缺氧条件下沉默解聚素-金属蛋白酶17基因对人乳腺癌MCF-7细胞增殖的影响

Effect of silencing ADAM17 gene on proliferation of human breast cancer MCF-7 cells under hypoxia

摘要:

目的  探讨在缺氧条件下靶向针对解聚素-金属蛋白酶17（ADAM17）基因的短发夹RNA（shRNA）对人乳腺癌MCF-7细胞增殖的作用机制。方法  针对ADAM17基因设计具有特异性ADAM17-shRNA，经电穿孔转染MCF-7细胞。依据细胞培养条件（常氧和缺氧）和细胞转染因素（空白PBS、转染无义序列ADAM17-shNC、转染ADAM17-shRNA），实验分为常氧对照组、常氧shNC组、常氧shRNA组、缺氧对照组、缺氧shNC组和缺氧shRNA组。通过充入1%氧O2、5%二氧化碳CO2和94%氮N2的混合气体培养箱模拟缺氧环境，分别采用实时荧光定量聚合酶链反应（qRT-PCR）、iCELLigence系统、流式细胞仪检测MCF-7细胞的ADAM1基因的表达水平、细胞生长曲线、增殖活性和细胞周期。结果  靶向针对ADAM17基因的ADAM17-shRNA在缺氧条件下可以有效沉默人乳腺癌MCF-7细胞ADAM17基因的表达（缺氧shRNA组2-△△CT=0.55±0.16）。从而导致MCF-7细胞生长速度减慢，细胞增殖能力降低，细胞周期延缓。结论  ADAM17 shRNA和缺氧存在协同作用，共同抑制肿瘤细胞的增殖。

Abstract:

Objective To investigate the effects of short hairpin RNA (shRNA) targeting at a disintegrin and metalloproteinase 17 (ADAM17) gene on proliferation of human breast cancer MCF-7 cells under hypoxia. Methods The specific ADAM17-shRNA expression vector was designed for ADAM17 gene. They were transfected into human breast carcinoma cell line MCF-7 cells by electroporation. Depending on cell culture conditions (normoxia or hypoxia) and cell transfection factors (blank control PBS, transfection with ADAM17-shNC, transfection with ADAM17-shRNA), experiment groups were divided into normoxic control group, normoxic shNC group, normoxic shRNA group, hypoxic control group, hypoxic shNC group and hypoxic shRNA group. Hypoxic environment was acquired by a three gas incubator filled with 1% O2, 5% CO2 and 94% N2. qRT-PCR was used to study the expression levels of ADAM17. The proliferative ability and cell growth curve of MCF-7 cells were detected by iCELLigence. Cell cycle distribution of MCF-7 cells was analyzed by flow cytometry. Results The specific ADAM17-shRNA expression vector could effectively silence the expression of ADAM17 gene in human breast cancer MCF-7 cells under hypoxia [hypoxic shRNA group 2-△△CT = (0.55 ± 0.16)]. The proliferative ability and cell growth speed of MCF-7 cells were inhibited and the cell cycle was delayed by shRNA transfection and hypoxic incubation. Conclusions It suggests that the synergistic effect of ADAM17-shRNA and hypoxia inhibits the proliferation of tumor cells.