Novel Alternatively Spliced Variants of Smad4 Expressed in TGF-β-Induced EMT Regulating Proliferation and Migration of A549 Cells
Novel Alternatively Spliced Variants of Smad4 Expressed in TGF-β-Induced EMT Regulating Proliferation and Migration of A549 Cells
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在 TGF-β 诱导的 EMT 调节 A549 细胞增殖和迁移中表达的 Smad4 新型选择性剪接变体
DOI：
10.2147/ott.s247015
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发表时间：
2020-01-01
影响因子：
4
通讯作者：
Feng, Jianguo
中科院分区：
文献类型：
--
作者：
Wan, Rongxue;Xu, Xichao;Feng, Jianguo
前言：非小细胞肺癌（Non-small cell lung cancer，NSCLC）是世界范围内严重威胁人类生命的恶性肿瘤。已知TGF-β/Smad信号传导调节细胞增殖、分化、迁移和生长。作为在TGF-β信号传导中起关键作用的唯一的co-Smad，据报道Smad 4在肿瘤细胞中频繁突变或以选择性剪接的形式发生。据报道，Smad 4参与TGF-β诱导的EMT过程。然而，在TGF-β诱导的NSCLC EMT过程中是否存在Smad 4的选择性剪接尚不清楚。方法：本研究探讨了TGF-β诱导A549细胞EMT过程中Smad 4的选择性剪接。使用10 ng/mL TGF-β诱导EMT。采用巢式PCR和琼脂糖凝胶电泳检测Smad 4变异体的表达，并测序得到变异体的DNA序列。为了在A549细胞中重组表达Smad 4变体，我们使用慢病毒变体感染细胞。通过MTT法、集落形成实验和创伤愈合实验，探讨突变体对A549细胞增殖和迁移的影响。结果：TGF-β诱导的A549细胞中有新的Smad 4基因片段表达，测序结果表明，这些基因片段确为Smad 4基因中未见报道的变异体。在A549细胞中重组表达Smad 4变异体，发现其对细胞的增殖和迁移有明显的影响，并对E-cad和Vim蛋白的表达有调节作用。结论：TGF-β诱导的EMT过程中表达了新的Smad 4变异体。功能研究表明，这些新的变体调节细胞增殖和迁移，并影响E-cad和Vim蛋白的表达，显示出作为癌症治疗靶点的潜力。
Introduction: Non-small cell lung cancer (NSCLC) is a worldwide malignance threatening human life. TGF-beta/Smad signaling is known to regulate cell proliferation, differentiation, migration and growth. As the only co-Smad playing crucial roles in TGF-beta signaling, Smad4 is reported to be frequently mutated or to occur as alternatively spliced in tumor cells. Smad4 was reported to be involved in the TGF-beta-induced EMT process. However, whether the alternative splicing occurs in the TGF-beta-induced EMT process in NSCLC was not clear.Methods: In our current study, we explored the alternative splicing of Smad4 during the process of TGF-beta-induced EMT in A549 cells. 10 ng/mL TGF-beta was used to induce EMT. Then, nest-PCR and agarose electrophoresis were performed to detect the expression of Smad4 variants and sequencing to get the variant DNA sequences. For recombinant expression of variants of Smad4 in A549 cells, we used lentiviral variants to infect cells. In order to explore the effects of variants on the proliferation and migration of A549 cells, the MTT assay, colony formation assay and wound-healing assay were done. The effects of variants on E-cad and VIM protein expression were explored through Western blot.Results: There were several novel gene fragments expressed in TGF-beta-induced A549 cells, and the sequencing results showed that they were indeed the Smad4 variants that were not reported. For recombinant expression of Smad4 variants in A549 cells, we found that they have significant effects on the proliferation and migration of cells, and also regulated the E-cad and VIM protein expression.Conclusion: Our results indicated that novel Smad4 variants were expressed in TGF-beta-induced EMT process. The functional study showed that these novel variants regulate cell proliferation and migration and affect E-cad and VIM protein expression, showing the potential as targets for cancer therapy.