Effect Of Changes In Senescence Marker Protein 30 Of Human Lens Epithelial Cell Line SRA01/04 Under Calcium Elevation And Acute Oxidative Stress

OBJECTIVE:To investigate the effects of changes in senescence marker protein-30 of human lens epithelial cell line SRA01/04 under calcium elevation and acute oxidative stress conditions.Theoretical basic is provided for further study on the pathogensis of age-related cataract and also provided for preventation of cataract formation.Methods:1.Senescence marker protein 30 over-expression and knock-down stable strain were established:SRA01/04 cells were seeded in 6-well plates at 5x10⁴/well,and over-expressed lentivirus（LV-RGN,19544-1）)was used according to MOI=5 when cells were grown to 20%and knock-down lentivirus（LV-RGN_RNAi,50743-1）and the corresponding negative control lentivirus-infected cells,which were replaced with normal medium after 24hours of routine culture.Transfection efficiency was observed under a fluorescence microscope after 72 hours.When the cells were grown to 90%,2μg/ml of puromycin was added to filtrate uninfected cells.RNA and protein were extracted from transfected cells for real-time quantitative polymerase chain reaction（RT-PCR）and Western blot（WB）to detect the transfection efficiency of the cell line,verify whether the model build was successful2.Construction of high calcium culture condition:When the transfected cells were cultured to 80%,15 mmol/L CaCl₂ was added for 24 hours,and the required RNA and protein were extracted to detect the expression level of SMP30.3.Construction of acute oxidative stress culture condition:The transfected cells were cultured to 80%and added with 300μmol/L H₂O₂ for 2hours,and RNA and protein were extracted to detect the expression level of SMP30 in this state.Results:1.Transfection efficiency of over-expression（OE）and knock-down（KD）stable strains:RT-PCR results showed that the expression abundance of the OE（over-expression）group（2.8330±0.201）was 2.83times than that of the NC-OE（over-expression negative control group）（0.987±0.888）（p<0.05）;knockdown rate of the KD group（0.090±0.040）was 91%（p<0.05）compared with the NC-KD group（Knock down negative control）group（1.009±0.006）;WB results show that:compared with NCOE group（0.599±0.013）,expression of SMP30 gene in OE group（0.923±0.013）was increased（p=0.000<0.05）,expression of SMP30 gene in KD组（0.610±0.002）was decreased when compared with NC-KD group（0.742±0.013）（p=0.000<0.05）.2.Expression level of SMP30 protein and RGN mRNA gene in calcium elevation condition:RT-PCR results showed that the expression level of RGN mRNA in the OE group（5.234±0.512）was higher than that in the NC-OE group（1.007±0.149）（p=0.000<0.05）,and that in the KD group（0.510±0.023）was lower than that in the NC-KD group（1.006±0.137）（p=0.022<0.05）;WB results showed that the expression level of SMP30 protein in the OE（over-expression）group（0.5668±0.0113）was lower than that in the NC-OE group（1.107±0.006）（p=0.000<0.05）,and that in the KD group（1.266±0.034）was higher than that in the NC-KD group（1.099±0.026）（p=0.003<0.05）.Compared with the expression of SMP30 in OE group and KD group under normal conditions,RGN mRNA expression level increased in both groups(p_OE=0.002<0.05,p_KD=0.000<0.05),while SMP30 protein expression level was down-regulated in the OE group（p=0.000<0.05）and up-regulated in the KD group（p=0.000<0.05）.3.Expression of genes and proteins in each group under acute oxidative stress condition:RT-PCR results showed that the expression level of RGN mRGN in the OE group（4.332±0.264）was higher than that in the NC-OE group（1.001±0.050）（p=0.002<0.05）,and the expression level of KD group（0.377±0.029）was lower than that in the NC-KD group（1.004±0.112）（p=0.001<0.05）;WB results showed that the expression level of SMP30 protein in the OE group（1.103±0.002）was up-regulated compared with that in the NC-OE group（0.876±0.010）（p=0.000<0.05）,and the expression level in the KD group（0.611±0.024）was not statistically significant compared with that in the NC-KD group（0.591±0.034）（p=0.46>0.05）.Compared with the normal condition,RGN mRNA gene expression increased in both the OE group and KD group(p_OE=0.001<0.05,p_KD=0.001<0.05),SMP30 protein expression was up-regulated in the OE group（p=0.000<0.05）,and the expression level in KD group was not statistically significant（p=0.967>0.05）.Conclusion:Compared with normal condition,the expression of SMP30in SMP30 overexpression group is more obvious under acute oxidative stress,SMP30 increased under acute oxidative stress condition,participate in the process of oxidative stress in human lens epithelial cells.In the calcium elevation conditon,the expression of SMP30 showed a reactive change,suggesting that SMP30 is closely related to intracellular calcium homeostasis and exerts its calcium regulation.