Regulation And Molecular Mechanism Of Long Non-coding RNA-HOXA11-AS On The Stemness Of Liver Cancer Stem Cells

Background: Hepatocellular carcinoma(HCC)is prone to relapse and metastasis.Cancer stem cells(CSCs)=play an important role in the metastasis and recurrence of tumor cells.Homobox A11 gene(HOXA11)regulates the occurrence and development of HCC by activating the Wnt/β-catenin signaling pathway.Long non-coding RNA(lnc RNA)plays an important role in maintaining the stemness of some tumor stem cells.HOXA11 antisense lnc RNA-HOXA11-AS can induce tumor progression and maintain the stemness of cervical cancer,but its role in HCC stem cells has not been reported.Objective: This study aims to explore the effects of lnc RNA-HOXA11-AS on HCC stem cell stemness and related mechanisms.Methods: 1.The expression levels of lnc RNA-HOXA11-AS in tumor tissues and paracancer tissues of HCC patients were detected by The Cancer Genome Atlas(TCGA)database and qRT-PCR.Kaplan-meier curve was used to analyze the effect of lnc RNA-HOXA11-AS expression level on the prognosis of HCC patients.2.qRT-PCR was used to detect the expression of lnc RNA-HOXA11-AS in hepatoma cell lines(SMMC-7721,HCCLM3,Hep3 B,Hep G2,Huh7)and normal liver cell lines(L02).Flow cytometry was used to sort and enrich stem cell spheres Hep3B-sphere and Huh7-sphere.qRT-PCR was used to detect the expression levels of stem cell surface markers CD133 and CD44 and stemness related factors Nanog,Sox2 and Oct4 in Hep3B-sphere and Huh7-sphere.3.The interference plasmid(sh-HOXA11-AS)or overexpression plasmid(oe-HOXA11-AS)were transfected into Hep3 B and Huh7 stem cells,respectively.qRT-PCR and Western blot were used to detect the expression levels of CD133,CD44,Nanog,Sox2 and Oct4.Cell sphere formation assay and soft AGAR colony formation assay were used to detect the self-renewal ability of HCC stem cells.Transwell assay was used to detect the invasion of HCC stem cells.The Ed U assay assays the proliferative ability of stem cells.4.The expression levels of HOXA11 in HCC tumor tissues and adjacent tissues were detected by qRT-PCR and Western blot,and the correlation between the expression levels of HOXA11 and lnc RNA-HOXA11-AS was analyzed by Person method.m RNA and protein expression levels of HOXA11 in Hep3 B and Huh7 cells,Hep3B-sphere and Huh7-sphere were detected by qRT-PCR and Western blot.Then,overexpression plasmid(oe-HOXA11)was transfected into Hep3 B and Huh7 stem cells,respectively,to investigate the effect of overexpression of HOXA11 on the stemness of HCC stem cells.Further,Hep3 B and Huh7 stem cells were co-transfected with oe-HOXA11 and oe-HOXA11-AS plasmids,respectively,to conduct functional rescue experiments.5.Cell localization of lnc RNA-HOXA11-AS in HCC stem cells was detected by fluorescence in situ hybridization,and Cp G island distribution of HOXA11 promoter region was predicted by methprimer website.After the tumor stem cells were treated with DNA methyltransferase inhibitor 5-aza-dc or methyltransferase Sss I(M.Sss I),the methylation status of HOXA11 gene promoter in Hep3 B and Huh7 stem cells was detected by MSP method.Western blot was used to detect the expression level of DNMT1 in HCC stem cells after silencing or overexpression of lnc RNA-HOXA11-AS and HOXA11.The binding of DNMT1 to HOXA11 promoter region was detected by chromatin immunoprecipitation(Ch IP),and the binding of lnc RNA-HOXA11-AS to DNMT1 was verified by RNA immunoprecipitation(RIP)and RNA Pull down assay.6.Western blot was used to detect the proteinlevels of Wnt signaling pathway marker factors NKD1,β-catenin,C-myc and cyclin D1 in HCC cells and stem cells.Oe-HOXA11-AS-transfected HCC stem cells were treated