| Objective: To investigate the mechanism of IRX5 promoting EMT by Wnt / ?-catenin signaling pathway in hepatocellular carcinoma(HCC).Methods:(1)Western blot was used to detect the expression levels of IRX5,?-catenin and E-cadherin in 3 pairs of HCC tissue samples;ELISA was used to detect the expression levels of IRX5 and ?-catenin in the serum of 100 patients with HCC and50 people of the control group.Spearman method was used to analyze the correlation between IRX5 and ?-catenin in HCC.(2)Hep G2 and SMMC7721 cells were selected as research objects.Each cell line was divided into two groups: control group(sh-NC)and knockdown group(sh-IRX5).Transient transfection technique was used to construct a cell model of IRX5 knockdown,the expression level of IRX5 was detected by Western blot and RT-q PCR.The morphology of hepatocellular carcinoma cells was observed by light microscope.The migration of HCC cells was detected by wound healing.The expressions of Wnt / ?-catenin signaling pathway proteins(?-catenin,phospho-?-catenin,GSK-3?,phospho-GSK-3?),downstream target genes of Wnt / ?-catenin signaling pathway(MMP2,MMP9,c-MYC,Cyclin D1)and EMT-related proteins(E-cadherin,N-cadherin,Vimentin,Snail,Twist1)were detected by Western blot.(3)To construct low-expressed IRX5 stable lines,HCC cells were infected lentivirus of IRX5 sh RNA.To verify the knockdown level of IRX5 by RT-q PCR.Above cells were selected as research objects,which were divided into control group(sh-IRX5+pc DNA3.1)and rescue group(sh-IRX5+pc DNA3.1-IRX5),and the expressions level of IRX5 were detected by Western blot.Wound healing were used to detect the migration levels of IRX5 in HCC cells.Western blot was used to detect Wnt / ?-catenin signaling pathway proteins(?-catenin,phospho-?-catenin),downstream target genes of Wnt / ?-catenin signaling pathway(MMP2,MMP9)and EMT-related proteins(E-cadherin,N-cadherin,Twist1).(4)Stably transfected cell lines were injected into subcutaneous tissue of nude mice,detecting the volume and weight of subcutaneous tumors in nude mice,the expression levels of IRX5,?-catenin and phospho-?-catenin in the subcutaneous tumorigenic tissue were detected by immunohistochemistry.Results:(1)Compared with adjacent tissues,the expression levels of IRX5 and ?-catenin in HCC tissues were significantly increased,and the expression levels of E-cadherin were decreased;Compared with the control group,the expressions of IRX5 and ?-catenin in HCC serum were significantly increased(P<0.01).In addition,IRX5 was positively correlated with ?-catenin expression(r=0.8997,P<0.01).(2)After the knockdown of IRX5,the levels of IRX5 m RNA in Hep G2 and SMMC7721 were 0.43?0.06 and 0.46?0.05,respectively(P <0.01).The levels of IRX5 protein were significantly decreased.After the knockdown of IRX5,the morphology of HCC cells was more epithelial and the migration ability of HCC cells was significantly reduced(P <0.01).After the knockdown of IRX5,the expression of Wnt / ?-catenin signaling pathway key proteins ?-catenin,downstream target gene proteins(MMP2,MMP9,c-MYC,Cyclin D1)and EMT-related proteins(N-cadherin,Vimentin,Snail,Twist1)were significantly decreased,the expressions of phospho-?-catenin and E-cadherin were increased(P <0.01),the expressions of GSK-3? and phospho-GSK-3? were not significantly changed(P> 0.05).(3)The expressions of IRX5 m RNA in stably transfected cell lines were significantly reduced(P<0.01);Over-expressed IRX5 in stably transfected cell lines,the migration ability was significantly enhanced compared with the control group(P <0.01);The expression of Wnt / ?-catenin signaling pathway key proteins ?-catenin,downstream target genes(MMP2,MMP9)and EMT-related proteins(N-cadherin,Twist1)were significantly increased,the expressions of phospho-?-catenin and E-cadherin were decreased(P <0.01).(4)Compared with the control group,the tumor volume and weight of the nude mice in the IRX5 knockdown group were significantly lower.The expressions of IRX5 and ?-catenin were significantly reduced and the expression of phospho-?-catenin was significantly increased in subcutaneous tumorigenic tissuecompared with the control group.Conclusion: The transcription factor IRX5 can promote EMT in HCC by activating the Wnt / ?-catenin signaling pathway,which is beneficial to the migration of HCC cells. |
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