COL1A1敲除抑制卵巢癌细胞 ES-2增殖  

Effect of COL1A1 deletion on proliferation of ovarian cancer ES-2 cells

作　　者：钱若文轩 杨柳青 石子晴 陈子彦 胡汉寅 邹尚朴 朱恒延 潘巍巍[1] QIAN Ruo-wen-xuan;YANG Liu-qing;SHI Zi-qing;Chen Zi-yan;HU Han-yin;ZOU Shang-pu;ZHU Heng-yan;Pan Wei-wei(Department of Biology,College of Medicine,Jiaxing University,Jiaxing 314001,China)

机构地区：[1]嘉兴学院医学院生物教研室

出　　处：《中国病理生理杂志》2020年第1期59-66,共8页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.31871402; No.81402162);浙江省自然科学基金资助项目(No.LY17H160060);浙江省大学生科技创新项目(No.2018R417024);嘉兴学院重点SRT项目(No.NH85178445; No.NH85179378; No.NH85179377; No.851719108; No.851719496)

摘　　要：目的:探讨Ⅰ型胶原α1链(collagen type I alpha 1 chain,COL1A1)基因缺失是否抑制卵巢癌细胞的增殖。方法:构建COL1A1基因敲除的载体PX459-COL1A1,转染人卵巢癌ES-2细胞并用嘌呤霉素筛选,获得COL1A1敲除的ES-2细胞。用基因组测序方法检测COL1A1基因的编辑;细胞增殖、集落形成和细胞迁移实验检测COL1A1缺失对卵巢癌细胞增殖、集落形成及迁移的影响;细胞周期和细胞凋亡实验检测COL1A1缺失对卵巢癌细胞周期和凋亡的影响。裸鼠荷瘤模型体内实验研究COL1A1基因敲除对卵巢癌细胞增殖的影响。结果:Western blot和DNA测序结果显示CRISPR/Cas9方法可以编辑ES-2细胞的COL1A1基因,从而抑制COL1A1蛋白表达。COL1A1缺失显著抑制细胞增殖、软琼脂集落形成能力和细胞迁移(P<0.01)。此外,COL1A1缺失将卵巢癌细胞阻滞于G2期(P<0.01),并促进细胞凋亡(P<0.01)。分子机制研究表明COL1A1缺失可以明显抑制成骨细胞特异性转录因子osterix和基质金属蛋白酶13(matrix metalloproteinase-13, MMP-13)表达(P<0.01),促进聚集蛋白聚糖酶1(aggrecanase-1)表达(P<0.01)。裸鼠荷瘤模型结果表明COL1A1缺失抑制肿瘤生长速度。结论:COL1A1缺失可以通过影响多种基因表达而抑制肿瘤细胞增殖,促进细胞凋亡。AIM: To explored the role of collagen type I alpha 1 chain(COL1 A1) deletion in proliferation and apoptosis of human ovarian cancer ES-2 cells. METHODS: CRISPR/Cas9-mediated COL1 A1 gene knockout vector was constructed and transfected into ES-2 cells, and the ES-2 cells with COL1 A1 knockout were obtained by puromycin screening. Genomic sequencing was used to verify the efficiency of knockout. Cell proliferation, colony formation and cell migration were detected in COL1 A1 knockout ovarian cancer cells. Cell apoptosis was analyzed by PI-annexin V staining and flow cytometry. Nude mouse model was used to detect the tumorigenic capacity of COL1 A1 knockout cells in vivo. RESULTS: COL1 A1 deletion inhibited the proliferation and migration of ovarian cancer cells(P<0.01). COL1 A1 deletion blocked the G2 phase of ovarian cancer cell cycle and promoted apoptosis(P<0.01). COL1 A1 deletion significantly inhibited the expression of osteoblast-specific transcription factor osterix and matrix metalloproteinase-13, and promoted the expression of aggrecanase-1(P<0.01). The results of tumor-bearing model in nude mice showed that COL1 A1 deletion inhibited the growth rate of tumor. CONCLUSION: COL1 A1 deletion inhibits the proliferation and promotes the apoptosis of tumor cells by affecting the expression of a variety of genes, thus reducing the malignant transformation of tumor.

关 键 词：I型胶原α1链 卵巢癌 细胞增殖 细胞凋亡 

分 类 号：R730.23[医药卫生—肿瘤] R737.31[医药卫生—临床医学]