with Wnt pathway inhibitor DDK1 to explore the effect of DDK1 on the stemness of HCC stem cells.7.The tumorigenic capacity of HCC cells and stem cells was compared through tumorigenesis experiment in nude mice.Stably transfected Hep3B-spheres were injected subcutaneously into BALB/C nude mice,and tumor volume changes and tumor weight were observed for 5 consecutive weeks,and the expression levels of Wnt pathway marker proteins in tumor tissues were evaluated.Results: 1.Lnc RNA-HOXA11-AS was highly expressed in HCC,and HCC patients with high expression of lnc RNA-HOXA11-AS had lower overall survival rate.2.Lnc RNA-HOXA11-AS was highly expressed in 5 HCC cells.Compared with Hep3 B and Huh7 cells,the expression levels of characteristic markers CD133,CD44,Nanog,Sox2 and Oct4 of Hep3B-sphere and Huh7-sphere stem cells were significantly increased,and the expression levels of lnc RNA-HOXA11-AS were significantly increased.3.After lnc RNA-HOXA11-AS interference,the expressions of CD133,CD44,Nanog,Sox2 and Oct4 in HCC stem cells were decreased,the number of cell spheres and clones was significantly reduced,and the ability of invasion and proliferation was decreased.Overexpression of lnc RNA-HOXA11-AS significantly increased the stemness characteristics of HCC stem cells.4.The expression of HOXA11 was low in HCC tissues and negatively correlated with the expression of lnc RNA-HOXA11-AS.The expression of HOXA11 was low in HCC stem cells,and the stemness characteristics of HCC stem cells were significantly weakened after overexpression of HOXA11.Compared with HCC stem cells transfected with oe-HOXA11-AS,co-transfection of oe-HOXA11 and oe-HOXA11-AS significantly reduced the stemness of HCC stem cells.5.Lnc RNA-HOXA11-AS was mainly distributed in the nucleus,and there were a large number of CPG islands in the promoter region of HOXA11.MSP results showed that the methylation level of HOXA11 promoter was increased in the oe-HOXA11-AS group compared with oe-NC group,and decreased in the sh-HOXA11-AS group compared with sh-NC group.lnc RNA-HOXA11-AS can promote the methylation of HOXA11 promoter.Ch IP results showed that DNMT1 enrichment of HOXA11 promoter increased in oe-HOXA11-AS group,while DNMT1 enrichment of HOXA11 promoter decreased in sh-HOXA11-AS group.Both RIP and RNA pull down experiments showed that the binding of lnc RNA-HOXA11-AS to DNMT1 was increased in oe-HOXA11-AS group,while the binding of sh-HOXA11-AS group was decreased.6.The NKD1 level was decreased in HCC stem cells,and the levels of β-catenin,C-myc,and cyclin D1 were increased.Overexpression of lnc RNA-HOXA11-AS resulted in the activation of the Wnt/β-catenin signaling pathway,while the Wnt/β-catenin signaling pathway was inhibited in HCC stem cells co-transfected with oe-HOXA11 and oe-HOXA11-AS.In DDK1-treated lnc RNA-HOXA11-AS overexpressed HCC stem cells,the Wnt/β-catenin signaling pathway was inhibited,and the stemness characteristics of HCC stem cells are also inhibited by DDK1.7.Tumorigenicity of Hep3B-sphere and Huh7-sphere was higher than that of Hep3 B and Huh7 cells.Overexpression of lnc RNA-HOXA11-AS accelerated the growth speed of Hep3B-spheres and increased tumor volume and weight,the mechanism of which was attributed to the the activation of Wnt signaling pathway.However,concurrent overexpression of HOXA11 in Hep3B-sphere overexpressing lnc RNA-HOXA11-AS decreased its tumorigenic ability,due to inhibition of the Wnt/β-catenin pathway by HOXA11.Conclusions: lnc RNA-HOXA11-AS regulates the expression of HOXA11 by regulating promoter methylation of HOXA11,and then promotes the activation of Wnt signaling pathway,thus participating in the regulation of HCC stem cell driness.