目的

探讨敲低miR-221/222表达上调p27^kip1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p27^kip1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p27^kip1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,p27^kip1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p27^kip1蛋白表达明显上调(P=0.000)。

结论

反义miR-221/222通过上调p27^kip1蛋白表达可以增加MCF-7人乳腺癌细胞系放射敏感性。

Objective

To study the effect of knocking down miR-221/222 expression and up-regulating p27^kip1 expression on the radiosensitivity of human MCF-7 breast cancer cell line.

Methods

The seed sequences of miRNA-221/222 and their relationship with p27^kip1 were determined by bioinformation analysis.Lipofectamine 2000 was used to transfect miRNA-221/222 antisense oligonucleotides.Northern blot was conducted to detect the expression level of transfected MCF-7 cells of miR-221 and miR-222.There were six groups used in this study,including a control group,a contrast irradiation group,a scramble group,a scramble irradiation group,an antisense miR-221/222 cotransfection group,and antisense miR-221/222 cotransfection irradiation group.Cell proliferation and radiation synergy were determined by MTT.The cell cycle distribution was detected by cell flow cytometry.Clonogenic assay was used to measure the mitotic cell death.Western blot assay was conducted to detect p27^kip1 expression alternation.Statistical analysis was performed using analysis of variance and LSD test.

Results

Bioinformation analysis showed that the seed sequences of miRNA-221/222 were almost the same,and p27^kip1 was a target gene of miRNA-221/222.Northern blot showed the expression levels of miR-221 and miR-222 were significantly knocked down in the antisense miR-221/222 cotransfection group(miR-221:P=0.000;miR-222:P=0.000),but no significant change was observed between the control group and the scramble group(miR-221:P=0.371;miR-222:P=0.284).MTT showed that 4 days after transfection,the growth of MCF-7 cells treated with antisense miR221/222 was dramatically suppressed in the cotransfection group compared with the scramble group(P=0.000),but there was no synergy with irradiation(P=0.091).The cell cycle was arrested in G₀/G₁ phase in the cotransfection group(P=0.000).The S phase rate reduced after the cells were treated with irradiation(P=0.002).Anti-miR-221/222 could enhance the radiosensitivity of MCF-7 cells.Western blot analysis showed the expression of p27^kip1 was obviously up-regulated in the antisense miR-221/222 cotransfection group(P=0.000).

Conclusions

Through up-regulating p27^kip1,anti-miR-221/222 can enhance the radio-sensitivity of human MCF-7 breast cancer cell line.