干扰黑色素瘤细胞A375中miR-210的表达对细胞转移及金属基质蛋白酶相关分子的影响

Effect of interfering the expression of miR-210 on cell metastasis and MMP related molecules in melanoma cell line A375

目的探究黑色素瘤中微小RNA-210(miR-210)的表达情况及干扰人黑色素瘤细胞A375中miR-210的表达对细胞转移能力及金属基质蛋白酶(MMP)表达的影响。方法收集西安交通大学第一附属医院病理科2016年3月1日至2017年5月31日的37例皮肤恶性黑色素瘤组织,17例交界痣组织及30例正常皮肤组织,荧光定量链式聚合酶反应(qPCR)检测组织中miR-210的表达情况。体外培养A375细胞,慢病毒转染空白载体及小干扰RNA(si RNA)介导的干扰miR-210载体于A375细胞中,分别作为对照组及干扰组细胞,qPCR检测两组细胞miR-210、MMP-2及MMP-9 mRNA表达水平,Western blot实验检测两组细胞MMP-2、MMP-9蛋白表达水平,Transwell侵袭实验检测两组细胞侵袭能力,Transwell迁移实验检测两组细胞迁移能力。结果与交界痣及正常皮肤组织相比,黑色素瘤组织miR-210表达显著增高[(0.165±0.060)vs(0.076±0.033)及(0.048±0.046)],差异均具有统计学意义(P<0.05);与对照组细胞相比,干扰组细胞miR-210[(0.153±0.037)vs(0.372±0.041)]、MMP-2 mRNA[(0.175±0.053)vs(0.424±0.038)]及MMP-9 mRNA[(0.323±0.068)vs(0.610±0.102)]表达水平显著下调,差异均具有统计学意义(P<0.05);与对照组细胞相比,干扰组细胞MMP-2[(0.114±0.016)vs(0.356±0.047)]及MMP-9[(0.263±0.030)vs(0.571±0.084)]蛋白表达水平显著下调,差异均具有统计学意义(P<0.05);与对照组细胞相比,干扰组细胞侵袭细胞数显著减少[(36.9±8.7)个vs(72.3±10.5)个],干扰组细胞迁移细胞数也显著减少[(45.3±9.4)个vs(78.3±12.6)个],差异均具有统计学意义(P<0.05)。结论恶性黑色素瘤中miR-210存在高表达,体外干扰A375细胞miR-210的表达可介导MMP分子表达下调,进而抑制细胞转移能力,是潜在的分子靶向治疗位点。

Objective To investigate the expression level of miR-210 in melanoma tissues and the effect of in-terfering the expression of mircoRNA-210 （miR-210） on the ability of cell metastasis and the expression of metal ma-trix protease （MMP） in human melanoma cell line A375. Methods A total of 37 cases of malignant melanoma of theskin, including 17 cases of junctional nevus and 30 cases of normal skin tissues, were collected from Department of Der-matology and Sexually Transmitted Diseases of the First Affiliated Hospital of Xi＇an Jiaotong University from March 1,2016 to May 31, 2017. Fluorescence quantitative polymerase chain reaction （qPCR） was used to detect the expression ofmiR-210 in each tissue. A375 cells were cultured and transfected with empty lentivirus vector and miR-210 lentivirussmall interference RNA vector, respectively, as the control group and interference group. qPCR was used to detect the ex-pression of miR-210, MMP-2 and MMP-9 mRNA in two groups of cells. Western blot assay was used to detect the ex-pressions of MMP-2 and MMP-9 protein in two groups of cells. Transwell invasion assay was used to detect the abili-ty of cell invasion, and Transwell migration assay was applied to detect the ability of cell migration. Results Com-pared with junctional nevus and normal skin tissue, the expression of miR-210 in melanoma tissue was significantly in-creased, （0.165±0.060） vs （0.076±0.033） and （0.048±0.046）, and the difference was statistically significant （P〈0.05）.Compared with the control group, the expression of miR-210 （0.153±0.037） vs （0.372±0.041）, MMP-2 （0.175±0.053）vs （0.424 ± 0.038） and MMP-9 （0.323 ± 0.068） vs （0.610 ± 0.102） mRNA in interference group were significantly de-creased, and the differences were statistically significant （P〈0.05）. Compared with the control group, the expressionsof MMP-2 （0.114±0.016） vs （0.356±0.047） and MMP-9 （0.263±0.030） vs （0.571±0.084） protein in interference groupwere significantly decreased, and the difference was statistically significant （P〈0.05）. Compared with the controlgroup, the number of cell invasion in interference group was significantly decreased （36.9±8.7） vs （72.3±10.5）, and thenumber of cell migration was significantly decreased （45.3±9.4） vs （78.3±12.6）, and the differences were statisticallysignificant （P〈0.05）. Conclusion High expression of miR-210 is found in malignant melanoma. The interference ofmiR-210 in A375 cells in vitro can down-regulate the expression of MMP and inhibit cell metastasis, which is a potentialmolecular targeted therapeutic site.