外泌体lncRNA TTN-AS1通过miR-524-5p介导TGFβ/Smads通路调控乳腺癌进展的机制研究

Extracellular lncRNA TTN-AS1 Mediates miR-524-5p through TGFβ/Smads Pathway Regulating the Progress of Breast Cancer

目的:探讨外泌体长链非编码RNA(lncRNA)TTN-AS1通过miR-524-5p介导转化生长因子β(TGFβ)/Smads信号通路,进而调控乳腺癌进展的分子机制。方法:首先采用实时荧光定量聚合酶链反应(qRT-PCR)检测人乳腺癌细胞MDA-MB-231、MCF-7、SKBR-3和乳腺上皮细胞MCF-10A中lncRNA TTN-AS1和miR-524-5p表达量,Western blot法检测TGFβ1和p-Smad2蛋白的表达量。然后采用细胞转染技术抑制lncRNA TTN-AS1表达,比较MDA-MB-231(阴性对照)和转染组lncRNA TTN-AS1、miR-524-5p、TGFβ1和p-Smad2表达量。CCK8法检测细胞增殖力,流式细胞术检测凋亡率,Transwell小室检测迁移力。结果:与MCF-10A相比,MDA-MB-231、MCF-7和SKBR-3中lncRNA TTN-AS1表达量显著升高,而miR-524-5p明显下降,TGFβ1和p-Smad2上调(P<0.05)。与阴性对照相比,转染组lncRNA TTN-AS1表达量显著降低,而miR-524-5p明显增加,TGFβ1和p-Smad2下调(P<0.05)。细胞增殖率和迁移细胞数目明显下降,而凋亡率增加(P<0.05)。结论:lncRNA TTN-AS1在乳腺癌的进展中可能发挥促癌效应,通过抑制miR-524-5p表达并激活TGFβ1/Smad2信号通路活性来参与调控乳腺癌的增殖与凋亡。lncRNA TTN-AS1有望成为靶向干预乳腺癌的新型分子位点。

Objective:To investigate the molecular mechanism of extracellular long chain non coding RNA(lncRNA)TTN-AS1 mediated miR-524-5p through transforming growth factorβ(TGFβ/Smads signaling pathway,then to regulate the breast cancer progression.Methods:Firstly,real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to detect the expressions of lncRNA TTN-AS1 and miR-524-5p in human breast cancer cell MDA-MB-231,MCF-7,SKBR-3 and breast epithelial cell MCF-10A,Western blot was to detect TGFβ1 and p-Smad2 proteins expression.Then,cell transfection technology was used to inhibit expression of lncRNA TTN-AS1,lncRNA TTN-AS1,miR-524-5p,TGFβ1 and p-Smad2 expressions were compared between MDA-MB-231(negative control)and transfection group.CCK8 method was to detect cell proliferation,flow cytometry was to detect cell apoptosis rate,and Transwell chamber was to detect cell migration.Results:Compared with MCF-10A,the expression levels of lncRNA TTN-AS1 in MDA-MB-231,MCF-7,SKBR-3 were significantly higher,while miR-524-5p were significantly less,TGFβ1 and p-Smad2 were upregulated(P<0.05).Compared with the negative control group,the expressions of lncRNA TTN-AS1 in the transfection group was significantly lower,while miR-524-5p was significantly more,resulting in TGFβ1 and p-Smad2 were downregulated(P<0.05).The cell proliferation rate and the number of migrating cells were significantly less,while the apoptosis rate was higher(P<0.05).Conclusion:lncRNA TTN-AS1 may play a cancer promoting role in the progression of breast cancer by inhibiting the expression of miR-524-5p and activating TGFβ1/Smad2 signaling pathway,which is involved in regulating the proliferation and apoptosis of breast cancer.LncRNA TTN-AS1 is expected to become a new molecular site targeting breast cancer intervention.