Hsa-miR-145调控Rab GTP酶家族成员27A基因对乳腺癌细胞株MDA-MB-231迁移和侵袭的影响  

Has-miR-145 suppresses MDA-MB-231 cell metastasis by directly targeting Rab GTPase family 27A

作　　者：唐莉[1] 葛梦圆[1] 韦达[2] 严枫[1] 

机构地区：[1]南京医科大学附属肿瘤医院检验科江苏省恶性肿瘤分子生物学与转化医学重点实验室, 210009 [2]南京医科大学附属肿瘤医院普外科, 210009

出　　处：《中华实验外科杂志》2014年第9期1929-1932,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（21075055）;江苏省科技厅临床医学专项（BL2013036）;江苏省医学领军人才与创新团队项目（LJ201131）;江苏省六大人才高峰项目（2010-WS-075）

摘　　要：目的 观察hsa-miR-145对Rab GTP酶（GTPase）家族成员27A（Rab27A）基因的靶向调控作用,以及对乳腺癌细胞体外迁移和侵袭能力的影响.方法 利用生物信息学软件筛选出hsa-miR-145与乳腺癌迁移侵袭能力相关的潜在的靶基因Rab27A,将含有hsa-miR-145结合位点的Rab27A3＆#39;端非编码区域（3＆#39;-UTR）片段（2 539 bp）连入psi-CHECK2载体,转染293T细胞48 h后检测hsa-miR-145与靶基因的结合特性.将hsa-miR-145模拟物和阴性对照10 μmol/L瞬时转染MDA-MB-231细胞,分别培养48、72 h检测Rab27A蛋白表达,18h检测细胞迁移率变化,24h检测细胞侵袭率的变化.结果 空白组双荧光测定比值为1.50±0.12,hsa-miR-145模拟物组为1.05±0.05,hsa-miR-145抑制物组为2.18-0.14.转染hsa-miR-145模拟物48、72 h后,与阴性对照组比较,Rab27A的相对蛋白表达量分别下降为59.07％、28.57％.MDA-MB-231细胞中过表达hsa-miR-145使细胞相对迁移率降低为（32.96±1.54）％,相对侵袭率降低为（43.89±2.80）％.结论 hsa-miR-145能与Rab27A直接结合,参与调控乳腺癌细胞的体外迁移与侵袭.Objective To investigate the function and possible action mechanisms of microRNA hsa-miR-145 in breast cancer cells.Methods In order to search the microRNA which suppresses metastasis of breast cancer,three well known microRNA target prediction programs were utilized to select the microRNAs that target the genes related to tumor metastasis.The binding site for hsa-miR-145 in the 3＆#39; untranslated region （3＆#39;-UTR）,of Rab GTPase family 27A （Rab27A） was amplified by polymerase chain reaction （PCR）.The wild and mutant 3＆#39;-UTR sequences of the Rab27A gene （2539 bp） were ligated into the psi-CHECK2 vector separately to construct dual-luciferase reporter vectors.The constructed vectors were transfected into 293T cells and uciferase activity was measured 48 h later.The highly metastatic cell line MDA-MB-231 was transfected with hsa-miR-145 mimic and negative control separately,to test whether hsa-miR-145 plays a role in breast cancer cell metastasis.Western blotting was performed to detect the protein expression of Rab27A,and cell migration assay and cell invasion assay were used to detect the changes in cell metastasis 24 h after Transwell assay.Results Sequencing results showed the successful construction of dual-luciferase reporter vectors.The luciferase assay revealed that overexpression of hsa-miR-145 could significantly inhibit the Rab27A 3＆#39;-UTR-mediated luciferase activit y（P 〈0.05）.The dual-fluorescence measurements ratio of blank control group,hsa-miR-145 mimic group,and hsa-miR-145 inbihitor group was 1.50 ±0.12,1.05 ± 0.05 and 2.18 ± 0.14 respectively.Western blotting showed the expression of Rab27A in MDA-MB-231 cells was down-regulated to 59.07% （48 h） and 28.57% （72 h） as compared with the negative control group.Cell migration assay and cell invasion assay confirmed that cell migration and invasion ability of MDA-MB-231 cells was obviously decreased after transfection of hsa-miR-145 mimic.Conclusion These results highlight the significance of hsa-miR-145

关 键 词：乳腺癌 微小RNA 迁移 侵袭 

分 类 号：R737.9[医药卫生—肿瘤]