miR-504靶向CHD1L抑制乳腺癌细胞侵袭的分子机制

The molecular mechanism of miR-504 targeting CHD1L inhibiting the invasion of breast cancer cells

目的探讨miR-504靶向染色质解旋酶DNA结合蛋白(chromodomain helicase/ATPase DNA binding protein 1-like gene,CHD1L)抑制乳腺癌细胞侵袭的分子机制。方法 qRT-PCR检测人正常乳腺上皮细胞(MCF-10A)与乳腺癌细胞(MDA-MB-231、MCF-7)中miR-504的表达量,同时检测miR-504在MDA-MB-231细胞中的转染效率;双荧光素酶实验检测miR-504与CHD1L是否存在结合靶点;qRT-PCR及Western blot法检测CHD1L mRNA和蛋白的表达水平;Transwell侵袭实验检测MDAMB-231细胞的侵袭能力;Western blot法检测p-Akt、MMP-2和MMP-9的表达情况。结果乳腺癌细胞中miR-504的表达量明显低于人正常乳腺上皮细胞(P <0. 05);miR-504在MDA-MB-231/miR-504组中的表达水平明显增高(P <0. 05);双荧光素酶实验检测显示miR-504能与CHD1L mRNA的3'-UTR结合;此外,过表达miR-504后CHD1L mRNA的表达水平无显著变化,但CHD1L蛋白表达水平显著降低(P <0. 05);与MDA-MB-231/NC组相比,MDA-MB-231/miR-504组侵袭能力明显降低,而MDA-MB-231/miR-504+CHD1L组侵袭能力比MDA-MB-231/miR-504+Con组明显增加;用表皮生长因子分别刺激转染不同质粒的MDA-MB-231细胞,与MDA-MB-231/NC组相比,MDA-MB-231/miR-504组中p-Akt、MMP-2和MMP-9蛋白表达水平明显降低(P <0. 05),而MDA-MB-231/miR-504+CHD1L组中p-Akt、MMP-2和MMP-9蛋白表达水平与MDA-MB-231/miR-504+Con组相比明显增加(P <0. 05)。结论 miR-504靶向CHD1L通过PI3K/Akt/MMP信号通路抑制乳腺癌细胞的侵袭。

Purpose To investigate the molecular mechanism of miR-504 targeting chromodomain helicase/ATPase DNA binding protein 1-like gene( CHD1 L) inhibiting the invasion of breast cancer cell. Methods The expression of miR-504 in normal breast epithelial cells( MCF-10 A) and breast cancer cells(MDA-MB-231,MCF-7) was detected by qRT-PCR,as well as the transfection efficiency of MDA-MB-231 cells. Double luciferase assay was conducted to detect whether there was binding target between miR-504 and CHD1 L. The mRNA and protein expression levels of CHD1 L were detected by qRT-PCR and Western blot. The invasion ability of MDA-MB-231 cells was detected by Transwell invasion assay. The expressions of MMP-2,MMP-9 and p-Akt proteins were detected by Western blot. Results The expression of miR-504 in breast cancer cells was significantly lower than that in normal human breast epithelial cells( P <0. 05). The expression level of miR-504 was significantly increased in the MDA-MB-231/miR-504 group( P < 0. 05).Double luciferase assay showed that miR-504 could bind to the3’-UTR of CHD1 L mRNA. In addition,there was no significant change in the expression of CHD1 L mRNA after overexpression of miR-504,but the protein expression level of CHD1 L was significantly decreased( P < 0. 05). Compared with the MDA-MB-231/NC group,the invasion ability of MDA-MB-231/miR-504 group was significantly reduced. But the invasion ability of MDA-MB-231/miR-504 + CHD1 L group was significantly increased compared with the MDA-MB-231/miR-504 + Con group.Epidermal growth factor was used to stimulate MDA-MB-231 cells after transfected with different plasmids. Compared with the MDA-MB-231/NC group,the expression levels of p-Akt,MMP-2 and MMP-9 protein were significantly decreased in MDA-MB-231/miR-504 group( P < 0. 05). However,the expression levels of p-Akt,MMP-2 and MMP-9 protein were significantly increased in MDA-MB-231/miR-504 + CHD1 L group compared with the MDA-MB-231/miR-504 + Con group( P < 0. 05).Conclusion miR-504 targeting CHD1 L inhibit the invasion of breast cancer cells via PI3 K/Akt/MMP signaling pathway.