MiR-4719通过靶向调控ARHGAP36抑制人乳腺癌细胞的迁移和侵袭

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。

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

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.