The Role And Mechanism Of CNPase In The Process Of Epithelial-Mesenchymal Transition During Posterior Capsular Opacification

Objective Epithelial-mesenchymal transition(EMT)is the primary cause of organ fibrosis as well as most cataracts.Cataract is responsible for much of the blindness worldwide,and surgery may be the most efficient treatment.However,postoperative complications such as posterior capsular opacification(PCO)may result in secondary visual impairment or even blindness.2’,3’-Cyclic-nucleotide 3’-phosphodiesterase(CNPase)is a key enzyme for the hydrolysis of 2’,3’-cyclic nucleotides.2’-adenosine monophosphates(AMPs)hydrolysised from corresponding 2’,3’-cyclic nucleotides are further converted into adenosine,which is involved in the purinergic signal transduction.Accumulating evidence suggests that purinergic signalling significantly promote EMT,accordingly,CNPase may play a crucial role in the process of EMT during PCO.This study aimed to explore the potential mechanism of CNPase in the development of EMT,and attempt to provide a novel strategy for preventing and treating PCO.Methods Part Ⅰ: In vitro experiment: The human lens epithelial cell line SRA 01/04 was treated with recombinant human TGF-β2 to induce EMT in vitro.The difference of gene expression between treatment group and control group was detected by RNA sequence(RNA-seq),q RT-PCR and Western blot analysis were used to assess the change of CNPase during EMT.In vivo experiment: A small incision was made in the lens anterior capsule of C57BL/6J mice with a 0.26-mm thick needle blade,examined the lens under a slit-lamp microscope and photographed.In order to evaluate the model,Haematoxylin-eosin(HE)staining and Masson Staining were performed at the 7 day after surgery.Besides,before the lens were removed and paraffin-embedded for immunofluorescence,the mice were allowed to heal for 5、7 and 14 days.Part Ⅱ: We silenced CNPase with small interfering RNA(si RNA)and overexpressed CNPase with lentivirus.We studied the effects of silencing or overexpressing of CNPase on LECs,respectively.The experimental groups are as follows: A1: normal control cell group;B1: si RNA transfected cell group;C1: normalcell+ TGF-β2 group;D1: si RNA transfected cell+ TGF-β2 group;A2: normal cell group;B2: lentivirus transfected cell group.Transwell migration assay,wound healing assay,Ed U staining assay,Western blot and immunofluorescence staining were used to detect the effect of CNPase on the migration,proliferation and EMT of human lens epithelial cells(LECs).Part Ⅲ: To find out the interaction between CNPase and transgelin 2(TAGLN2),coimmunoprecipitation(Co-IP),mass spectrometry and Western blot were performed.Silenced CNPase with si RNA and overexpressed CNPase with lentivirus,then explored the regulation effect of CNPase on TAGLN2 by Western blot and immunofluorescence staining.Further silenced TAGLN2 in CNPase overexpressing cells,Transwell migration assay,wound healing assay,Ed U staining assay,Western blot and immunofluorescence staining were used to detect the role of TAGLN2 in the process of CNPase mediated EMT.Part Ⅳ: Overexpressed TAGLN2 with lentivirus,using Western blot,immunofluorescence staining,Transwell migration assay,wound healing assay and Ed U staining assay to observe the effect of TAGLN2 on TGF-β/Smad signalling pathway and EMT.After that,TAGLN2 overexpressing cells were treated with Smad3 inhibitor(SIS3),Western blot,immunofluorescence staining,Transwell migration assay,wound healing assay and Ed U staining assay were used to reveal the role of Smad3 in the process of TAGLN2 mediated EMT.Results 1.Pierced the lens anterior capsule of mice by needles,the results showed that the expression of CNPase was remarkably increased,and the expression of vimentin,a key marker of EMT,showed a similar trend with CNPase.2.Overexpression of CNPase noticeably promote proliferation,migration and EMT of LECs in vitro,while silencing of CNPase can effectively reverse these changes.3.In vitro experiments revealed the interaction between CNPase and TAGLN2 in LECs,meanwhile,silencing of TAGLN2 remarkably inhibited the progression of CNPase mediated EMT.4.Overexpression of TAGLN2 significantly activated TGF-β/Smad signalling pathway,however,promotion of EMT induced by TAGLN2 was noticeably inhibited by SIS3.Conclusions We confirmed that CNPase significantly promote the progress of EMT in LECs.What is more,the EMT-promoting mechanism of CNPase may be achieved by interacting with TAGLN2 and further targeting the TGF-β/Smad signalling pathway.In conclusion,CNPase may be a therapeutic target for the treatment of fibrosis such as PCO.