Effect Of Smad4Gene Silencing By Small Interfering Rna On Apoptosis Of Porcine Granulosa Cells

Smads proteins, which were newly found as intracellular signaltransduction proteins, play important roles in signal transduction pathway of TGF-β. As the important member of the Smads proteins family, Smad4was the only Co-smad founpurudond in mammals, which considered to be the necessary central transduction molecules in signal transmission of TGF-β. Smad4play a key role in TGF-β/Smad signaling pathway, the expression deletion or mutation would caused to TGF-β/Smad signal transduction disorder, and then impact on tissues or cells. In recent years, study has demonstrated that Smad4signal transduction pathway exist in mouse ovarues, TGF-β regulation of follicle differentiation was possible to be achieved through this way.In this study, RNAi was used to explore the effect of silencing Smad4gene expression on apoptosis of porcine Granulosa Cells (PGC). Designed and synthesized small interference RNA targeting to Smad4was transfected into PGC through lipidosome. Smad4mRNA expression level was assayed by real-time fluorescence quantitative PCR. Cell survival rate, apoptotic and apoptotic index were analyzed by MTT colorimereic analysis, TUNEL and flow cytometry, respectively. Expression level of Bcl-2and Bax mRNA was analyzed by real-time fluorescence quantitative PCR.1Smad4-siRNA transfection of procine Granulosa Cells and detection efficiencyAccording to the cDNA sequence of porcine Smad4gene, small interference RNA was designed, then Smad4-siRNA were transfected into PGC using lipidodome. Smad4mRNA expression level was assayed by real-time fluorescence quantitative PCR, Smad4-siRNA inhibitory most effective at8pmoL for36h, and the expression level in siRNA group decreased by84%than compared with control group(p<0.01). Cell survival rate was analysised by MTT colorimereic after transfection, the result showed the cytoactive significantly decreased (0.31to0.27)(p<0.05). The result suggested that the silencing of Smad4gene can apparently inhibited the proliferation of ovarian granulose cells, and suppose that knockdown of endogenous expression of Smad4may cause apoptosis of PGC.2Effects of Smad4gene silencing by small interfering RNA on apoptosis of Granulosa CellsApoptotic of cells were analyzed by TUNEL and flow cytometry. The result show the apoptoic cells in siRNA group significantly increased than control group, and the apoptotic rate increased(17.0%to22.1%) after expression of Smad4mRNA was inhibited (p<0.05). These shows that silencing of Smad4gene can increase apoptosis of PGC.3Effects of Smad4gene silencing by small interfering RNA on Bcl-2/Bax of Granulosa CellsExpression level of Bcl-2and Bax mRNA was analyzed by real-time fluorescence quantitative PCR. The result show the bcl-2mRNA expression level in siRNA group decreased79%than in control group (p<0.01). The result further shows that silencing of Smad4gene can increase apoptosis of PGC, which possibly relate to regulation of the expression of Bcl-2.