Study On The Expression And Biological Function Of LncRna LINC02561 On Hepatocellular Carcinoma Cells

Primary hepatic carcinoma was reported as the sixth commonest cancer in the world,as well as the third leading explanation for cancer death.China has a high incidence of liver cancer.In 2020,hepatocellular carcinoma(HCC)caused deaths accounted for 47.12% of the world in China,which has seriously affected the lives and health of our people.Primary liver cancer includes hepatocellular carcinoma(HCC)(75%-85%),intrahepatic cholangiocarcinoma(10%-15%)and other rare types.Clinical data have demonstrated that HCC patients who received early-stage diagnosis and treatment might expect to relatively long survival.However,patients are frequently detected at an advanced stage because of HCC’s complex and quick progression.Chemotherapy is significant palliative care choice.Long non-coding RNAs(lncRNAs),a subgroup of non-coding RNAs(ncRNAs),are longer than 200 nucleotides in length and with little protein-coding potential.LncRNAs not only sponge a variety of micro RNAs,but also interact with one or more RNA-binding proteins(RBPs),and participate in the regulation of biological processes such as cell proliferation,apoptosis,migration,and invasion.In recent years,the influence of lncRNA including tumor occurrence,metastasis,prognosis,recurrence and drug resistance has become a research hotspot.Nonsense-mediated mRNA decay(NMD),as an important RNA monitoring mechanism in eukaryotic cells,identify and degrade mRNAs containing premature termination codons(PTC)in the open reading frame to avoid Toxic to cells due to accumulation of truncated protein product.Nonsense transcript regulator 1(Upframeshift suppressor 1,UPF1),as a highly conserved RNA helicase and ATPase,plays a key role in NMD.We first screened out a novel lncRNA LINC02561,which is specifically highly expressed in liver cancer,by bioinformatics methods,and verified its expression level in liver cancer tissues.In HepG2 and Hu H7 liver cancer cells,under the condition of overexpression and knock down of LINC02561,LINC02561 can promote the migration and invasion ability of liver cancer cells.Liver cancer is one of the most obvious cancers regulated by hypoxia.We found that hypoxia induced the expression of LINC02561,predicted the potential hypoxia-inducible factor-1(HIF-1α)binding motif in the promoter region of LINC02561 by bioinformatics methods.We verified that HIF1-α can be enriched in the LINC02561 promoter region,thereby promoting the transcriptional activation of LINC02561.After knocking down of LINC02561,the cell samples were sent to RNA-sequence analysis,and we obtained lots of genes related to LINC02561.Bioinformatics analysis showed that LINC02561 may be related to focal adhesions,RNA transcription and translation.Among them,we selected N-Myc downstream regulated gene(N-myc downstream regulated 1,NDRG1),which is closely related to EMT in liver cancer.Further research found that LINC02561 enhanced the migration and invasion ability of liver cancer cells by regulating the protein NDRG1.Under hypoxic conditions,hypoxia inducible factor-1(HIF-1α)can bind to the promoter of LINC02561,thereby promoting the upregulation of LINC02561 expression in liver cancer cells.By Western blot analysis and fluorescent real-time quantitative PCR,LINC02561 can up-regulate the protein level and mRNA level of NDRG1,and the half-life of NDRG1 mRNA was significantly shortened after interfering with LINC02561.We demonstrated that NDRG1 mRNA can directly bind to LINC02561 by RNA-RNA pull down.Moreover,dual luciferase assays showed that LINC02561 specifically binds to the 5’UTR region of NDRG1 and promotes its translational activity.UPF1 can combine with NDRG1 and degrade NDRG1 mRNA,and the overexpression of LINC02561 significantly inhibited the combination of UPF1 and NDRG1 mRNA.It suggests that LINC02561 can compete with UPF1 for binding to NDRG1 mRNA,preventing its degradation process.Finally,in vivo experiments,we established a mouse model of lung metastasis by injecting the sh LINC02561 lentiviral hepatocellular carcinoma cell line into the tail vein,indicating that down-regulating the expression of LINC02561 can inhibit the metastatic ability of hepatocellular carcinoma cells and reduce the metastatic foci of hepatocellular carcinoma cells in the lungs of mice.Immunohistochemical results showed that low expression of LINC02561 could significantly inhibit the lung metastasis process of liver cancer cells,and highly expressed E-cadherin protein and low expression of NDRG1 protein.Taken together,the HIF1α-LINC02561-UPF1-NDRG1 regulatory axis may be a potential target for HCC.