Roles Of LncRNA-MEG3 And The Diagnostic Values Of Exosome Derived-lncRNA In Hepatocellular Carcinoma

As one of the most common malignancies in the world,hepatocellular carcinoma(HCC)has a veryhigh morbidity and mortality.It is a major global health challenge that affects almost 500000 people worldwide every year.Despite recent improvements in surgery and chemotherapy,the prognosis for HCC remains grim.Therefore,there is a pressing requirement to identify new prognostic biomarkers and therapeutic targets for HCC.Furthermore,the underlying pathophysiological mechanisms of HCC development remain elusive.In recent years,long non-coding RNAs(lncRNAs),which are defined as transcripts of greater than 200 nucleotides with no or little protein coding function,havedrawnmore andmore attentioninmany researchfields.The long non-coding RNA maternally expressed gene 3(MEG3)with abnormal expressions in severalmalignancies have been documented to be involved in the pathogenesis of malignant tumors including hepatocellular carcinoma,cervical carcinoma,bladder cancer,and gastric cancer.Although some evidences have proved that hypermethylation of differentiallymethylated regions(DMRs)exerts a vital role in the silence of MEG3 in malignancies,the underlying mechanisms inducing the down-regulation of MEG3 expression still remain rarely unclear.In the present study,we found the expression pattern of MEG3 was significantly associated with clinical features of HCC patients.Researchers have demonstrated the inhibition affection of MEG3 in proliferation in vitro,however,no evidence was indicated in vivo.Here,we performed the loss-function and gain-function experiments to further demonstrate the anti-cancer effect of MEG3 as well as the potential up-stream factors of MEG3 and we also confirmed that MEG3 regulates proliferation and apoptosis partially via activation of p53.Subsequently,UHRF1,a new identified oncogene,was found to be negatively correlated with the expression ofMEG3 in HCC tissues.To demonstrate whether UHRF1 could regulate MEG3 expression in HCC,we altered the expression of UHRF1.The data showed the expression of UHRF1 was negatively associated with MEG3 expression.However,UHRF1 was never reported to be a regulatory factor of MEG3.Interestingly,we found that hypermethylation of MEG3 promoter,which may be responsible to the down-regulation of MEG3,has been detected in many malignancies.Besides,UHRF1 was reported acting a crucial role in DNAmethylation by recruiting DNMT1 to hemimethylated DNA during DNA replication.Thus,we hypothesized that UHRF1 may regulate MEG3 expression via modulation ofDNMT1.To confirmthe hypothesis,we altered the expression of UHRF1 in HCC cells,and found the DNMT1 was positively associated with the alterations of UHRF1.Furthermore,we also obtained that MEG3 expression was increased with the inhibition of DNMT1 by RNA interference.Therefore,we illuminated a UHRF1/DNMT1/MEG3/p53 axis signalingmight be involved in HCC progression.Some evidences suggested that inhibiting effect of MEG3 in proliferation still remains in the p53-/-mice therefore,we supposed that the effect of MEG3 inHCCmay not only be attributed to the regulation of p53,but also be ascribed to the other downstream function alteration of MEG3.To further exolore the diagnostic value of lncRNA including lncRNA-MEG3 in the plasma of HCC patients.We performed the high-throughput sequencing to check the lncRNA expression pattern in the plasma and plasma-exosome of HCC patients/control.As we got two lncRNA expression patterns,we performed the venny analysis to found the IncRNA which exerted the similar expression level in the above two IncRNA expression pattern.Then we got the the the candidatel IncRNAs:IncRNA-MALAT1,lnc-RP11-571m6.73 and lnc-RP11-1143G9.4.We performed RT-PCR to check the expression status of the three lncRNAs in clinical samples and confirmed the results of high-throughput sequencing.Then we explored the diagnostic value of the three lncRNAs via using the ROC cure analysis and found the lncRNA-MALAT1,lnc-RP11-571m6.7,and lnc-RP11-1143G9.4 had the correlation between the expression levels and the tumor metasitasis.By using the co-expression analysis,we also found lneRNA-MALAT1 expression was related with the GPC3 expression.To further confirm the potential regulation correlation between GPC3 and MALAT1,via using the information biology prediction technology we found MALAT1 could bind to the amino acid sequence of GPC3.Then we performed the RIP and confirmed the predication results.By using the Glycosylation level measurement experiments,we got the results that upregulation of MALAT1 could upreuglate the glycosylation level of GPC3.Besides,we also found the GPC3-MALAT1 overexpression exosome could promote the differentiation of naive T cells to Treg.By using the protein chip technology we found upregulation of GPC3 and MALAT1 could inhibit the PI3K/AKT/mTOR pathway.By using Western blotting,we confirmed the results of protein chips.In conclusion,we found the lncRNA-MALAT1,lnc-RP11-571m6.7,and lnc-RP11-1143G9.4 had the diagnostic value related with the HCC metasitasis,and the lncRNA-MALAT1 could bind to the amino acid sequence of GPC3 and promoted the naive T cells to Treg via inhibiting the PI3K/AKT/mTOR pathway.