微小RNA-326靶向CARD10抑制乳腺癌细胞的增殖和侵袭

microRNA-326 inhibits the proliferation and invasion of breast cancer cells by targeting CARD10

目的探讨乳腺癌中微小RNA-326(miR-326)靶向CARD10(caspase recruitment domain family member 10)抑制乳腺癌细胞增殖和侵袭的分子机制。方法 q PCR检测乳腺癌细胞株MCF7、BT549、MB-MDA-468、HCC1937、SKBR3和人正常乳腺上皮细胞MCF10A中miR-326的表达。通过microRNA.org预测网站显示miR-326靶向结合CARD10基因。以miR-326表达量最低的乳腺癌细胞株为研究对象,分为阴性对照组(转染miR-NC)和实验组(转染miR-326模拟物),q PCR检测miR-326、CARD10 mRNA的表达,Western blotting检测CARD10、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、锌指转录因子Snail、锌指转录因子Slug蛋白的表达,双荧光素酶报告基因系统检测荧光素酶活性以验证miR-326与CARD10的靶向调控关系,MTT法检测细胞增殖能力,Transwell侵袭实验检测miR-326的表达对乳腺癌细胞侵袭能力的影响。结果与人正常乳腺上皮细胞相比,乳腺癌细胞株中miR-326低表达(P<0.05),BT549细胞表达量最低(P<0.01)。与miR-NC组相比,miR-326组BT549细胞中miR-326表达显著上调(P<0.01),CARD10 mRNA表达显著下降(P<0.01)。E-cadherin蛋白表达升高,CARD10、N-cadherin、Snail、Slug蛋白表达降低。双荧光素酶实验结果表明miR-326能靶向结合CARD10的3'UTR端。与miRNC组相比,miR-326组细胞的增殖能力显著下降(P<0.05),细胞侵袭能力明显降低(P<0.01)。结论过表达miR-326可抑制乳腺癌细胞BT549的增殖和侵袭,其机制可能是靶向抑制CARD10基因的表达。

Objective To investigate the effect of microRNA-326（miR-326） in breast cancer targeting CARD10 on the proliferation and invasion of breast cancer cells.Methods The expression of miR-326 was detected by qPCR in breast cancer cell lines MCF7, BT549, MB-MDA-468, HCC1937, SKBR3 and human normal mammary epithelial cells MCF10A. The microRNA.org prediction site shows that miR-326 binds to the CARD10 gene. Breast cancer cell lines with the lowest expression of miR-326 were divided into negative control group（transfected with miR-NC） and experimental group（transfected with miR-326 mimics）. The expression of miR-326 and CARD10 mRNA was detected by qPCR. The expression of CARD10, E-cadherin, N-cadherin, Snail and Slug proteins was detected by Western blotting. The luciferase reporter gene was used to detect luciferase activity for verifying the targeted regulatory relationship between miR-326 and CARD10. MTT assay was used to detect the cell proliferation ability. Transwell invasion assay was used to detect the effect of miR-326 expression on the invasive ability of breast cancer cells.Results Compared with normal human breast epithelial cells, the expression of miR-326 was decreased in breast cancer cell lines（ P 〈0.05）, and the expression in BT549 cells was the lowest（ P 〈0.01）. Compared with miR-NC group, the expression of miR-326 was significantly up-regulated in miR-326 group（ P 〈0.01）, CARD10 mRNA expression was significantly decreased（ P 〈0.01）, and the expression of E-cadherin protein increased, while the expression of CARD10, N-cadherin, Snail, Slug proteins decreased. The double luciferase assay showed that miR-326 was capable of targeting the 3′UTR side of CARD10 mRNA. Compared with miR-NC group, the proliferation ability of cells in miR-326 group was significantly decreased（ P 〈0.05）, and the invasive ability was significantly decreased（ P 〈0.01）.Conclusion Over-expression of miR-326 can inhibit the proliferation and invasion of breast cancer cell line BT549 by inhibiting the expression of CARD10 gene.