干预miR-206/MECOM/TGF-β/smad3轴对三阴性乳腺癌恶性进展的影响机制

Effect and Mechanism of miR-206/MECOM/TGF-β/smad3 Axis in Inhibiting the Malignant Progression of Triple-negative Breast Cancer

目的以三阴性乳腺癌细胞株231为研究对象,探讨干预miR-206/MECOM/TGF-β/smad3轴对三阴性乳腺癌恶性进展的影响及机制。方法使用ualcan与Kaplan Meier Plotter在线分析MECOM、TGF-βR1、TGF-βR2、smad3、smad4、HDAC1、HDAC2及CTBP在乳腺癌组与正常乳腺组之间差异表达和与预后的关系。采用细胞共培养及共转染技术,在MDA-MB-231细胞中分别沉默转录因子MECOM、过表达和沉默miR-206,以及将二者共转染,采用RT-qPCR法检测TGF-βR1、TGF-βR2、smad3、smad4、HDAC1、HDAC2及CTBPmRNA表达水平;结果ualcan在线分析在TCGA-BRCA中MECOM、TGF-βR2乳腺癌组低于正常乳腺组,差异有统计学意义(P<0.05);Kaplan Meier Plotter在线分析MECOM、TGF-βR1、TGF-βR2、smad3、smad4、HDAC1、HDAC2及CTBP差异表达与预后的关系。在TCGA数据库结果提示MECOM低表达较高表达患者中位生存期(overall survival,OS)长(P<0.05),smad3与smad4高表达较低表达患者OS长,差异有统计学意义(P<0.05),TGF-βR1、TGF-βR2、HDAC1、HDAC2及CTBP高表达与低表达患者OS差异无统计学意义(P>0.05)。单纯沉默MECOM后,与空白对照组相比,Si-MECOM组的TGF-βR2、HDAC1、HDAC2、smad3、smad4、CTBP mRNA表达水平降低(P<0.05);单纯干预miR-206后,MECOM、TGF-βR1、TGF-βR2、HDAC1、HDAC2、smad4、CTBP mRNA表达水平降低(P<0.05);共转染MECOM与miR-206后,与Si-MECOM组相比,siMECOM+inhibitor miR-206组的TGF-βR1、HDAC1、smad3、smad4mRNA表达水平降低,差异有统计学意义(P<0.05),TGF-βR2、HDAC2、CTBP mRNA表达水平升高,差异有统计学意义(P<0.05)。结论miR-206对MECOM的调控可能是通过影响TGF-βR2、HDAC2、CTBPmRNA的表达来抑制TGF-β/SMAD通路活化阻止TNBC进展。

Objective Taking triple-negative breast cancer cell line 231 as the study object,to investigate the effect and mechanism of miR-206/MECOM/TGF-β/smad3 axis in inhibiting the malignant progression of triple-negative breast cancer.Methods Ualcan online analysis and Kaplan Meier Plotter online analysis were used to analyze the differential expression of MECOM,TGF-βR1,TGF-βR2,smad3,smad4,HDAC1,HDAC2 and CTBP between breast cancer group and normal breast cancer group and their prognostic relationship.Using cell co-culture and co-transfection techniques,transcription factor MECOM,overexpression and silencing miR-206 in MDA-MB-231 cells were respectively silenced,and both were co-transfected.The mrna expression levels of TGF-βR1,TGF-βR2,smad3,smad4,HDAC1,HDAC2 and CTBP were detected by RT-qPCR.Results Ualcan online analysis showed that MECOM and TGF-βR2 breast cancer groups were lower than normal breast cancer groups in TCGA-BRCA,and the difference was statistically significant(P<0.05);Kaplan Meier Plotter online analyzed the differential expression of MECOM,TGF-βR1,TGF-βR2,smad3,smad4,HDAC1,HDAC2 and CTBP and their prognostic relationship.The results in the TCGA database indicated that patients with low and high MECOM expression had longer median survival(OS)(P<0.05).Patients with high expression of smad3 and smad4 had longer median survival(OS)than those with low expression(P<0.05).There was no significant difference in median survival(OS)between patients with high expression of TGF-βR1,TGF-βR2,HDAC1,HDAC2 and CTBP(P>0.05).After simple silence of MECOM,mRNA expression levels of TGF-βR2,HDAC1,HDAC2,smad3,smad4 and CTBP in Si-MECOM group were decreased compared with blank control group(P<0.05);after simple intervention with miR-206,mRNA expression levels of MECOM,TGF-βR1,TGF-βR2,HDAC1,HDAC2,smad4 and CTBP were decreased(P<0.05);after co-transfection with MECOM and miR-206,mrna expression levels of TGF-βR1,HDAC1,smad3 and SMad4 of siMECOM+inhibitor miR-206 group were decreased compared with those of Si-MECOM group(P<0.05).The mRNA expression levels of TGF-βR2,HDAC2 and CTBP were increased(P<0.05).Conclusion The regulation of miR-206 on MECOM may inhibit the activation of TGF-β/SMAD pathway and prevent TNBC progression by affecting the expression of TGF-BR2,HOAC2,and CTBPm RNA.