Mechanisms Of RB And TSC2 Gene Induce Synergistic Effects On Vascular Smooth Muscle Cell Proliferation

Object:Uncontroled proliferation of VSMC (vascular smooth muscle cell) is the main reason of restenosis after vascular remolding. RB/E2F pathway and mTOR signaling pathway have close relationship with cell cycle regulation and cell proliferation, RB and TSC2 gene were the core of these two signaling pathway, which regulated the activation of these two pathway. This study was to explore the association of RB/E2F and mTOR signaling system and VSMC proliferation; this study was also investigates RB and TSC2 gene double knockdown induced synergistic effect inhibite cell proliferation; explore whether ROS is the reason which RB and TSC2 double knockout induced synergistic effect mediated cell growth arrest.Methods:1, VSMC cultured in vitro, applied PDGF-BB intervention, simulation VSMC proliferation in vivo; 2, After PDGF-BB intervention, RT-PCR and Western-Blot were used to test the level of downstream target gene of RB/E2F and TSC2/mTOR signaling pathway.3, After lentiviral transfection of Rb and TSC2 gene, MTT were used to detect cell proliferation level of the empty lentiviral group (as the empty vector control group) and Rb gene knockout group, TSC2 gene knockout group, gene RB/TSC2 double knockout group.4, Brdu staining was used to detected DNA synthesis level of control group, RB gene knockout group, TSC2 gene knockout group, RB/TSC2 double knockout group.5, Detect the ROS level of control group, RB gene knockout group, TSC2 gene knockout group, RB/TSC2 double knockout group.6, Detect the level of cell proliferation by MTT after applying the NAC (oxygen free radical scavenger) intervention in control group, RB knockout group, TSC2 gene knockout group, RB/TSC2 double knockout.Results:1, The levels of gene CCNA2 and CDK1 were increased about 8 folds after PDGF-BB treatment. The level of pS6K was clearly increased after PDGF-BB treatment.2, Knockdown of single Rb or TSC2 increased cell proliferation. However, knockdown of both Rb and TSC2 decreased cell proliferation induced by PDGF-BB.3, The DNA synthesis is significantly faster after single knockout Rb or TSC2. However, double knockout Rb and TSC2 inhibited DNA synthesis compared to single knockout.4, The ROS level is significantly higher in double knockout Rb and TSC2. No significant change in RB or TSC2 single knockout group and control group.5, NAC treatment rescues inhibiting cell proliferation in double knockout Rb and TSC2 under PDGF-BB treatment.Conclusion:The increased level of E2F target suggests that the proliferation of VSMC requires Rb/E2F signaling. The increased level of phosphorylation of S6K indicates that TSC/mTOR signaling plays important roles in VSMC proliferation, single knockdown of Rb or TSC2 significantly increased cell proliferation. Double knockdown of Rb and TSC2 showed synergistic effect in inhibiting cell proliferation. Single knockdown of Rb or TSC2 caused significantly increased DNA synthesis. Double knockdown of Rb and TSC2 caused decreased DNA synthesis. The inhibiting cell proliferation effect was supported by the findings in DNA synthesis study. Double knockdown of Rb and TSC2 increased ROS level.NAC rescues the inhibiting cell proliferation in double KO cells. It suggests that the inhibiting cell proliferation effect might due to increased ROS level. We found the mechanism to explain the inhibiting cell proliferation effect.