摘要
AIM: To study the effect of senescence marker protein 30(SMP30) on the proliferation and apoptosis of human lens epithelial cell(HLEC) SRA01/04.METHODS: SMP30 overexpression(OE) and knock down(KD) type cell lines were cultivated by using two groups regucalcin(RGN; SMP30) lentiviral vectors(LVRGN, LV-RGN-RNAi) and the respective negative control virus infect SRA01/04 cells. Western blot and real-time quantitative polymerase chain reaction(q-PCR) analysis were used to determine RGN overexpression and knock down efficiency. We use cell counting kit-8(CCK8) assay to measure cell viability and 5-bromodeoxyuridine(Brd U) assay to test cell proliferation. Cell cycle was measured by PI FACS assay and cell apoptosis was tested by Annexin V-APC assay through flow cytometry. We use Western blot to measure the content of caspase-3 in SRA01/04.RESULTS: We used PCR and Western blot techniques to determine the successful transfection of SMP30 OE and KD SRA01/04 cell lines. By CCK8, Brdu and PI FACS cell cycle assay, it was found that the SMP30 OE group promoted cell proliferation(P<0.05) compared with the control group, and the KD group inhibited cell proliferation(P<0.05). 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(P<0.05) but lower in OE group(P<0.01). The expression of caspase-3 was down-regulated in OE group through Western blot assay and up-regulated in KD group compared with respective control group. CONCLUSION: Proliferation of SRA01/04 was promoted by SMP30 OE and apoptosis was suppressed. Increasing the expression of SMP30 may protect HLEC SRA01/04 against apoptosis in cataract.
AIM： To study the effect of senescence marker protein 30（SMP30） on the proliferation and apoptosis of human lens epithelial cell（HLEC） SRA01/04.METHODS： SMP30 overexpression（OE） and knock down（KD） type cell lines were cultivated by using two groups regucalcin（RGN; SMP30） lentiviral vectors（LVRGN, LV-RGN-RNAi） and the respective negative control virus infect SRA01/04 cells. Western blot and real-time quantitative polymerase chain reaction（q-PCR） analysis were used to determine RGN overexpression and knock down efficiency. We use cell counting kit-8（CCK8） assay to measure cell viability and 5-bromodeoxyuridine（Brd U） assay to test cell proliferation. Cell cycle was measured by PI FACS assay and cell apoptosis was tested by Annexin V-APC assay through flow cytometry. We use Western blot to measure the content of caspase-3 in SRA01/04.RESULTS： We used PCR and Western blot techniques to determine the successful transfection of SMP30 OE and KD SRA01/04 cell lines. By CCK8, Brdu and PI FACS cell cycle assay, it was found that the SMP30 OE group promoted cell proliferation（P〈0.05） compared with the control group, and the KD group inhibited cell proliferation（P〈0.05）. 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（P〈0.05） but lower in OE group（P〈0.01）. The expression of caspase-3 was down-regulated in OE group through Western blot assay and up-regulated in KD group compared with respective control group. CONCLUSION： Proliferation of SRA01/04 was promoted by SMP30 OE and apoptosis was suppressed. Increasing the expression of SMP30 may protect HLEC SRA01/04 against apoptosis in cataract.
基金
Supported by the National Natural Science Foundation of China(No.81360146)
