MicroRNA-145在乳腺癌中的表达及其对乳腺癌侵袭转移的影响研究

Study on the Expression of Microrna-145 in Breast Cancer and its Effect on Breast Cancer Invasion and Metastasis

目的探讨miR-145在乳腺癌中的表达及其对乳腺癌侵袭转移的影响。方法选择2016年7月至2018年7月辽宁省人民医院确诊的乳腺癌患者48例作为研究资料,取术中癌组织及相应癌旁组织作为标本,采用实时荧光定量PCR法检测miR-145的表达,如将pSIF-miR-145、pSIF空白载体转染人乳腺癌细胞系SK-BR-3,检测对细胞迁移及侵袭能力的影响及对ANGPT2蛋白表达的影响。结果经实时FQ-PCR检测乳腺癌组织和癌旁组织中miR-145的相对表达量分别为(49.85±21.02)、(185.04±42.64);实时FQ-PCR检测对数生长期乳癌细胞系的miR-145表达,MCF-7为(0.50±0.04)、MDA-MB-231(0.06±0.01)、MDA-MB-468(0.35±0.03)、SK-B-3(0.04±0.01),均偏低,呈现为低表达。转染pSIF-miR-145质粒48 h后,SK-BR-3细胞miR-145的表达量与阴性对照相比增高了445.42倍;划痕实验24 h后转染pSIF-miR-145质粒细胞化和愈合速度较低,侵袭实验显示,转染pSIF-miR-145质粒后穿过基质胶的平均细胞数为(132±45)个,对照组穿过基质胶的平均细胞数为(622±76)个。经双荧光报告基因分析,HEK293T细胞转染pSIF-miR-145、pRL-TK-ANGPT2野生质粒后,荧光素酶的活性显著降低;而共转染了pSIF-miR-145、pRL-TK-ANGPT2-mut质粒后,细胞中荧光素酶活性无变化,即miR-145直接靶向ANGPT2。SK-BR-3中转染pSIF-miR-145或空载质粒,检测ANGPT2的表达,ANGPT2蛋白较阴性对照组显著下调,即认为miR-145对ANGPT2蛋白表达有抑制作用。结论miR-145在乳腺癌中的表达为低表达,可通过靶向调控ANGPT2蛋白从而抑制乳腺癌的侵袭转移。

Objective To investigate the expression of miR-145 in breast cancer and its effect on breast cancer invasion and metastasis.Methods Choose during July 2016 to July 2018 in liaoning province people's hospital diagnosis of 48 cases of breast cancer patients as research materials,take the intraoperative tissue adjacent to carcinoma tissue and corresponding as samples,using real-time fluorescent quantitative PCR method to detect the expression of miR-145,such as the pSIF-miR-45,pSIF blank carrier transfection human breast cancer cell line SK-BR-3,the effect of detection of cell migration and invasion ability and influence to ANGPT2 protein expression.Results The relative expression levels of miR-145 in breast cancer tissues and paracancer tissues detected by real-time FQ-PCR were(49.85±21.02)and(185.04±42.64),respectively.Real-time FQ-PCR was used to detect the expression of miR-145 in logarithmically growing breast cancer cell lines,and the expressions of MCF-7(0.50±0.04),MDA-MB-231(0.06±0.01),MDA-MB-468(0.35±0.03),and SK-B-3(0.04±0.01)were all low,showing a low expression.After 48h of pSIF-miR-145 plasmid transfection,the expression level of miR-145 in SK-BR-3 cells was 445.42 times higher than that of the negative control.After 24 h of scratch test,pSIF-miR-145 plasmids were transfected with pSIFmiR-145 plasmids at a low rate of granulocytosis and healing.The invasion test showed that the average number of cells transfected with pSIF-miR-145 plasmids that crossed the matrix glue was(132±45),and the average number of cells transfected with pSIF-miR-145 plasmids that crossed the matrix glue in the control group was(622±76).Double fluorescent reporter gene analysis showed that luciferase activity was significantly decreased after transfection of pSIF-miR-145 and prl-tk-angpt2 wild plasmids into HEK293T cells.When pSIF-miR-145 and prl-tk-angpt2-mut plasmids were co-transfected,there was no change in luciferase activity in the cells,that is,miR-145 directly targeted ANGPT2.Psif-miR-145 or no-load plasmid was transfected in SK-BR-3 to detect the expression of ANGPT2,and ANGPT2 protein was significantly down-regulated compared with the negative control group,suggesting that miR-145 had an inhibitory effect on the expression of ANGPT2 protein.Conclusion The expression of miR-145 in breast cancer is low and can inhibit the invasion and metastasis of breast cancer by targeting the regulation of ANGPT2 protein.