收稿日期: 2023-08-28
网络出版日期: 2023-12-11
基金资助
贵州省科技计划项目（编号：黔科合基础-ZK［2022］一般419）
FGF2 gene knockout inhibits MCF⁃7 cell proliferation， motility and invasion while promotes cell apoptosis
Received date: 2023-08-28
Online published: 2023-12-11
目的 探索成纤维细胞生长因子2（FGF2）敲除对人乳腺癌细胞株MCF-7增殖、迁移侵袭和凋亡的影响及作用机制。 方法 将FGF2-sgRNA基因序列转入载体lenticrispv2 构建CRISPR-cas9-FGF2稳转细胞系，Western blot 检测CRISPR-cas9-FGF2稳转细胞系FGF2的蛋白敲除情况及相关凋亡蛋白指标和周期蛋白指标，将MCF-7细胞分为CRISPR-cas9-NC组（对照组）和CRISPR-cas9-FGF2组（FGF2基因敲除组），使用CCK-8实验和细胞集落形成实验检测细胞增殖能力，细胞划痕实验和transwell实验检测细胞迁移侵袭能力，流式细胞仪检测细胞周期的变化。 结果 （1）设计的三条FGF2-sgRNA序列均与载体lenticrispv2 连接成功。（2）用Western blot 检测表明FGF2-sgRNA1敲除作用更明显（P < 0.000 1）。（3）与对照组细胞相比，FGF2基因敲除组细胞的增殖、迁移侵袭能力明显降低（P < 0.01）；周期蛋白CDK2和cyclinA蛋白表达显著降低（P < 0.001），Bax促凋亡蛋白表达升高（P < 0.001），Bcl-2抑凋亡蛋白表达降低（P < 0.001），Bcl-2/Bax的比值减低（P < 0.000 1）促进细胞凋亡。 结论 FGF2基因敲除后明显抑制乳腺癌细胞的增殖和迁移侵袭，将细胞阻滞在S期，抑制细胞的增殖与分裂，并促进细胞凋亡，提示FGF2基因具有成为乳腺癌治疗的潜在分子靶点。
关键词： 成纤维细胞生长因子2; CRISPR/cas9; 乳腺癌; 细胞迁移; 细胞凋亡
张正花 , 龚荣府 , 方文 . 敲除成纤维细胞生长因子2基因抑制乳腺癌MCF-7细胞的增殖、迁移侵袭并促进细胞凋亡[J]. 实用医学杂志, 2023 , 39(22) : 2885 -2890 . DOI: 10.3969/j.issn.1006-5725.2023.22.005
Objective To explore the effect and mechanism of fibroblast growth factor 2 （FGF2） knockout on proliferation， migration， invasion and apoptosis of human breast cancer cell line MCF?7. Methods FGF2?sgRNA gene sequence was inserted into the vector lenticrispv2 to create CRISPR?cas9?FGF2 stably transfected cell line， and FGF2 knockout in the cell lines and related apoptotic protein and cyclin indicators were detected by using Western blot. MCF?7 cells were separated into two groups： CRISPR?cas9?NC （control） and CRISPR?cas9?FGF2 （FGF2 knockout）. CCK?8 assay and cell colony formation assay were used to detect cell proliferation ability， so were cell scratch assay and Transwell assay to detect cell migration and invasion ability， and flow cytometry to detect changes in cell cycles. Results （1） All three designed FGF2?sgRNA sequences were successfully linked to the carrier lenticrispv2. Western blot showed that FGF2?sgRNA1 knockout effect was more obvious （P < 0.000 1）. The cellular proliferation， migration， and invasion ability were markedly lowered in the FGF2 knockout group as compared with the control group （P < 0.01）. Expressions of Cyclin CDK2 and cyclinA were dramatically reduced （P < 0.001）， the expression of pro?apoptotic protein Bax was increased （P < 0.001）， whereas the expression of anti?apoptotic protein Bcl?2 was decreased （P < 0.001）. A decline in the ratio of Bcl?2/Bax promoted apoptosis. Conclusions FGF2 gene knockout significantly inhibited proliferation， migration， and invasion of breast cancer cells， blocked cells in the S phase， inhibited cell division， and promoted cell apoptosis， indicating that FGF2 gene has become a potential molecular target for breast cancer therapy.
Key words： fibroblast growth factor 2; CRISPR/cas9; breast cancer; cell migration; apoptosis
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