XIAP 3'UTR Serves As A CeRNA For HMGA2 To Activate Hepatocellular Carcinoma Progression By Arresting Endogenous Let-7a-5p

Background Hepatocellular carcinoma(HCC)is the leading cause of deaths in cirrhotic patients,and the global incidence is still growing.The prognosis remains extremely poor due to late diagnosis and ineffective treatment.Thus,the treatment of hepatocellular carcinoma is still a major problem in medicine.X-linked inhibitor of apoptosis protein(XIAP)is a member of the inhibitor of apoptosis family of proteins(IAP).XIAP has been demonstrated to be powerfully anti-apoptotic by directly binding to caspases and inhibiting their activity,and then take part in the development and progression of human HCC.High-mobility group AT-hook 2(HMGA2)serves as a structural transcription factor that regulates gene transcription.HMGA2 protein is present at high levels in human cancer,but is greatly decreased in various normal adult human tissues.A growing body of literature has demonstrated that micro RNAs(mi RNAs)lead to the degradation or translational repression of messenger RNAs(m RNAs)by binding to the non-coding region of m RNA at the 3'-untranslated region(3'UTR).The interaction between m RNA and mi RNA plays an important role in the occurrence and progression of cancer,and also plays a guiding role in the prognosis of the disease.Aim: To explore the effects of XIAP 3'UTR on cell proliferation,migration and invasion capacity in HCC cell lines;To observe whether the effect of XIAP 3'UTR expression in HCC cell lines on the progression is directly mediated by HMGA2;To analyze the mechanism that XIAP 3'UTR influence biological behaviour of HCC cell lines by let-7a-5p.Methods 1)We selected two human HCC cell lines(low aggressive: Hep G2;high aggressive: SMMC-7721);2)The expression levels of XIAP and HMGA2 in XIAP 3'UTR overexpressed HCC cell lines were detected by real-time quantitative PCR(q PCR)and western blot assays;3)The common target mi RNA of XIAP and HMGA2 was discussed;4)After overexpressed XIAP 3'UTR in two HCC cell lines,cell function assays were performed by proliferation assay,cell viability assay,colony formation assay,migration and invasion assay,and we observed the changes of cells' proliferation,migration and invasion capacity;5)HMGA2 si RNA was introduced in functional studies to determine whether the effects of XIAP 3'UTR expression in HCC cell lines on the progression were directly mediated by HMGA2;6)Malignant biological properties were observed by cell function assays after HCC cells with forced expression of XIAP 3'UTR were transfected with let-7a-5p mimics.Result 1)q PCR showed that XIAP 3'UTR transfected SMMC-7721 and Hep G2 cells were successfully established(both p<0.05);2)q PCR assays and western-blot assays showed up-regulation of XIAP and HMGA2 at RNA and protein expression levels in XIAP 3'UTR transfected SMMC-7721 and Hep G2 cells(both p<0.05);3)Luciferase reporter gene assay demonstrated that let-7a-5p could work as a common binding site of XIAP 3'UTR and HMGA2 3'UTR.The expression of let-7a-5p was down-regulated by q PCR analysis of total RNA from XIAP 3'UTR and negative control cells(p<0.05);4)Cells biological behaviors analysis showed that overexpression of XIAP 3'UTR promoted proliferation,viability,colony formation,migration and invasion in SMMC-7721 and Hep G2 cells(both p<0.05);5)HMGA2 si RNA was transiently transfected in cells with forced expression of XIAP 3'UTR and the efficacy of HMGA2 depletion were verified using western blot assays;6)Knockdown of HMGA2 repressed malignant potential of XIAP 3'UTR stable transfected HCC cell lines(SMMC-7721-XIAP 3'UTR and Hep G2-XIAP 3'UTR)relative to the control cells by cell function assays(both p<0.05);7)Expression of let-7a-5p down-regulated the expression of XIAP and HMGA2 protein in SMMC-7721-XIAP 3'UTR cells or Hep G2-XIAP 3'UTR cells by western blot assay;8)Overexpression of let-7a-5p down-regulated proliferation,migration and invasion capacity in XIAP 3'UTR stable transfected HCC cell lines compared with negative control(both p<0.05).Conclusion XIAP 3'UTR serves as a competitive endogenous RNA(ce RNA)for HMGA2 to activate HCC progression by arresting endogenous let-7a-5p.