Effect of ILK SiRNA on the proliferation and apoptosis of human Tenon fibroblasts induced by transforming growth factor-β2

Abstract:

AIM: To investigate the role of small interference RNA interference targeted Integrin-linked kinase(ILK SiRNA)on the proliferation and apoptosis of human Tenon fibroblasts(HTFs)induced by transforming growth factor-β₂ (TGF-β₂).

METHODS: The HTFs were identified by immunofluorescence analysis with Vimentin and keratin. HTFs with no other addiction was as normal control; H+T group: HTFs+5μg/L TGF-β₂; H+T+NC SiRNA group: HTFs+5μg/L TGF-β₂+50nmol/L negtive SiRNA; H+T+ILK SiRNA group: HTFs+5μg/L TGF-β₂+50nmol/L ILK SiRNA. The ILK SiRNA were transfected into HTFs by lipofectamine 2000, then the cells were stimulated with 5μg/L TGF-β₂. The protein expression of ILK were analyzed by Western Blot. The proliferation levels of HTFs were analyzed by CCK-8, the apoptosis of HTFs were analyzed by Hoechst 33342/PI double staining.

RESULTS: The protein ILK were expressed in both TGF-β₂ treated and control groups, and TGF-β₂ up-regulated the expression of ILK, ILK SiRNA inhibited the protein expression of ILK(P<0.05). CCK-8 analysis showed that compared with the normal control group, the cell proliferation rate of HTFs in TGF-β₂ treated group increased, and in ILK SiRNA group the cell proliferation rate was suppressed after exposured to ILK SiRNA for 48h(P<0.05). Hoechst 33342/PI double staining showed that there was no change on the apoptosis of TGF-β₂ stimulated group(P>0.05), compared with the normal control group, however in the ILK SiRNA group, we found lots of apoptosis cells and a few of necrotic cells(P<0.05).

CONCLUSION: The ILK SiRNA attenuates the abnormal proliferation of HTFs induced by TGF-β2, thereby enhancing the apoptosis of HTFs.