The Effect Of Senescence Marker Protein 30 On The Proliferation And Apoptosis Of Human Lens Epithelial Cells SRA01/04

Purpose:This study aims to test the change of proliferation and apoptosis induced by senescence marker protein 30(SMP30)in human lens epithelial cell(HLEC)SRA01/04,to provide new ideas for the study of etiology and prevention mechanism of cataract.Methods:①To determine whether SRA01/04 is susceptible to lentivirus transfection,we carried out pre-experiment in which cells were seeded at a density of 2×10~3/well in 96-well plate,transfected with GV115-NC(pGcsil-004).Cells were divided into four groups of different transfection conditions:Normal medium,Normal medium+Polybrene(5μg/ml),Eni.s,Polybrene(5μg/ml)+Eni.s,and divided into three groups of different MOI(Multiplicity of Infection):1,10,100 to find out the best infection conditions.②With the most moderate condition,cells which seeded at 5×10~4/well in 6-well plate were infected with LV-RGN(Lentivirus-Regucalcin)in OE(Overexpression)group,LV-RGN-RNAi(Lentivirus-Regucalcin-RNA interference)in KD(Knock Down)group,and the control group were infected with the respective negative control virus.After 24h,the infected solution was changed into normal complete medium and the green fluorescence intensity was observed by fluorescence microscope at 72h.When the cells were grown to about 90%,2μg/ml of Puromycin was added to remove uninfected cells,and then 1μg/ml of Puromycin was maintained in subcultured cells to obtain stably transfected cells.WB(Western blot)and q-PCR(Real-time Quantitative Polymerase Chain Reaction)analysis were used to determine RGN(Regucalcin,SMP30)overexpression and knock down efficiency.③A Cell Counting Kit-8(CCK8)assay was used to measure cell viability.5-bromodeoxyuridine(BrdU)assay was used to test the cell proliferation.Apoptosis and cell cycle were respectively measured by Annexin V-APC and PI FACS assay through flow cytometry.The differences of expression of caspase-3 in SRA01/04 were detected by western blot assay.Results:①The most moderate transfection condition was Normal+Polybrene(5μg/ml)and MOI=5.②We used PCR and WB techniques to determine the successful transfection of SMP30 overexpression(OE)and knock down(KD)SRA01/04 cell lines,RGN gene expression in OE group(19.373±1.445)was 19.373 times higher than that of NC-OE(Overexpression negative control group)group(1.023±0.253)(t=-21.659,p<0.01).The knock down rate of KD group(0.039±0.003)was 96%compared with NC-KD(Knock down negative control group)(1.003±0.1)(t=16.729,p<0.01)by q-PCR assay.Compared with respective negative control group,the expression of SMP30proteins were significantly higher in OE group(1.338±0.186)(t=-9.039,p<0.01)and lower in KD group(0.137±0.048)(t=13.006,p<0.01)by WB.③By CCK-8,cell viability of KD group(0.355±0.008)was decreased compared with the NC-KD group(0.398±0.011)(t=7.466,p<0.01).However,there was no difference between OE group(0.415±0.01)and NC-OE group(0.402±0.009)(t=-2.189,p>0.05).By Brdu,the proliferation of OE group(0.857±0.023)was promoted compared with NC-OE group(0.682±0.006)(t=6.49,p<0.05).However,there was no difference between KD(0.664±0.01)and NC-KD(0.676±0.008)group(t=1.61,p>0.05).By PI FACS cell cycle assay,compared with NC-KD group,the cells in the KD group were decreased in the S phase(t=11.18,p<0.01),and increased in the G1 phase(t=-12.97,p<0.01),and there was no significant change in the G2/M phase(t=0.288,p>0.05);Compared with NC-OE group,the cells in the OE group were decreased in the S phase(t=12.643,p<0.01),and increased in the G1 phase(t=-11.849,p<0.01)and G2/M phase(t=-12.447,p<0.01).The results of Annexin V APC signal staining detection indicated that compared with respective control group,the cell apoptosis rate was higher in KD group(t=-5.986,p<0.01)but lower in OE group(t=13.181,p<0.05).The expression of caspase-3 was decreased in OE group through Western Blot assay and increased in KD group compared with respective control group.Conclusion:These data indicate that changes in SMP30 content play an essential role in regulating cell proliferation and apoptosis.Improving the content of SMP30 in human lens may protect HLEC against apoptosis in age-related cataract(ARC).