摘要
旨在构建SGK3基因RNAi慢病毒表达载体,观察其对人乳腺癌细胞MB-474增殖和凋亡的影响。构建靶向SGK3的shRNA序列慢病毒载体,将其转染至人乳腺癌MB-474细胞以沉默SGK3基因,Real-time PCR、Western bloting方法检测MB-474细胞SGK3 mRNA及蛋白表达,MTT法和流式细胞术检测SGK3基因沉默对MB-474细胞增殖、细胞周期和凋亡的影响。测序结果显示,成功构建4组SGK3基因RNAi慢病毒表达载体,经293T细胞包装,病毒滴度为(3-8)×108 TU/m L;将慢病毒转染MB-474细胞后,Real-time PCR、Western bloting结果显示,干扰组SGK3的表达水平较对照组均降低,且PGC-LV3-SGK3-1序列对其干扰效果最佳,SGK3基因沉默可抑制MB-474细胞增殖、促进凋亡,但其对细胞周期进程无明显影响。成功构建靶向SGK3基因的RNAi慢病毒载体,SGK3基因沉默可影响乳腺癌细胞增殖和凋亡等生物学行为。
This study is aimed to construct the lentiviral RNA interference （ RNAi ） vector of SGK3 gene, and to observe its effect on the proliferation and apoptosis of human breast cancer cell line MB-474. The lentiviral vector of shRNA sequence targeting SGK3 was constructed and transfected into human breast cancer MB-474 cells to silence the SGK3 gene. The expression of SGK3 mRNA and protein in transfected MB-474 ceils was detected by real-time PCR and Western bloting, and the effect of SGK3 gene silencing on the proliferation, cell cycle and apoptosis of MB-474 cells was detected by M33＂ assay and flow cytometry. Gene sequencing showed that 4 RNAi lentiviral vectors targeting the SGK3 gene were successfully constructed. The lentiviral vectors were packed successfully by 293T cells and the virus titer was（ 3-8 ） × 108 TU/mL. After the recombinant lentiviral vector was transfected into MB-474 cells, real-time PCR and Western bloting demonstrated that the expression of SGK3 in interference group was lower than that of control group. Among the 4 lentiviral vectors, the one with best interference effect is the PGC-LV3-SGK3-1 sequence. SGK3 silencing inhibited MB-474 cell proliferation and promoted apoptosis, but had no significant effect on cell cycle progression. In short, RNAi lentivirus vector targeting SGK3 gene was successfully constructed, and SGK3 gene silencing affected cell proliferation and apoptosis of breast cancer.
出处
《生物技术通报》
CAS
CSCD
北大核心
2018年第1期247-252,共6页
Biotechnology Bulletin
基金
黑龙江省教育厅科学技术研究项目(2016-KYYwF-0836)
