Novel Alternatively Spliced Variants of Smad4 Expressed in TGF-β-Induced EMT Regulating Proliferation and Migration of A549 Cells.

introductionNon-small cell lung cancer (NSCLC) is a worldwide malignance threatening human life. TGF-β/Smad signaling is known to regulate cell proliferation, differentiation, migration and growth. As the only co-Smad playing crucial roles in TGF-β 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-β-induced EMT process. However, whether the alternative splicing occurs in the TGF-β-induced EMT process in NSCLC was not clear.

methodsIn our current study, we explored the alternative splicing of Smad4 during the process of TGF-β-induced EMT in A549 cells. 10 ng/mL TGF-β 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.

resultsThere were several novel gene fragments expressed in TGF-β-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.

conclusionOur results indicated that novel Smad4 variants were expressed in TGF-β-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.