Kindlin-2 RNA interference inhibits vascular smooth muscle cell proliferation via the Wnt signaling pathway

Abstract:

Objective：To investigate the effect of Kindlin-2 RNA interference（RNAi） on rat vascular smooth muscle cell（VSMC） pro－liferation and related mechanism of action. Methods：Kindlin-2 small interfering RNA（siRNA） lentiviral vectors were constructed and then used to infect rat VSMCs. CCK-8 assay and the BrdU technique were used to assess the proliferation of VSMCs induced by re－combinant Wnt3a protein；quantitative real-time PCR was used to measure the mRNA expression of Kindlin-2，c-myc，and cyclinD1 in VSMCs；co-immunoprecipitation was used to evaluate the association between Kindlin-2 and β-catenin；Western blot was used to measure the protein expression of Kindlin-2，β-catenin，phosphor-β-catenin（Ser675），glycogen synthase kinase-3β（GSK-3β），and phosphor-GSK-3β（Ser9） in VSMCs. Results：Kindlin-2 siRNA lentiviral vectors effectively infected rat VSMCs. The CCK-8 and BrdU results indicated that Kindlin-2 RNAi significantly inhibited the proliferation of VSMCs induced by Wnt3a（1.12±0.14 vs. 2.25±0.15，P=0.000；0.162±0.017 vs. 0.288±0.019，P=0.000）. Compared with the negative control group，the Kindlin-2 RNAi group had significantly lower mRNA expression of Kindlin-2，c-Myc，and cyclin D1（Kindlin-2：0.530±0.029 vs. 0.964±0.014，P=0.000；c-Myc：0.572±0.022 vs. 0.980±0.025，P=0.000；cyclin D1：0.590±0.035 vs. 0.979±0.009，P=0.002），and the Kindlin-2 RNAi+Wnt3a group also had significantly lower expression than the negative control group（Kindlin-2：0.569±0.027 vs. 0.964±0.014，P=0.000；c-Myc：0.741±0.026 vs. 0.980±0.025，P=0.001；cyclin D1：0.769±0.017 vs. 0.979±0.009，P=0.023）. Co-immunoprecipitation confirmed that Kindlin-2 could bind to β-catenin in VSMCs，and the Kindlin-2 RNAi+Wnt3a group had significantly lower protein expression of Kindlin-2，phosphor-β-catenin（Ser675），and phosphor-GSK-3β（Ser9） than the nega－tive control+Wnt3a group（Kindlin-2：0.468±0.029 vs. 0.725±0.033，P=0.001；phosphor-β-catenin：1.058±0.109 vs. 1.478±0.045，P=0.001；phosphor-GSK-3β：0.624±0.048 vs. 0.809±0.067，P=0.020）. There were no significant changes in the total levels of β-catenin and GSK-3β protein in each group（β-catenin：F=0.638，P=0.647；GSK-3β：F=0.781，P=0.563）. Conclusion：Kindlin-2 RNAi can inhibit VSMC proliferation via the Wnt signaling pathway.