Study On The Function And Mechanism Of Oncofetal LncRNA Ptn-dt In The Mouse Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the most common malignancies in the world.Although the treatment and the treatment strategies are continually improved,there is still a low 5-year survival rate.One of the most important reasons is the poor understanding of the specific mechanism of HCC,leading to the lack of effective means for early diagnosis of HCC patients.A large number of patients in early stage of liver cancer can not be diagnosed timely,therefore they lose the best opportunity for treatment.So,it is an urgent task to strengthen the study of the molecular mechanism of HCC in the process of development and search for more effective marker molecules for HCC diagnosis.Long noncoding RNAs(lncRNAs)are a kind of RNA molecules with the length more than 200 nucleotides.Recent researches show that lncRNAs likely play crucial roles in the development of HCC.In 1970 s,some scientists had found cancer clues from embryonic development,for example,alpha-fetoprotein which is specific high expression in embryonic stage is currently the most effective molecular marker for diagnosis and prognosis of HCC.Therefore,we begun with the oncofetal traits of lncRNAs to search for some valuable lncRNA molecules by screening and functional analysis of oncofetal lncRNA.In this research,we found a lot of differentially expressed lncRNAs by genome-wide screening using microarrays in fetal liver tissues,adult liver tissues and liver cancer tissues of mice.There is a kind of lncRNAs moleculars called oncofetal lncRNAs,which normally expressed in fetal liver and adult liver,and silenced in adult liver.We have identified a new oncofetal lncRNA,named as lncRNA Ptn-dt(Pleiotrophin downstream transcript)because of located in the downstream transcript of Pleiotrophin(Ptn).We defined the oncofetal trait of Ptn-dt by real-time Polymerase Chain Reaction(PCR),Northern blot analysis and RNA fluorescence in situ hybridization(FISH).Meanwile,the results of Northern blot suggested that Ptn-dt existed independently in the transcriptome of mice,and Ptn-dt was mainly distributed in the cytoplasm by RNA FISH.After that,we determined the full length of Ptn-dt by 5’,3 ’ RACE analysis,and then established stable cell lines of Ptn-dt over-expression and knock-down Ptn-dt in Hepa1-6 cells.We found that Ptn-dt could promote the proliferation of mouse liver cancer cells by a series of experiments in vitro and in vivo.On the basis of making clear that Ptn-dt could promote the proliferation of mouse hepatoma cells,we also explored the mechanism of action of Ptn-dt preliminarily.RNA pull-down assays and RNA Immunoprecipitation(RIP)indicated that Ptn-dt related to HuR protein,which is a RNA binding protein that could stabilize many RNAs and regulate their transcription.Meanwile,RIP assay showed that there was a correlation between miR-96 and HuR protein.However,our results showed that over-expression of Ptn-dt could induce the expression of miR-96.Some studies had reported that the fungal natural product azaphilone-9 binded to HuR and inhibited HuR-RNA interaction in vitro.Therefore,we hypothesized that the binding between Ptn-dt and HuR effected the binding of miR-96 to HuR,reducing the stability and expression of miR-96.Among the numerous target molecules of mi R-96,we found that Alk(Anaplastic lymphoma kinase)was a receptor of Ptn.Besides,studies had reported that Ptn could activate Alk,and Ptn promoted tumor growth and vascular abnormalization via Alk signaling.Our study showed that over-expression of Ptn-dt could promote the expression of Alk in both mRNA and protein levels.Therefore,based on the above findings and research reports,we preliminarily concluded that the binding between Ptn-dt and HuR effected the binding of miR-96 to HuR,so that the stability and expression of miR-96 was reduced,and then weakened the effect of miR-96 on the post-transcriptional suppressor of Alk,finally achieved its own role in promoting proliferation.That is,Ptn-dt might exerted its function of promoting proliferation through managing HuR/mi R-96/Alk pathway and pleiotrophin-Alk axis.Generally speaking,our study elaborated the function and possible molecular mechanism of Ptn-dt in tumors for the first time.Meanwile,we reported a new model of the mechanism of lncRNAs,that is,lncRNA played its role by regulate the reception of the adjacent coding gene.All in all,this study improve the understanding of the gene regulatory network of liver cancer,and provide some references for the study of the molecular mechanism of HCC.Therefore,our study complemented the function and mechanism of lncRNAs in HCC,and provided some reference value for the study of the molecular mechanism of HCC.