BRCA1 R1325K突变对胆囊癌细胞增殖及凋亡的影响

doi:10.3969/j.issn.1674-8115.2023.09.001

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (9): 1071-1079.doi: 10.3969/j.issn.1674-8115.2023.09.001

• 论著 · 基础研究 •

BRCA1 R1325K突变对胆囊癌细胞增殖及凋亡的影响

杨婧潇¹(), 贾子尧¹, 吴文广¹, 吴向嵩²^,³, 张飞²^,³, 李怀峰²^,³, 朱逸荻²^,³, 李茂岚¹()

^1.上海交通大学医学院附属仁济医院胆胰外科，上海 200127
^2.上海交通大学医学院附属新华医院普外科，上海 200092
^3.上海市胆道疾病研究中心，上海 200092

收稿日期:2023-01-03 接受日期:2023-08-29 出版日期:2023-09-28 发布日期:2023-09-28
通讯作者: 李茂岚 E-mail:yang_jingxiao@126.com;limaolan6@163.com
作者简介:杨婧潇（1998—），女，硕士生；电子信箱：yang_jingxiao@126.com。
基金资助:
国家重点研发计划(2021YFE0203300);国家自然科学基金面上项目(82073206);上海市卫生健康委员会健康产业临床研究专项(20224Z0014);上海市教育委员会曙光项目(20SG14);上海市科学技术委员会基础研究项目(20JC1419100);上海市申康医院发展中心临床科技创新项目(SHDC12019110);上海市消化疾病临床医学研究中心分中心(19MC1910200);上海交通大学医学院“双百人”项目(20181808)

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

YANG Jingxiao¹(), JIA Ziyao¹, WU Wenguang¹, WU Xiangsong²^,³, ZHANG Fei²^,³, LI Huaifeng²^,³, ZHU Yidi²^,³, LI Maolan¹()

^1.Department of Biliary-Pancreatic Surgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
^2.Department of General Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
^3.Shanghai Key Laboratory of Biliary Tract Disease Research, Shanghai 200092, China

Received:2023-01-03 Accepted:2023-08-29 Online:2023-09-28 Published:2023-09-28
Contact: LI Maolan E-mail:yang_jingxiao@126.com;limaolan6@163.com
Supported by:
National Key Research and Development Program of China(2021YFE0203300);National Natural Science Foundation of China(82073206);Shanghai Municipal Health Commission Health Industry Clinical Research Special Project(20224Z0014);Shuguang Program of Shanghai Education Development Foundation and Shanghai Municipal Education Commission(20SG14);Basic Research Project of Science and Technology Commission of Shanghai Municipality(20JC1419100);Clinical Science and Technology Innovation Project of Shanghai Shenkang Hospital Development Center(SHDC12019110);Shanghai Clinical Medical Research Center for Digestive Diseases(19MC1910200);“Two-hundred Talents” Program of Shanghai Jiao Tong University School of Medicine(20181808)

