The Function Of Smad7 Gene In The Process Of Human Bronchial Epithelial Cells Malignant Transformation

Lung cancer is one of the most common cancers throughout the world, and its incidence is still increasing.However, the molecular mechanism invovled in lung carcinogenesis is still unknown.In this study, Northern blot was performed to investigate the expression of Smad7 in cell models; stable gene transfection was performed to investigate the effect of Smad7 on cells proliferation and on transforming growth factor-P (TGF-P) -mediated growth suppression in human bronchial carcinogenesis induced by alpha particles. siRNAs specific to Smad7 gene were prepared by transcription in vitro. Tumorigenic BERP35T2 cell line was transciently transfected with Smad7 siRNAs and Smad7 expression plasmid. Northern blot was performed to detected the variation of Smad7 mRNA expression level in BERP35T2 cells. BERP35T2 cell line was transciently transfected with Smad7 siRNA and exogenous TGF-β1 was added to the cells , the effect of siRNA on cells proliferation and TGF-β1- mediated growth suppression was investigated. The results are as follow:1. The expression of Smad7 in cell models and its responsiveness to TGF-β1: the expression of Smad7 was higher in tumorigenic BERP35T2 cell line than its parental BEP2D cells, a human papillomavirus-18 immortalized human bronchial epithelial cell line. The TGF-β1-induced Smad7 expression was normal in BEP2D cells but disordered in BERP35T2 cells.2. Effects of Smad7 overexpression on proliferation and TGF-β1- mediated growth suppression in BEP2D and BERP35T2 cells: BERP35T2 cells with higher expression level of Smad7 have more superiority in proliferation and less responsiveness to TGF-β1-mediated growth suppression than BEP2D cells. Stable transfection of BEP2D and BERP35T2 cells with the full length Smad7 cDNA construct resulted in markedly increasing the proliferation of cells and the loss of growth inhibitory response to TGF-β1.3. The suppression of Smad7 gene expression by the technique of RNA interference and its effects on proliferation and TGF-β1- mediated growth suppression in BERP35T2 cells: The result of Northern blotting showed that without the interference of siRNA, the expressionlevel of endogenous Smad7 mRNA was high in BERP35T2 cells. At the same time, the expression level of exogenous Smad7 mRNA was also high. But when added with siRNA, endogenous Srnad? mRNA was almost degraded completely, and the expression of exogenous Smad7 mRNA was also suppressed in part. The degradation of 542 siRNA and 701 siRNA to target mRNA had no distinct difference. Both siRNA and TGF-pi can suppress the proliferation of BERP35T2 cells respectively, combination of both factors showed stronger growth suppression.These findings indicate that in the process of human bronchial epithelial cells malignant transformation, the expression of Smad7 is increased. Its overexpression may facilitate cells proliferation and contribute to the process of cells carcinogenesis by negative regulating TGF-P signal transduction pathway. The expression of Smad7 gene can be suppressed effectively by the technique of RNA interference in tumorigenic human bronchial epithelial cell line. The decreased expression of Smad7 gene can also feedback regulate TGF-(3 signal transduction pathway, increasing the sensitivity of cells to growth inhibitory response to TGF-pi, facilitating TGF-(31- mediated growth suppression and resulting in markedly decreased proliferation in malignant cells .