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Subbulakshmi Chidambaram, Suganya Sivagurunathan, Karthikka Palanisamy, Sulochana Natarajan, Pukhraj Rishi, Jayamuruga Pandian Arunachalam; Expression of Piwi/piRNA in human ocular tissues: Role in maintaining functional integrity of retinal pigment epithelial cells and implication in proliferative diabetic retinopathy. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2953.
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© ARVO (1962-2015); The Authors (2016-present)
Piwi clade comprises of 4 proteins namely Piwil1-4 which specifically bind a novel group of non coding small RNAs, Piwi-interacting RNA (piRNA). Expression of Piwi/piRNA is believed to be confined to germline and stem cells. Serendipitously we found the presence of Piwil1 in vitreous from donor eye ball during MALDI ToF analysis. Intrigued by the result we set out to examine the presence of Piwi/piRNA in ocular tissues and their possible role.
Human primary cells were isolated from donor eye balls. NGS was done in Illumina GAIIX. VEGF, PEDF and adiponectin cDNA were cloned in pACGFPC1 vector. Panaroma protein antibody cell signalling array (Sigma) was scanned in Perkin Elmer system. The study was done with approval from Institution Ethics Board.
RT-PCR and qPCR showed that the transcripts of piwil1-4 were expressed in ocular tissues and cells. Western blot for Piwil1-4 showed stronger expression in retina followed by RPE/choroid. In addition, IHC of human retinal section confirmed the presence of Piwi proteins in RPE and RGC layer. Piwil1-4 localized in the cytoplasm of hRPE cells as shown by IF. The presence of piRNA in human retina was identified by NGS and validated by RIP and qPCR. Piwil4 was knocked down in hRPE using DsiRNA and expression of around 37 genes, involved in the vital functions of RPE was screened. Decreased levels of tight junction proteins (ZO1, CLDN1 and OCLN), Phagocytosis and trafficking proteins (Syn1A, Syn16, Rab5 and Vamp8) were observed in Piwil4 knockdown. Interestingly TGFβ and VEGF levels were also decreased. When VEGF was overexpressed in hRPE, Piwil4 was upregulated. In contrast, Adiponectin and PEDF downregulated Piwil4. Protein antibody array was used to analyse the changes in the proteome of Piwil4 knockdown cells. Further, western blot of vitreous from normal donor eye ball (n=6) and PDR (n=3) showed reduced level of Piwil4 in PDR.
The novelty of current finding is the demonstration of widespread presence of Piwi/piRNA in human somatic (ocular) tissue. Transdifferentiation of RPE into fibroblast like cells leads to pathological fibrosis in PDR. Our preliminary results suggest that Piwil4/piRNA pathway may have a significant role in regulating the epithelial mesenchymal transition of RPE.
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