论文部分内容阅读
目的探讨敲低miR-221/222表达上调p27kip1对MCF-7人乳腺癌细胞系放射敏感性的影响。方法经生物信息学分析查询miR-221/222成熟体序列和它们与p27kip1的关系。脂质体共转染反义寡聚核苷酸(反义miR-221/222)后,用Northern blot法检测转染后MCF-7细胞miR-221、miR-222表达水平;将实验细胞分为6组:对照组、对照照射组、无义序列组、无义序列照射组、反义miR-221/222共转染组和反义miR-221/222共转染照射组。用MTT法检测细胞增殖及放射协同作用,流式细胞仪分析细胞周期,克隆形成实验检测细胞增殖,Western blot分析p27kip1蛋白的表达变化。数据间的方差分析采用F检验;两两比较采用LSD-t检验。结果经生物信息学分析显示miR-221/222成熟体序列的种子序列几乎一致,p27kip1是miR-221/222的靶基因。Northern blot显示反义miR-221/222共转染组miR-221、miR-222的表达水平明显下降(miR-221:P=0.000;miR-222:P=0.000)。对照组及无义序列组之间miR-221、miR-222表达水平的差异无统计学意义(miR-221:P=0.371;miR-222:P=0.284)。MTT结果显示转染后第4天共转染组肿瘤细胞生长抑制效果最好,细胞增殖率明显低于对照组和无义序列组(P=0.000),但与放射治疗无协同作用(P=0.091)。流式细胞术检测可见共转染组细胞周期存在G0/G1期阻滞(P=0.000)。经放射治疗后,可明显降低S期比例(P=0.002)。克隆形成实验表明反义miR-221/222可增加MCF-7细胞的放射敏感性。Western blot显示反义miR-221/222共转染组的p27kip1蛋白表达明显上调(P=0.000)。结论反义miR-221/222通过上调p27kip1蛋白表达可以增加MCF-7人乳腺癌细胞系放射敏感性。
Objective To investigate the effect of knockdown of miR-221/222 expression on the radiosensitivity of p27kip1 in MCF-7 human breast cancer cell lines. Methods The bioinformatics analysis was used to investigate the sequence of miR-221/222 mature body and their relationship with p27kip1. Lipofectamine was transfected into antisense oligonucleotides (antisense miR-221/222). Northern blot was used to detect the expression of miR-221 and miR-222 in MCF-7 cells. Control group, control group, nonsense group, nonsense group, antisense miR-221/222 co-transfection group and antisense miR-221/222 co-transfection group. Cell proliferation and radiosensitivity were detected by MTT assay. Cell cycle was analyzed by flow cytometry. Clone formation assay was used to detect cell proliferation. The expression of p27kip1 protein was analyzed by Western blot. Analysis of variance between the data using F test; pairwise comparison using LSD-t test. Results Bioinformatics analysis showed that the seed sequence of miR-221/222 was almost identical, and p27kip1 was the target gene of miR-221/222. Northern blot showed that miR-221 and miR-222 expression were significantly decreased in antisense miR-221/222 co-transfected cells (miR-221: P = 0.000; miR-222: P = 0.000). There was no significant difference in the expression levels of miR-221 and miR-222 between the control group and the nonsense sequence group (miR-221: P = 0.371; miR-222: P = 0.284). The MTT results showed that the tumor cells in the cotransfected group had the best growth inhibitory effect on day 4 and the cell proliferation rate was significantly lower than that of the control group and the nonsense sequence group (P = 0.000), but no synergistic effect with radiotherapy (P = 0.091). Flow cytometry showed that the cell cycle of co-transfection group had G0 / G1 phase arrest (P = 0.000). After radiotherapy, can significantly reduce the proportion of S phase (P = 0.002). Clonal formation experiments showed that antisense miR-221/222 increased the radiosensitivity of MCF-7 cells. Western blot showed that the expression of p27kip1 in antisense miR-221/222 co-transfection group was significantly up-regulated (P = 0.000). Conclusion Antisense miR-221/222 can increase the radiosensitivity of MCF-7 human breast cancer cell lines by up-regulating p27kip1 protein expression.