MiR-4719通过靶向调控ARHGAP36抑制人乳腺癌细胞的迁移和和侵袭

MiR 4719 inhibits migration and invasion of human breast cancer cells via targeting ARHGAP36

目的检测miR-4719在乳腺癌组织和细胞中的表达,研究其对乳腺癌细胞侵袭迁移的影响及其分子机制。方法采用实时荧光定量PCR检测30对人乳腺癌组织和癌旁组织,乳腺癌细胞株(BT549和MDA-MB-231)以及正常乳腺上皮细胞(MCF-10A)中miR-4719和ARHGAP36的表达水平。生物信息手段分析miR-4719对乳腺癌患者生存率的影响,并预测miR-4719的潜在靶基因。将miR-4719模拟物,靶向ARHGAP36的shRNA、ARHGAP36过表达质粒分别转染乳腺癌细胞。Western blot和免疫组化实验检测ARHGAP36的蛋白水平;细胞划痕实验和Transwell实验检测癌细胞的迁移和侵袭能力;双荧光素酶报告实验验证miR-4719与ARHGAP36的mRNA 3'-非翻译区(3'-UTR)直接结合。结果与癌旁组织、正常乳腺上皮细胞相比,miR-4719和ARHGAP36分别在乳腺癌组织(P<0.001)和乳腺癌细胞(P<0.01)中显著降低和增高。miR-4719低表达与乳腺癌患者不良预后密切相关(P<0.01)。过表达miR-4719或敲低ARHGAP36可明显抑制乳腺癌细胞的侵袭和迁移(P<0.01)。乳腺癌组织中miR-4719和ARHGAP36表达水平呈负相关(P<0.01),双荧光素酶报告实验显示miR-4719可靶向调控ARHGAP36的表达(P<0.01),向癌细胞外源性导入miR-4719可明显抑制ARHGAP36的表达(P<0.01)。此外,过表达ARHGAP36可逆转miR-4719模拟物对癌细胞迁移和侵袭能力的抑制作用(P<0.01)。结论乳腺癌组织和癌细胞中miR-4719的丧失,导致靶基因ARHGAP36的异常高表达,促进了人乳腺癌细胞的迁移和侵袭。

Objective To detect the expression of miR-4719 in breast cancer tissues and cells and explore its role in regulating invasion and migration of breast cancer cells.Methods qRT-PCR was used to detect the expression of miR-4719 and ARHGAP36 in 30 pairs of human breast cancer tissues and adjacent tissues,two breast cancer cell lines(BT549 and MDA-MB 231)and normal breast cells(MCF-10A).Bioinformatic methods were utilized to analyze the relationship between miR-4719 expression and overall survival of breast cancer patients and predict the potential target gene miR 4719.miR-4719 mimics,ARHGAP36 shRNA and ARHGAP36 plasmids were transfected into breast cancer cells to test the effects of miR-4719 overexpression,ARHGAP36 knockdown and ARHGAP36 overexpression on cell migration and invasion using wound healing assay and Transwell assay.A dual-luciferase reporter assay was used to verify the direct binding between miR-4719 and 3'-UTR of ARHGAP36.Results Compared with those in adjacent tissues or normal breast cells,the expressions of miR-4719 were significantly decreased and the expression of ARHGAP36 was increased in breast cancer tissues(P<0.001)and breast cancer cell lines(P<0.01).A low expression of miR-4719 was correlated with a poorer overall survival of breast cancer patients(P<0.05).Overexpression of miR-4719 and ARHGAP36 knockdown both significantly attenuated the invasion and migration abilities of breast cancer cells(P<0.05).The expression of miR-4719 was inversely correlated to that of ARHGAP36 in breast cancer tissues(P<0.01).Dual-luciferase reporter assay confirmed that ARHGAP36 was the target gene of miR-4719(P<0.01),and exogenous miR-4719 could significantly lower the expression of ARHGAP36(P<0.05).ARHGAP36 overexpression significantly reversed the inhibitory effects of miR-4719 mimics on migration and invasion of breast cancer cells(P<0.05).Conclusion The expression of miR-4719 is aberrantly decreased in breast cancer tissues to promote migration and invasion of breast cancer cells by up-regulating ARHGAP36 expression.