| In recent years, ncRNA which is found in large quantities and different withknown mRNA, rRNA and tRNA, are not directly involved in protein synthesis.NcRNA are widely distributed in a variety of organisms and contains many categories,including miRNA (microRNA), piRNA(Piwi-RNA), gRNA and lncRNA (longnoncoding RNA). LncRNA is a very important class of noncoding RNA molecule,which is longer than200nt, with a number of investigations show that they play animportant role in cell physiological activities and participate in the pathogenesis of avariety of tumors and other diseases.First, we studied the relationship between aberrenttly expressed lncRNAs andhepatocellular carcinoma (HCC). Using high-throughput sequencing technology andreal-time quantitative PCR to detect expression of lncRNAs in HCC samples, wefound that GAS5, NCRNA00107, NCRNA00115, SNHG6and MEG3weredifferently expressed in HCC compared with adjacent normal tissues. Construction ofthe eukayotic expression vector of these lncRNAs and screening high expression ofstable cell lines, along with qRT-PCR, Western Blot, RNA-pulldown, RIP andmicroarray, we demonstrated that MEG3was associated with p53to form complexesto play regulatory roles. Taken together, our data suggest that MEG3non-coding RNAfunctions as a tumor suppressor, whose action is mediated by interacting with p53protein to active p53target genes.This study also focused on the other differentially expressed lncRNAs in HCC.Our study determined the genome-wide lncRNAs and mRNAs expression profile inHCC by microarray analysis and constructed lncRNA-mRNA co-expression network.The results showed that clusters of lncRNAs were deregulated in HCC, whichindicated that these differentially expressed lncRNAs may exert a partial or importantrole in tumor development. The main progresses are listed:1. Some lncRNAs were aberrantly expressed in HCC compared to adjacent tissues.Using high-throughput sequencing and qRT-PCR, we found that GAS5,NCRNA00107, NCRNA00115and SNHG6were up-regulated, while MEG3wasdown-regulated in most of HCC samples compared with adjacent normal tissues.2. MEG3inhibites cell proliferation. Overexpression of MEG3resulted in a decreasein HepG2cell growth rate according to the results of growth curve and colonyformation. Meanwhile, MEG3introduces cell apoptosis but has no effect on cellcycle.3. MEG3could promote p53-mediated transcriptional activity and regulate partialtarget genes of p53. MEG3overexpression leads to increased levels of p53proteinand promotes the transcriptional activity of p53.4. Association between MEG3and p53. The in vivo RIP and in vitro RNA pull-downresults demonstrate a specific association between p53and MEG3. DNA bindingdomain (DBD) of p53is responsible for the direct association with MEG3. Fulllength of MEG3and intact structure are critical for it to activate p53-mediatedtransactivation.5. Expression profile of lncRNAs and mRNAs in HCC. By microarray analysis,214lncRNAs and338mRNAs were found to be significantly differentially expressedin three HCC samples compared with their adjacent NT samples. Using DAVIDFunctional Annotation Chart, we analyzed the enrichment of these338differentially regulated mRNAs. The result showed that the most significantfunctional groups consisted of metabolic process, cellular component organization,cell cycle and response to chemical stimulus.6. To verify the microarray data, we randomly selected fifty differentially expressedlncRNAs including twenty up-regulated lncRNAs and thirty down-regulated onesand validated their expression levels by Quantitative RT-PCR (qRT-PCR) in threesets of HCC tissues. The result showed forty of fifty lncRNAs matched themicroarray data. Eight lncRNAs from these forty have also been evaluated in nineteen pairs of HCC and adjacent NT samples using qPCR The qRT-PCR resultsare basically identical with the microarray. Additionally, the change trend oflncRNAs and their nearby coding genes is basically identical by qPCR analysis ofa selected number of differentially expressed lncRNAs and their nearby genes.7. LncRNA-mRNA co-expression network. Our study determined the genome-widelncRNAs and mRNAs expression profile in HCC by microarray analysis. In orderto determine the correlation between differentially expressed lncRNA and mRNAin HCC, we constructed a lncRNA-mRNA co-expression network.In conclusion, this paper reveals MEG3which is aberrantly expressed in HCCcould interact with p53, affecting its half-life and transcriptional activity. Therefore,MEG3inhibits cell proliferation and induces apoptosis. Meanwhile, accordingbioinformatics analysis of lncRNA microarray, we found the aberrantly expressedlncRNA in HCC and construted lncRNA-mRNA co-expression network. |
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