摘要
目的利用RNA干扰(RNA interference,RNA i)技术下调结肠癌细胞THC-8307中REV3表达,观察细胞凋亡并探讨p53在该凋亡过程中的作用。方法以人高分化结肠癌细胞系(THC-8307)为研究对象,进行REV3及p53干扰质粒的转染。通过实时荧光定量PCR技术(qRT-PCR)和免疫荧光技术检测干扰效率。利用流式细胞仪检测细胞凋亡率,以qRT-PCR和Western blot检测各组p53的表达量。同时,对REV3及p53进行两质粒共转染实验,同时下调REV3和p53后两组细胞的凋亡率。结果下调REV3诱导THC-8307细胞早期凋亡率和晚期凋亡率明显增加(P<0.01),且实验组p53表达量明显增高;REV3-p53同时下调与REV3单独下调相比,细胞凋亡率明显减低。结论下调REV3通过激活p53诱导结肠癌细胞THC-8307凋亡。
Objective To down- regulate the expression of REV3 through RNA interference in colorectal carcinoma cells and THC- 8307,and to observe cellar apoptosis and to explore the roles of p53 in the process of apoptosis. Methods The human well- differentiated colon cancer cell lines（ THC- 8307） were targeted.The interfering fragments of REV3 and p53 were designed and the plasmids of REV3- miRNA- GFP and p53- miRNA- RFP were transfected through lipo 2000. The interfering efficiency was detected via quantitative real- time polymerase chain reaction（ qRT- PCR） and immunofluorescence（ IF）. The cell apoptosis was examined with flow cytometry and the expression of p53 was detected through qRT- PCR and Western blot. To further clear the functions of p53 in this process of apoptosis,the experiment of co- transfection of REV3-siRNA and p53- siRNA were designed to compare the apoptosis of this group to the group of single transfection of REV3- miRNA. Results The early apoptosis and advanced apoptosis increased significantly after REV3 down regulation（ P〈0. 01）,and the expression of p53 raised significantly. Compared REV3- p53 co- inhibition to REV3 single- inhibition,the rate of apoptosis decreased significantly. Conclusion The down- regulation of REV3 induces apoptosis in THC- 8307,which is the results of activating p53.
出处
《宁夏医科大学学报》
2015年第3期237-241,F0004,共6页
Journal of Ningxia Medical University
基金
国家自然科学基金(31360251)
