摘要
目的探讨miR-345影响胃癌细胞生长周期的作用机制。方法采用脂质体介导转染miR-345反义寡核苷酸至胃癌细胞MGC803、MKN45以沉默miR-345表达,采用实时英光定量PCR(Real-time PCR)、蛋白免疫印迹(Western blotting)检测胃癌细胞中p21Cip1水平,随后检测CDK4、CDK6、Cyclin D1、p-Rb、Rb蛋白水平,进一步克隆形成实验比较其增殖能力,流式细胞术检测miR-345对胃癌细胞周期的影响。结果脂质体介导转染miR-345反义寡核苷酸至胃癌细胞MGC803、MKN45可以抑制miR-345的表达;p21Cip1的mRNA和蛋白水平在低miR-345表达的胃癌细胞系中升高;下调miR-345的表达后MGC803、MKN45细胞中CDK4、CDK6、Cyclin D1和总Rb蛋白的表达水平无明显变化,但Rb蛋白的磷酸化水平显著降低。结论沉默miR-345的表达能够明显阻滞胃癌细胞从G1期向S期的进程,减慢细胞生长速度;其机制与上调p21Cip1进而抑制Rb蛋白的磷酸化有关。
Objective To investigate the the effect of miR-345 in the growth cycle of gastric cancer cell .Methods miR-345 antisense oligonucleotides were transfected into MGC 803, MKN45 cells.Real-time PCR was conducted to de-tect the expression of miR-345 in transfected cells.The expression of p21^Cip1, CDK4, CDK6, Cyclin D1, p-Rb, Rb were examined by Western blotting and Real-time PCR.Then the cell cycle progression was examined by flow cytome-try.Results The expression of miR-345 in MGC803, MKN45 cells transfected with antisense miR-345 was significantly reduced.miR-345 knockdown could up-regulate the expression of p21^Cip1 and the level of p-Rb was significantly re-duced, but no obvious change in the levels of the CDK 4, CDK6, Cyclin D1, Rb.The cell proliferation activity of MGC803, MKN45 cells was inhibited.The cell cycle was arrested in G1 phase.Conclusion miR-345 knockdown can obviously block the process of gastric cancer cells from G 1 phase to S phase .Its mechanism is associated with up-regula-ting p21^Cip1 and suppressing Rb protein phosphorylation .
出处
《胃肠病学和肝病学杂志》
CAS
2015年第4期398-401,共4页
Chinese Journal of Gastroenterology and Hepatology
基金
广东省科技计划资助项目(2012B061700026)