked nuclear translocation of epidermal growth factor receptor(EGFR).Then,we found that when PANDA was overexpressed in liver cancer cells,it can promote liver cell proliferation in vitro and subcutaneous tumor growth in vivo.Besides,we found that overexpression of PANDA impaired the cellular senescence featured by SABG staining,p16,?-H2AX.The PANDA downstream target genes IL 8,a senescence-assocatied inflammatory factor,was significantly decreased.Overexpression of IL8 was able to rescue the phenotype of PANDA in liver cancer.And IL8 and PANDA had a negative correlation in clinical samples.Finally we found that the PANDA can restrain the transcription activity of IL8 directly,but it does not combine with IL8 protein.Conclusions:LincRNA p21 and PANDA are regulated by p53 in liver cancer,and significantly downregulated in liver cancer.Knockdown of lincRNA-p21 can promote liver cancer cell vitality,cell cycle progress,migration and invasion,which might attribute to lincRNA-p21's effect on cytoskeleton plasticity and subcellular localization of EGFR.Overexpression of PANDA can promote cell proliferation and inhibit cellular senescence,which might result from PANDA's suppression on senescence associated inflammatory factor IL8.p53 associated lincRNA-p21 and PANDA play important roles in liver cancer,and may serve as novel biomarkers for HCC diagnosis and potential targets for HCC treatment. |
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