摘要/Abstract

摘要：

目的·探究乳腺癌易感基因1（breast cancer susceptibility gene 1，BRCA1）第1325位精氨酸变为赖氨酸（R1325K突变）对胆囊癌细胞系GBC-SD和NOZ增殖和凋亡的影响。方法·使用BRCA1野生型过表达慢病毒、BRCA1 R1325K突变过表达慢病毒以及阴性对照慢病毒载体构建胆囊癌细胞系GBC-SD和NOZ稳转株。细胞分为不含目的基因的对照组、BRCA1野生型组及BRCA1突变组，并通过Western blotting验证目的蛋白BRCA1的表达情况。选用针对BRCA1突变的抑制剂奥拉帕利（Olaparib）20 μmol/L处理BRCA1突变组胆囊癌细胞，并根据目的蛋白的表达情况和加药与否将胆囊癌细胞系分为对照组、BRCA1野生型组、BRCA1突变组和BRCA1突变+Olaparib组。通过CCK8实验和克隆形成实验观察BRCA1 R1325K突变对胆囊癌细胞系GBC-SD和NOZ增殖能力及克隆形成能力的影响，通过TUNEL实验观察BRCA1 R1325K突变对胆囊癌细胞系GBC-SD和NOZ凋亡情况的影响，并通过Western blotting检测凋亡相关蛋白cleaved PARP、Bcl-2和Bax的表达情况。使用抑制剂Olaparib处理BRCA1 R1325K突变过表达胆囊癌细胞系GBC-SD和NOZ，并检测BRCA1 R1325K突变引起的相应表型改变（促进增殖、增强克隆形成能力和抑制凋亡）是否能被抑制剂所逆转。结果·通过CCK8实验和克隆形成实验发现：相较于对照组及BRCA1野生型组，BRCA1 R1325K突变能够促进胆囊癌细胞系GBC-SD和NOZ的增殖，并提高其克隆形成能力；抑制剂Olaparib处理则能够抑制BRCA1突变胆囊癌细胞系的增殖（均P<0.05）。通过TUNEL和Western blotting实验发现：相较于对照组，野生型BRCA1基因过表达能够诱导胆囊癌细胞系GBC-SD和NOZ的凋亡；BRCA1突变组相较于对照组和BRCA1野生型组，有抵抗凋亡的作用，且升高了凋亡抑制蛋白Bcl-2的表达并降低了促凋亡蛋白Bax的表达（P<0.05）。结论·BRCA1 R1325K突变能够促进胆囊癌细胞系GBC-SD和NOZ的增殖并抑制其凋亡。

关键词: 胆囊癌, BRCA1, 突变, 增殖, 凋亡

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

中图分类号:

R735.8

杨婧潇, 贾子尧, 吴文广, 吴向嵩, 张飞, 李怀峰, 朱逸荻, 李茂岚. BRCA1 R1325K突变对胆囊癌细胞增殖及凋亡的影响[J]. 上海交通大学学报（医学版）, 2023, 43(9): 1071-1079.

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.

图/表 5

图1 BRCA1 野生型及 BRCA1 R1325K突变过表达慢病毒感染后胆囊癌细胞系GBC-SD和NOZ中BRCA1蛋白的表达情况Note： A. Western blotting was used to detect BRCA1 protein expression in GBC-SD cell line. B. Western blotting was used to detect BRCA1 protein expression in NOZ cell line. ①P=0.000, ②P=0.003, ③P=0.001.

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

图2 BRCA1 R1325K突变对胆囊癌细胞系GBC-SD和NOZ增殖能力的影响Note： A. Proliferation curves of GBC-SD cell line. B. Proliferation curves of NOZ cell line. ①P=0.002, ②P=0.000, ③P=0.001.

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

图3 BRCA1 R1325K突变对胆囊癌细胞系GBC-SD和NOZ克隆形成能力的影响Note： A. The representative pictures of colony assay in GBC-SD cell line. B. The representative pictures of colony assay in NOZ cell line. C. The statistical graph of colony numbers for GBC-SD cell line. D. The statistical graph of colony numbers for NOZ cell line. ①P=0.015, ②P=0.008, ③P=0.000, ④P=0.001.

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

图4 BRCA1 R1325K突变对胆囊癌细胞系GBC-SD和NOZ凋亡的影响Note： A. The representative pictures of TUNEL assay in GBC-SD cell line. B. The representative pictures of TUNEL assay in NOZ cell line. C. The statistical graph of apoptosis ratio for GBC-SD cell line. D. The statistical graph of apoptosis ratio for NOZ cell line. ①P=0.001, ②P=0.000.

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

图5 BRCA1 R1325K突变对胆囊癌细胞系GBC-SD和NOZ凋亡相关蛋白表达情况的影响Note： A. Effects of BRCA1 R1325K mutation on expression of apoptosis-related proteins in GBC-SD cell line. B. Effects of BRCA1 R1325K mutation on expression of apoptosis-related proteins in NOZ cell line. cPARP—cleaved PARP. ①P=0.000, ②P=0.004, ③P=0.002, ④P=0.009.

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

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BRCA1 R1325K突变对胆囊癌细胞增殖及凋亡的影响

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

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