Effect of BRCA1 R1325K mutation on proliferation and apoptosis of gallbladder cancer cells

doi:10.3969/j.issn.1674-8115.2023.09.001

Abstract

Abstract:

Objective ·To investigate the effects of breast cancer susceptibility gene 1 (BRCA1) R1325K mutation [arginine (R) to lysine (K) mutation at amino acid 1325] on the proliferation and apoptosis of gallbladder cancer cell lines GBC-SD and NOZ. Methods ·BRCA1 wild-type overexpression lentivirus, BRCA1 R1325K mutation overexpression lentivirus, and negative control lentivirus were used to construct the stable transgenic strains of gallbladder carcinoma, cell lines GBC-SD and NOZ. The cells were divided into the control group without the target gene, the BRCA1 wild-type group, and the BRCA1 R1325K mutation group. The expression of target protein was verified by Western blotting. The BRCA1 R1325K mutant gallbladder cancer cells were treated with 20 μmol/L Olaparib, a BRCA1 mutation inhibitor. Gallbladder cancer cell lines were divided into the control group, the BRCA1 wild-type group, the BRCA1 R1325K mutation group, and the BRCA1 R1325K mutation+Olaparib group according to the target gene expression and whether or not the inhibitor was added. The effect of BRCA1 R1325K mutation on proliferation and clonogenesis ability of gallbladder cancer cell lines GBC-SD and NOZ was observed by CCK8 assay and clonogenesis assay, respectively. The effect of BRCA1 R1325K mutation on apoptosis of gallbladder cancer cell lines GBC-SD and NOZ was observed by TUNEL assay. The expressions of apoptosis-related proteins, cleaved PARP, Bcl-2 and Bax, were detected by Western blotting. The inhibitor Olaparib was used to treat the BRCA1 R1325K mutant gallbladder cancer cell lines GBC-SD and NOZ. The phenotypic changes (promoting proliferation, enhancing clonogenesis and inhibiting apoptosis) induced by BRCA1 R1325K mutation were tested in the presence of Olaparib to determine whether the changes could be reversed by the inhibitor. Results ·The results of CCK8 assay and clonogenesis assay showed that BRCA1 R1325K mutation could promote the proliferation of gallbladder cancer cell lines GBC-SD and NOZ, and improve their clonal formation ability, compared with the control group and the BRCA1 wild-type group. Olaparib inhibited the proliferation of gallbladder cancer cell lines overexpressing BRCA1 R1325K mutation (P<0.05). Through TUNEL and Western blotting, it was found that overexpression of wild-type BRCA1 could induce the apoptosis of gallbladder cancer cell lines GBC-SD and NOZ, compared with the control group. Compared with the control group and the BRCA1 wild-type group, the BRCA1 R1325K mutation group had anti-apoptotic effect, in which the expression of apoptosis-inhibiting protein Bcl-2 increased and the expression of pro-apoptotic protein Bax decreased (P<0.05). Conclusion ·BRCA1 R1325K mutation can promote the proliferation of GBC-SD and NOZ cell lines and inhibit their apoptosis.

Key words: gallbladder cancer, BRCA1, mutation, proliferation, apoptosis

CLC Number:

R735.8

YANG Jingxiao, JIA Ziyao, WU Wenguang, WU Xiangsong, ZHANG Fei, LI Huaifeng, ZHU Yidi, LI Maolan. Effect of BRCA1 R1325K mutation on proliferation and apoptosis of gallbladder cancer cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(9): 1071-1079.

Figures/Tables 5

Fig 1 Expression of BRCA1 protein in gallbladder cancer cell lines GBC-SD and NOZ after BRCA1 wild-type and BRCA1 R1325K mutation overexpression lentivirus infection

Fig 2 Effects of BRCA1 R1325K mutation on proliferation of gallbladder cancer cell lines GBC-SD and NOZ

Fig 3 Effects of BRCA1 R1325K mutation on colony formation ability of gallbladder cancer cell lines GBC-SD and NOZ

Fig 4 Effects of BRCA1 R1325K mutation on apoptosis of gallbladder cancer cell lines GBC-SD and NOZ

Fig 5 Effects of BRCA1 R1325K mutation on expression of apoptosis-related proteins in gallbladder cancer cell lines GBC-SD and NOZ

TrendMD

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