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Archive > Volume 24 Issue 6 > 2017,24(6):608-614. DOI:10.3872/j.issn.1007-385X.2017.06.006 Prev Next
Basic Research
Stable overexpression of human MGST1 gene inhibits apoptosis of lung adenocarcinoma cell line SPCA1
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Abstract:
Objective:Lung adenocarcinoma SPCA1 cell line that stably overexpresses microsomal glutathione Stransferase 1 (MGST1) was constructed to explore the function and mechanism of MGST1 in lung adenocarcinoma.Methods:The recombinant plasmid pcDNA3MGST1 and the empty vector pcDNA3 were transfected into SPCA1 by Lipofectaminemediated method; after being screened by G418, stably transfected cells were labeled and divided into pcDNA3MGST1 group and empty vector pcDNA3 group. qRTPCR and Western blotting were used to detect the expression of MGST1 at mRNA and protein level in stable cells. MTS was used to detect the viability of cells. Flow cytometry and Western blotting were used to detect the cell apoptotic rate and the downstream apoptotic proteins induced by H2O2, respectively. Results: The results of sequencing showed that the recombinant plasmid pcDNA3MGST1 was successfully constructed, and the stable cell line with G418 resistance was obtained. The mRNA and protein level of MGST1 in pcDNA3MGST1 group were all significantly elevated (P<0.01), and the cell viability was significantly increased (P<0.05). Under the induction of H2O2, the early apoptosis rate of pcDNA3MGST1 group was significantly lower than that of pcDNA3 group (\[3.30±0.40\]% vs \[6.50±0.95\]%, P<0.05). The expressions of apoptotic proteins (caspase 9, caspase3 and PARP) increased, and the expressions of cleavedcaspase 9, cleavedcaspase 3 and cleavedPARP were significantly decreased in pcDNA3MGST1 group. Conclusion: The lung adenocarcinoma cell line SPCA1, which stably overexpresses human MGST1, has been successfully established. It was found that MGST1 could inhibit the apoptosis of lung adenocarcinoma cells by regulating caspase apoptosis pathway, which laid the experimental foundation for the followup study.
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Project Supported:
Project supported by the National Natural Science Foundation of China(No.U1502222,No.81470005,No.81260307), and the key Scientific Research Equipment Development Project of the National Natural Science Foundation of China(No.61427807)
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