Abstract:AIM: To explore the effect of low expression of senescence marker protein 30(SMP30)on proliferation and oxidative stress of human lens epithelial cells(LECs)line SRA01/04 under high calcium conditions.
METHODS: Three RNAi sequences were designed to knock down SMP30 target gene RGN expression(KD1-3), and the blank-load sequence was used as the negative control group(NCKD), all of which were used to construct lentiviral vectors to infect SRA01/04 cells. Meanwhile, the uninfected SRA01/04 cells was used as the blank control group(CON). After transfecting SRA01/04 cells, the lentiviral vector with the highest knockdown efficiency was selected by RT-PCR for subsequent experiments. Cells were treated with 15mmol/L CaCl2 for 24h to simulate a high calcium conditions. BrdU-Elisa assay was used to measure cell proliferation, superoxide dismutase(SOD)assay kit and oxidized glutathione/total glutathione(GSSG/T-GSH)assay kit were used to detect the level of intracellular oxidative stress.
RESULTS: KD1-3 and NCKD lentiviral vectors were successfully constructed to infect SRA01/04 cells with an infection efficiency of about 80%. The knockdown efficiency of KD1-3 group was 93%, 60% and 74%, respectively, KD1 group was selected for follow-up experiment. Under the high calcium conditions, the activity of relative cell proliferation and SOD in KD1 group \〖(2.42±0.08)and(11.69±0.52U/mg)\〗 were significantly lower than that in NCKD group \〖(2.95±0.08)and(31.10±2.24U/mg)\〗 and CON group \〖(2.96±0.25)and(26.33±1.04U/mg)\〗, the ratio of GSSG/T-GSH in KD1 group(70.80±2.34)was significantly higher than that in NCKD group(15.93±3.47)and CON group(20.05±2.45)(P<0.05); there was no significant difference between NCKD group and CON group(P>0.05).
CONCLUSION: Under high calcium conditions, SRA01/04 cells(HLECs)with low expression of SMP30 mediated by shRNA lentivirus resulted in the decrease of the proliferation activity and antioxidant capacity, suggesting that SMP30 may play a protective role in regulating cell proliferation and anti-oxidative stress in HLECs.
