Abstract:
Objective
To study the effect of knocking down miR-221/222 expression and up-regulating p27kip1 expression on the radiosensitivity of human MCF-7 breast cancer cell line.
Methods
The seed sequences of miRNA-221/222 and their relationship with p27kip1 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 p27kip1 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 p27kip1 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 p27kip1 was obviously up-regulated in the antisense miR-221/222 cotransfection group(P=0.000).
Conclusions
Through up-regulating p27kip1,anti-miR-221/222 can enhance the radio-sensitivity of human MCF-7 breast cancer cell line.
Key words:
miR-221,
miR-222,
Breast neoplasms,
p27kip1,
Radiosensitivity
Mei MEI, Yu REN, Xuan ZHOU, Yan-bin QI, Hong-mei WANG, Hao ZHANG, Xiao-yong SHEN, Chuan ZHAO, Zheng SU, Zhi YAO. Up-regulating p27kip1 by antisense miR-221/222 enhances the radiosensitivity of MCF-7 breast cancer cell line[J]. Chinese Journal of Breast Disease(Electronic Edition), 2009, 03(06): 622-632.