Role Of SnoN Proteins In RPE Cells TGF-β/Smad Signaling Pathways

Myopia is currently the world’s highest incidence of refractiveerrors,paticularly marked in particular population in certain Asian countries andregions, which shows an increasing trend.But its pathogenesis is not very clear. Therole of retina-retinal pigment epithelium-choroid signal transduction system becomean eye research hot spot in the pathogenesis of myopia.Transforming growth factor beta(TGF-β) /Smad signaling pathway canregulate cell growth, differentiation, apoptosis, migration, invasion and metastasis,etc. Also,it is one of the most important signaling pathways of myopia.Recentstudies showed that Smad transcription inhibitor(Ski-related protein N, Sno N) canregulate signal transmission in renal tissues of diabetic rats. Sno N is an importantcytokine in TGF-β/Smad signaling pathway, and its expression level has a largeeffect on the biological effects of TGF-β.However, is there existence of Sno Nprotein in human retinal pigment epithelial cells, and its role in the TGF-β/Smadsignaling pathway still unclear.Objective: This experiment tests Sno N protein expression in human retinalpigment epithelial ARPE-19 cell line and changes of Sno N content in ARPE-19 cells under the effect of recombinant human TGF-β1(rh TGF-β1). Also, itexplorers the expression level of the nuclear transcription inhibiting factor Sno N inARPE-19 cells and its larvaceous function in TGF-β/Smad signaling pathway.Methods: Sno N protein expression levels and location in ARPE-19 cells wereexamined by immunofluorescence; intervene ARPE-19 cells with differentconcentration of recombinant human TGF-β1(0ng/ml,1ng/ml,2ng/ml, 5ng/ml,10ng/ml,20ng/ml) at 2h,intervene ARPE-19 cells with the same concentration ofrecombinant human TGF-β1(5ng/ml) at 0min, 30 min, 1h, 2h, 3h, 6h, 12 h, 24 h,48h,extract RNA and protein respectively, RT-PCR was used to examine thedifference of Sno N’s m RNA expression; protein immunoblot(Western-blot) wasused to examine the difference of Sno N protein expression.Results:①The immunofluorescence showed that Sno N protein was expressingin ARPE-19 cells, mainly in the cytoplasm; ②The Sno N m RNA levels weresignificantly increased in ARPE-19 cells incubated for 2 hours with differentconcentrations of rh TGF-β1(0ng/ml,1ng/ml,2ng/ml,5ng/ml,10ng/ml, 20ng/ml),compared with the blank control group(P<0.05). However,the Sno N protein levels,had no statistical difference(P>0.05);③In the same concentration of rh TGF-β1(5ng/ml), acting on RPE cells after different time(0min,30 min,1h,2h,3h,6h,12 h,24h,48h), the Sno N m RNA levels decreased in 30 minutes group, but there was nostatistical difference(P>0.05), and the Sno N m RNA levels rised in other groups,had statistically significant diference(P<0.05).The Sno N protein levels still had nostatistical difference(P>0.05).Conclusion: Sno N protein is expressing in the ARPE-19 cells, and may havebeen regulation in RPE cells TGF-β/Smad signaling pathways.