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Yue Li, Yan Wang; CNPase targets TAGLN2 to promote epithelial-mesenchymal transition of lens epithelial cells through activation of TGF-β/Smad signaling pathway. Invest. Ophthalmol. Vis. Sci. 2020;61(7):5030.
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We reported previously that lens epithelial cells (LECs) expressed 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) both in vivo and in vitro. What's more, CNPase significantly promoted proliferation, migration and epithelial-mesenchymal transition (EMT) of LECs. In this study, we further explored the EMT-promoting mechanism of CNPase.
Human LECs were treated with TGF-β2 and the total RNA was isolated for RNA Seq analysis. Interaction of CNPase, transgelin-2 (TAGLN2) and Smad3 was identified by coimmunoprecipitation (co-IP) and mass spectrometry. Subsequently, lentivirus was used to overexpress CNPase in cultured LECs, meanwhile, siRNA was used to knock down TAGLN2. Transwell migration assay, wound healing, EdU staining assays, Western blotting and immunofluorescence were used to assess the role of TAGLN2 and TGF-β/Smad signaling pathway in the regulatory fuction of CNPase during EMT.
RNA-seq analysis revealed that 998 mRNAs were elevated while 728 were downregulated in TGF-β2-induced LECs–EMT, of which CNPase increased 2.8 times. KEGG analysis showed that differentially expressed genes were mainly concentrated in several signaling pathways including TGF-β/Smad signaling pathway. Gene Ontology (GO) enrichment analysis illustrated that such difference may concern protein binding and integral component of cytoplasm. Co-IP and mass spectrometry demonstrated an interaction between CNPase and TAGLN2, as well as TAGLN2 and Smad3. Overexpression of CNPase significantly upregulated the expression of EMT markers and promoted the proliferation and migration of LECs. Interestingly, the EMT-promoting mechanism of CNPase may achieved by interacting with TAGLN2 and further targeting the TGF-β/Smad signaling pathway.
The present results have proved that CNPase targets TAGLN2 to promote EMT of LECs through activation of TGF-β/Smad signaling pathway. This may provide novel therapeutic targets for the treatment of cataracts.
This is a 2020 ARVO Annual Meeting abstract.
Differential expression of genes during EMT. (A) RNA-seq analysis. (B) GO analysis. (C) KEGG analysis.
(A) Co-IP demonstrated an interaction between CNPase and TAGLN2, as well as TAGLN2 and Smad3. (B, C) Western Blotting showed that overexpression of CNPase upregulated EMT labels (Slug, Snail etc.) and promoted TGF-β/Smad signaling pathway, while silencing of TAGLN2 can inhibit such effect.
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