miR-1246靶向StarD13对乳腺癌细胞影响的研究

Proliferation and invasion of breast cancer cells by targeting microRNA-1246 to StarD13

目的探讨miR-1246促进乳腺癌细胞增殖、迁移与侵袭能力的分子机制.方法生物信息学分析miR-1246的表达及其与乳腺癌患者生存率的关系.实时荧光定量聚合酶链反应(qRT-RCR)评估miR-1246在乳腺癌细胞和乳腺癌组织中的表达,Transwell小室检测细胞侵袭能力,EdU实验和克隆实验检测细胞增殖能力,划痕实验检测细胞迁移能力,生物信息学方法确定miR-1246的靶蛋白StarD13在乳腺癌中表达情况及生存率曲线,双荧光素酶报告基因检测miR-1246与StarD13的关系,蛋白质印迹法实验检测下调miR-1246后相关靶蛋白StarD13的表达情况.采用独立样本t检验分析两组之间的定量数据,采用单一因素方差分析进行多组之间的比较.结果miR-1246在乳腺癌组织和乳腺癌细胞MDA-MB-231中均高表达.Transwell实验结果显示,下调miR-1246后miR-1246 inhibitor组细胞较NC inhibitor组细胞侵袭能力降低,t=9.14,P<0.01.EdU实验结果显示,下调miR-1246后细胞增殖能力下降,t=6.81,P<0.01.克隆形成实验表明,下调miR-1246后细胞的增殖能力下降,t=5.41,P<0.01.划痕实验结果显示,miR-1246 inhibitor和NC inhibitor组的细胞迁移率分别为(0.21±0.02)%和(0.50±0.04)%,差异具有统计学意义,t=13.13,P<0.01.生物信息学预测miR-1246与StarD13之间存在互补序列;StarD13在乳腺癌组织中低表达,且低表达StarD13的乳腺癌患者预后较差,P<0.01.转染miR-1246抑制物时StarD13的表达量增加.双荧光素酶报告基因实验证实StarD13可作为miR-1246的靶基因.EdU实验结果显示,miR-1246 inhibitor+siStarD13组细胞[(55.89±1.95)%]比miR-1246 in-hibitor+NC组细胞[(25.24±6.17)%]EdU阳性率有提高,差异有统计学意义,t=8.21,P<0.01.Transwell实验表明,与miR-1246 inhibitor+NC组(127.67±23.46)比较,miR-1246 inhibitor+siStarD13组(385.67±11.59)穿过基底膜细胞数量明显增多,差异具有统计学意义,t=17.08,P<0.001.结论miR-1246可通过靶向调控StarD13促进乳腺癌细胞增殖、迁移与侵袭能力.

Objective To investigate the molecular mechanism of miR-1246 promoting the proliferation,migration and inva-sion of breast cancer cells.Methods The expression of miR-1246 and its relationship with the survival rate of breast cancer patients were analyzed by bioinformatics.Real-time fluorescence quantitative polymerase chain reaction(qRT-RCR)was used to evaluate the expression of miR-1246 in breast cancer cells and breast cancer tissues.Transwell chamber was used to detect cell invasion ability;EdU assay and cloning assay were used to detect cell proliferation.Scratch test was used to detect cell migration ability;the expression and survival curve of StarD13,the target protein of miR-1246,in breast cancer were determined by bioinformatics.The relationship between miR-1246 and StarD13 was detected by dual luciferase reporter gene.Western blot was used to detect the expression of related target protein StarD13 after down-regu-lation of miR-1246.Independent sample t test was used to analyze the quantitative data between the two groups,and one-way analysis of variance was used to compare multiple groups.Results miR-1246 was highly expressed in breast cancer tissues and breast cancer cell MDA-MB-231.Transwell assay showed that the invasion ability of miR-1246 inhibitor group was lower than that of NC inhibitor group(t=9.14,P<0.01).The results of EdU experiment showed that the cell proliferation ability decreased after down-regulation of miR-1246(t=6.81,P<0.01).Colony formation assay showed that the proliferation ability of cells decreased after down-regulation of miR-1246(t=5.41,P<0.01).The results of scratch test showed that the cell migration rates of miR-1246 inhibitor and NC inhibitor groups were 0.21±0.02 and 0.50±0.04,respectively,and the difference was statistically significant(t=13.13,P<0.01).Bioinformatics predicted that there was a complementary sequence between miR-1246 and StarD13;breast cancer patients with low expression of StarD13 in breast cancer tissues and low expression of StarD13 had a poor prognosis(P<0.01).The expression of StarD13 was increased when miR-1246 inhibitor was transfected.Dual-luciferase reporter assay confirmed that StarD13 could be used as a target gene of miR-1246.At the same time,the results of EdU assay showed that the positive rate of EdU in miR-1246 inhibitor+siStarD13 group[(55.89±1.95)%]was higher than that in miR-1246 inhibitor+NC group[(25.24±6.17)%],and the difference was statistically significant(t=8.21,P<0.01).Trans well assay showed that compared with the miR-1246 inhibitor+NC group(127.67±23.46),the number of cells passing through the basement membrane in the miR-1246 inhibitor+siStarD13 group(385.67±11.59)was significantly increased,and the difference was statistically significant(t=17.08,P<0.001).Conclusion miR-1246 can promote the proliferation,migration and invasion of breast cancer cells by targeting StarD13.