Effect Of Mfn2 On TGF-?2-induced Epithelial-mesenchymal Transition In Lens Epithelial Cells

Purpose:Mitochondrial fusion protein gene 2?mitofusin2,Mfn2?is an indispensable conserved protein in the outer membrane of mitochondria and the endoplasmic reticulum that regulates the mitochondrial morphology and regulates the mitochondrial fusion.It is closely related to various biological processes such as energy metabolism,cell proliferation and apoptosis.At the same time,many common diseases in ophthalmology,such as cataract,glaucoma,retinal diseases and corneal diseases,are closely related to mitochondrial function changes and cell proliferation and apoptosis.This study mainly investigated the expression and role of Mfn2 in the epithelial-mesenchymal transition?EMT?of human lens epithelial cells?HLECs?induced by transforming growth factor-?2?TGF-?2?.Methods:In this study,different concentrations(10ng·m L^-1?50ng·mL^-1?100ng·mL^-1)of TGF-?2 were first applied to HLECs respectively to establish posterior capsular opacification?PCO?model of posterior cataract and treated as TGF-?2 treatment group.Real-time quantitative PCR was used to respectively measure the difference of Mfn2expression in mRNA level in HLECs treated with different concentration gradient of TGF-?2(10ng·mL^-1?50ng·mL^-1?100ng·mL^-1)24 hours later compared with the control group(0 ng·mL^-1),then select the group with the most significant changes in Mfn2expression and choose the concentration as the best concentration in the follow-up experiment,immunofluorescence cytochemistry detection was used to detect the differences of Mfn2 expression in protein level between the TGF-?2 treatment group and the control group,Real-time quantitative PCR and immunofluorescence cytochemistry detection were used to validate the differences of E-cadherin and Vimentin expression between the TGF-?2 treatment group and the control group in mRNA and protein levels,respectively.HLECs were transfected with Mfn2 gene fluorescent expression vector?pEGFP-mfn2?as the transfection group,and real-time quantitative PCR and immunofluorescence cytochemical tests were performed to verify the differences in mRNA and protein levels of Mfn2,E-cadherin and Vimentin between the transfection group and the control group,respectively.Results:Real-time quantitative PCR results showed that the expression of E-Cadherin in the HLECs treated with TGF-?2 was decreased and the Vimentin and Mfn2 were up-regulated with statistically significant differences?all P<0.0001?in comparison with the control group in m RNA level.Both the brightness and quantity of immunofluorescence showed that the expression of E-cadherin in protein level was reduced,Vimentin expression was increased,and Mfn2 expression was increased in the TGF-?2 treatment group compared with the control group.After Mfn2 transfection,The results of Real-time quantitative PCR showed that the expression of E-cadherin was down-regulated and the expression of Vimentin was up-regulated in mRNA level in the transfection group,compared with the control group,with statistically significant differences?all P<0.0001?.Both the brightness and quantity of immunofluorescence showed that,compared with the control group,the expression of E-cadherin was reduced in the transfection group and the expression of Vimentin was increased in protein level.Conclusion:TGF-?2 successfully induced EMT in HLECs in vitro,and the expression of Mfn2 was increased in the presence of EMT in HLECs.The overexpression of Mfn2can cause EMT in HLECs.Mfn2 is involved in the TGF-?2-induced EMT process in HLECs,and Mfn2 is also an important factor that influence the TGF-?2-induced EMT in HLECs...