Influence Of Rna Interference Mediated Samd4Gene Silencing On The Proliferation And Cell Cycle Of Porcine Granulosa Cells

Granulosa cells, which are one of the important cells in ovary, synthesize and secrete steroid hormones and a variety of growth factors, which play a role through autocrine/paracrine way, in their various developmental stages. The growth, development, proliferation and differentiation of granulosa cells significantly influence the development of follicles. TGF-β/Smad signaling pathway is an important one in granulosa cells. Smad4is an important downstream signal transduction molecule of the pathway, and is also a key signal molecule in TGF-β superfamily signaling transmission from the cytoplasm to the nucleus. Anomalo-expression of Smad4of granulosa cells not only have the effect on their own growth, but also result in abnormal follicular development or even cancer, thereby influence the reproductive function of female animals.In order to investigate the effect of Smad4gene silencing by using small interfering RNA(siRNA) on the proliferation and cell cycle of pig granulosa cells in vitro, one siRNA sequence targeting Smad4gene was designed and synthetized. The siRNA was transfected into granulosa cells with LipofectamineTM RNAi Mix (the final concentration of Smad4-StealthTM RNA was13.3nM). Smad4mRNA expression was detected by real-time PCR. The effect of proliferation of granulosa cells was detected by MTT assay. Flow cytometry were used for evaluation of cell cycle. The alteration of Cyclin D1, Cyclin B, Cyclin A2, Cdk1, Cdk2, Cdk4(which are cell cycle related genes) mRNA expression were also detected by real-time PCR. The results showed that:(1) Smad4expression in granulsoa cells was effectively suppressed by the sequence-specific Smad4-StealthTM RNA.36h after transfection, the mRNA levels of Smad4was significantly reduced with the inhibition rate being79.85%(P<0.01);(2) The silencing of Smad4effectively inhibited the proliferation of granulosa cells. 12h,24h,36h,48h and72h after interference, and the survival rates of granulosa cells compared with controls were90.6%,87.2%,84.8%,82.7%,80.9%respectively (P<0.05).(3) The silencing of Smad4resulted in the cell cycle arrest of granulosa cells.36h after transfection, the percentage of G0/G1phase cells increased from86.84%(control group) to92.82%(transfection of Smad4-StealthTM RNA group)(P<0.05), S phase cells decreased from6.13%(control group) to1.64%(transfection of Smad4-StealthTM RNA group)(P<0.05), and G2/M phase cells had no change. The mRNA level of several cell cycle related genes Cyclin D1, Cyclin B, Cyclin A2, Cdk1, Cdk2and Cdk4decreased to41.9%,77.3%,22.3%,64.3%,48.0%and61.1%respectively, which were detected by real-time PCR. The mRNA level of Cyclin Dl, Cdkl decreased significantly (P<0.05) and Cyclin A2, Cdk2, Cdk4decreased very significantly (P<0.01), and Cyclin B had no obvious difference as compared to the control groups.It could be concluded that Smad4is an important gene that affect porcine granulosa cell proliferation and cell cycle.