Wip1基因重组慢病毒表达载体的构建及其对乳腺癌MCF-7细胞生物学行为的影响研究

发布时间：2018-04-20 01:21

本文选题：Wip + 乳腺肿瘤　；参考：《重庆医学》2017年33期

【摘要】：目的构建Wip1基因重组慢病毒表达载体,研究其对乳腺癌细胞生物学行为的影响。方法以慢病毒感染方法将Wip 1的短发夹状RNA(shRNA)转入乳腺癌MCF-7细胞。采用qRT-PCR和蛋白质印迹法(Western blot)检测转染前后细胞Wip1mRNA、蛋白的表达,MTT法、流式细胞术及Transwell侵袭实验检测Wip1-shRNA对MCF-7细胞增殖、凋亡、周期及侵袭转移的影响。筛选具有抑制p53基因表达的干扰RNA分子p53dsRNA,并分析其干扰后对乳腺癌MCF-7细胞侵袭转移的影响。结果转染48h后,Wip1-shRNA组细胞荧光较强,NC-shRNA组较弱。Wip1-shRNA组细胞Wip1mRNA和蛋白的表达分别为0.291±0.025、0.203±0.021与NC-shRNA组0.954±0.090、0.963±0.092比较,差异有统计学意义(P0.05)。两组各时间点细胞存活率比较,差异有统计学意义(P0.05)。NC-shRNA组早期与晚期细胞的凋亡数少于Wip1-shRNA组,差异有统计学意义(P0.05)。NC-shRNA组细胞G0+G1期、S期细胞数分别为53.5±3.6、27.3±1.5,Wip1-shRNA组分别为72.3±5.2、14.6±0.8,差异有统计学意义(P0.05)。Transwell侵袭转移结果表明,NC-shRNA组细胞的穿膜数均多于Wip1-shRNA组(P0.05)。对照组细胞中p53mRNA的表达、细胞侵袭转移的穿膜数与p53dsRNA组比较,差异有统计学意义(P0.05)。结论 RNA干扰可有效抑制乳腺癌MCF-7细胞Wip1表达,Wip1可能通过调控蛋白表达影响乳腺癌细胞增殖、凋亡、周期与侵袭转移,p53dsRNA干扰p53基因下调后增加乳腺癌MCF-7细胞侵袭转移的能力。
[Abstract]:Objective to construct the recombinant lentivirus expression vector of Wip1 gene and study its effect on the biological behavior of breast cancer cells. Methods the short hairpin RNAs of Wip 1 were transfected into breast cancer MCF-7 cells by lentivirus infection. Wip1mRNAs before and after transfection were detected by qRT-PCR and Western blot. The effects of Wip1-shRNA on proliferation, apoptosis, cell cycle, invasion and metastasis of MCF-7 cells were detected by flow cytometry and Transwell invasion assay. To screen the interfering RNA molecule p53dsRNAs which can inhibit p53 gene expression and analyze the effect of the interference on the invasion and metastasis of breast cancer MCF-7 cells. Results the expression of Wip1mRNA and protein in Wip1-shRNA group was 0.291 卤0.025 卤0.021 and 0.954 卤0.090 卤0.963 卤0.092 in Wip1-shRNA group, respectively. There was significant difference in the expression of Wip1mRNA and protein between Wip1-shRNA group and NC-shRNA group (P 0.05). The cell survival rate at each time point in the two groups was significantly lower than that in the Wip1-shRNA group, and the number of apoptosis in the early and late stage of NC-shRNA group was lower than that in the Wip1-shRNA group. The number of S phase cells in G0 G 1 phase of NC-shRNA group was 53.5 卤3.6N 27.3 卤1.5% Wip1-shRNA group was 72.3 卤5.2U 14.6 卤0.8, respectively. The difference was statistically significant (P 0.05) .Transwell invasion and metastasis results showed that NC-shRNA group had more penetrating membrane number than Wip1-shRNA group (P 0.05). The expression of p53mRNA and the number of cell invasion and metastasis in the control group were significantly higher than those in the p53dsRNA group (P 0.05). Conclusion RNA interference can effectively inhibit the expression of Wip1 in breast cancer MCF-7 cells and may affect the proliferation, apoptosis, cell cycle and invasion and metastasis of breast cancer MCF-7 cells after p53dRNA interference p53 gene down-regulates the ability of invasion and metastasis of breast cancer MCF-7 cells.
【作者单位】：河北省唐山市工人医院乳腺外科;河北省唐山市妇幼保健院妇产科;河北省唐山市人民医院放化疗科;
【基金】：2015年度河北省医学科研重点课题计划(20150953)
【分类号】：R737.9

【相似文献】