下调胰岛素样生长因子抑制miR-767-5p的表达对乳腺癌细胞增殖、迁移、侵袭及上皮间充质转化的影响

Suppression of miR-767-5p expression of inhibits breast cancer cell proliferation,migration,invasion and EMT through down-regulating IGF1

目的研究抑制miR-767-5p对乳腺癌细胞增殖、迁移、侵袭和上皮间充质转化(EMT)的影响。方法体外培养乳腺癌细胞,将miR-767-5p inhibitor和inhibitor-NC转染到乳腺癌细胞中。细胞分为3组:空白对照组(Control组),miR-767-5p inhibitor组和inhibitor-NC组,采用CCK-8法、Transwell法、划痕实验检测miR-767-5p抑制剂对乳腺癌细胞增殖、侵袭和迁移能力的影响。Western blot检测迁移相关蛋白基质金属蛋白酶-2(MMP-2)和MMP-9的表达水平。双荧光素酶报告基因实验验证miR-767-5p和胰岛素样生长因子-1(IGF1)的靶向结合作用。RT-qPCR和Western blot实验检测IGF1 mRNA和蛋白的表达水平。采用Western blot检测EMT相关蛋白的表达水平。结果CCK-8实验结果显示,与Control组和inhibitorNC组相比,miR-767-5p inhibitor组显著抑制MCF-7细胞的增殖(P<0.05)。Transwell实验结果表明,与Control组和inhibitor-NC组相比,miR-767-5p inhibitor组乳腺癌细胞侵袭细胞数显著下降(P<0.05)。划痕实验结果显示,与Control组和inhibitor-NC组相比,miR-767-5p inhibitor组乳腺癌细胞迁移率显著下降(P<0.05)。荧光素酶报告基因实验结果显示,miR-767-5p能够靶向结合IGF1。Western blot结果表明,与Control组和inhibitor-NC组相比,miR-767-5p inhibitor组的E-cadherin蛋白表达水平显著增高(P<0.05),Ncadherin、MMP-2和MMP-9蛋白的表达水平显著降低(P<0.05)。与Control组和inhibitor-NC组相比,miR-767-5p inhibitor组中IGF1的mRNA和蛋白表达水平显著降低(P<0.05)。结论抑制miR-767-5p的表达可通过下调IGF1抑制乳腺癌细胞的增殖、侵袭、迁移及EMT的发生。

Objective To investigate the effect of inhibiting miR-767-5p on migration,invasion and epithelial-mesenchymal transition(EMT)of breast cancer cells.Methods Breast cancer cells were cultured in vitro,and miR-767-5p inhibitor and inhibitor NC were transfected into breast cancer cells.The cells were divided into three groups:Control group,miR-767-5p inhibitor group and inhibitor-NC group.The effects of miR-767-5p inhibitor on the proliferation,invasion and migration of breast cancer cells were detected by CCK-8 assay,Transwell Migration assay and scratch test.Western blot was used to detect the expression levels of migration related proteins MMP-2 and MMP-9.The dual luciferase reporter gene experiment verifies the targeted binding effect of miR-7675p and IGF1.RT-qPCR and Western blot experiments were used to detect the expression levels of IGF1 mRNA and protein.Western blot was used to detect the expression level of EMT related proteins.Results CCK-8 assay showed that compared with the control group and inhibitor-NC group,the miR-767-5p inhibitor group significantly inhibited the proliferation of MCF-7 cells(P<0.05).Transwell assay showed that the number of invasive cells of breast cancer cells in miR-767-5p inhibitor group was significantly lower than that in control group and inhibitor NC group(P<0.05).Scratch test showed that compared with the control group and inhibitor NC group,the migration rate of breast cancer cells in miR-767-5p inhibitor group decreased significantly(P<0.05).Luciferase reporter gene assay showed that miR-767-5p targeted IGF1 binding.Western blot showed that compared with the control group and inhibitor NC group,the expression level of E-cadherin protein in the miR-767-5p inhibitor group was significantly increased(P<0.05),while the expression levels of N-cadherin,MMP-2,and MMP-9 protein were significantly reduced(P<0.05).Compared with the control group and inhibitor-NC group,the mRNA and protein expression levels of IGF1 in the miR-767-5p inhibitor group were significantly reduced(P<0.05).Conclusion Suppressing the expression of miR-767-5p may inhibit the proliferation,invasion,migration and EMT of breast cancer cells by down-regulating IGF1.