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

Construction of Wip1 gene recombinant lentiviral expression vector and its effects on breast cancer MCF-7 cell biological behavior

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(P<0.05)。两组各时间点细胞存活率比较,差异有统计学意义(P<0.05)。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(P<0.05)。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(P<0.05)。Transwell侵袭转移结果表明,NC-shRNA组细胞的穿膜数均多于Wip1-shRNA组(P<0.05)。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(P<0.05)。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。

Objective To construct the Wip1 gene recombinant lentiviral expression vector and to investigate its effects on breast cancer cell biological behaviors.Methods Wip1 gene short hairpin RNAs（shRNA）was transfected into breast cancer MCF-7 cells through lentiviral infection method.Wip1 mRNA and protein expressions before and after transfection were detected by using qRT-PCR and Western blotting.The effects of Wip1-shRNA on the proliferation,apoptosis,cell cycle,invasion and metastasis in MCF-7 cells were determined by using the MTT assay,flow cytometry and transwell invasion test.Interfering RNA molecule p53 dsRNA inhibiting p53 gene expression（p53 dsRNA）was screened,and the effect of p53 inhibition on MCF-7 cells invasion and metastasis was analyzed.Results After transfection for 48 h,the cellular fluorescence in the Wip1-shRNA group was stronger,while which in the NC-shRNA group was weaker.Cellular Wip1 mRNA and protein expressions in the Wip1 shRNA group were0.291±0.025 and 0.203±0.021 respectively,which in the NC-shRNA group were 0.954±0.090 and 0.963±0.092 respectively,the difference between the two groups was statistically significant（P〈0.05）.The cellular survival rate at various time points had statistical difference between the two groups（P〈0.05）.The early and late cell apoptosis number in the NC-shRNA group was less than that in the Wip1-shRNA group,the difference was statistically significant（P〈0.05）.The cells numbers at phase G0＋G1 and phase S in the NC-shRNA group were 53.5±3.6 and 27.3±1.5 respectively,which in the Wip-shRNA group were 72.3±5.2 and14.6±0.8 respectively,the difference was statistically significant（P〈0.05）.The Transwell invasion and metastasis results showed that the cell transmembrane number in the NC-shRNA group was more than that in the Wip1-shRNA group（P〈0.05）.The cellular p53 mRNA and protein expression had statistical difference between the control group and p53 dsRNA group（P〈0.05）.Conclusion RNA interference can effectively suppress Wip1 expression in MCF-7 cells.Wip1 may affect the proliferation,apoptosis,cell cycle,invasion and metastasis of breast cancer cells by modulating protein expression.p53 dsRNA increases the invasion and metastasis ability of breast cancer MCF-7 cells by interfering p53 gene down-regulation.