miRNA-222调控CDKN1b和CDKN1c表达促进乳腺癌细胞增殖和迁移的机制研究

The Mechanism of miRNA-222 Regulating CDKN1b and CDKN1c to Promote Proliferation and Migration in Breast Cancer Cells

通过检测乳腺癌样本和正常组织样本中miR-222表达量,转染miR-222模拟物(mimics)和抑制物(inhibitors),用CCK-8和Transwell检测miR-222对乳腺癌细胞增殖能力和迁移能力的影响.进一步预测miRNA-222的靶基因,构建其过表达载体和预测靶基因的荧光素酶报告载体.通过双荧光素酶检测系统鉴定靶基因,点突变实验验证与靶基因的结合位点.RT-PCR检测miRNA-222过表达或抑制后对靶基因表达的影响.分析CDKN1b和CDKN1c高、低表达水平与乳腺癌患者生存率的关系.结果表明与正常乳腺组织比较,肿瘤组织中miR-222表达显著上升(P<0.0001),miR-222的表达与淋巴结转移呈正相关.miR-222过表达后能促进乳腺癌细胞的增殖和迁移,反之则会抑制乳腺癌细胞的增殖和迁移.双荧光素酶报告系统显示miRNA-222过表达能显著抑制CDKN1b和CDKN1c的荧光素酶活性,点突变实验证实miR-222通过“种子区”与CDKN1b、CDKN1c的靶位点结合,从而负向调控CDKN1b和CDKN1c的表达.过表达miRNA-222后CDKN1b和CDKN1c mRNA的表达量显著降低(P<0.01).相反,抑制miRNA-222表达后CDKN1b和CDKN1c mRNA的表达量上升(P<0.05).CDKN1b和CDKN1c高表达患者的总体生存率显著高于低表达患者(P<0.05).本研究表明miRNA-222通过负向调控CDKN1b和CDKN1c基因表达,进而影响乳腺癌细胞的增殖和迁移.

In this study,miR-222 expression level in breast cancer samples and normal tissue samples were detected.miR-222 mimics and inhibitors were transfected,the effects of miR-222 on the proliferation and migration ability of breast cancer cells were detected by CCK-8 and Transwell,respectively.The target genes of miRNA-222 were further predicted,the overexpression vector and the luciferase reporter vector were constructed,identified by the dual luciferase detection system,and the binding sites between miRNA-222 and the target genes were verified by point mutation experiment.The effect of miRNA-222 overexpression or inhibition on the expression of target genes was detected by RT-PCR.The relationship between the expression levels of CDKN1b,CDKN1c and survival rate of breast cancer patients was analyzed in effect.The results showed that compared with normal breast tissue,the expression of miR-222 in tumor tissue significantly increased(P<0.0001),and the expression of miR-222 was positively correlated with lymph node metastasis.The overexpression of miR-222 can promote the proliferation and migration of breast cancer cells.The dual luciferase reporting system showed that the overexpression of miRNA-222 could significantly inhibit the luciferase activities of CDKN1b and CDKN1c.Point mutation experiments confirmed that miR-222 binds to the target sites of CDKN1b,CDKN1c through the“seed region.”The expressions of CDKN1b and CDKN1c mRNA significantly decreased after miRNA-222 mimics transfection(P<0.01).On the contrary,the expression levels of CDKN1b and CDKN1c mRNA increased after miRNA-222 expression was inhibited(P<0.05).The overall survival rate of patients with high CDKN1b and CDKN1c expression was significantly higher than that of patients with low expression(P<0.05).This study showed that miRNA-222 negatively regulates the expression of CDKN1b and CDKN1c genes,thereby affecting the proliferation and migration of breast cancer cells.