Effect And Mechanism Of Rat Hyperplasia Suppressor Gene On Proliferation And Apoptosis Of Rat Glioma Cells

ObjectiveTo investigate the expression of rat hyperplasia suppressor gene (rHSG) in rat gliomacells (C6cells) with the effect on proliferation and apoptosis of C6cells, and explore thepotential mechanism.Method1. The rat glioma cells (C6cells) were cultured in vitro and infected with recombinantadenovirus vectors containing rHSG(Adv-rHSG-GFP). Based on the MOI values, the cellswere divided into group A(MOI=0), group B(MOI=10), group C(MOI=50), group D(MOI=80)and group E(MOI=100). Cells of different groups were collected and tested after infection for24h. Western blot and immunohistochemical methods were applied for determining theexpression of rHSG protein; flow cytometry (FCM) and inverted fluorescence microscopewere used for observing GFP positive expression to assess the infection efficiency ofadenovirus vectors. Then C6cells were infected with Adv-rHSG-GFP(MOI=100) for differenttime periods(12h,24h,36h,48h) and collected. rHSG protein expression was detected andinfection efficiency was evaluated in the same manner.2. C6cells were divided into3groups: uninfected, infected with Adv-GFP and infectedwith Adv-rHSG-GFP. Western blot and immunohistochemical method were applied fordetermining the expression of rHSG protein; the effect of rHSG on cell cycle was analyzedby flow cytometer, together with MTT, cytometry and plate cloning formation method wereused for identifying the effect of rHSG on cell proliferation; the expression of proliferatingcell nuclear antigen (PCNA), cell cycle regulatory proteins p27Kip1and p21Cip1weredetermined by western blot.3. The morpHological characteristics of apoptosis was observed by Hoechst33342/PIdouble staining, while effect of rHSG on DNA damage in C6cells was determined via cometassay; Western blot was applied to detect expression level of PARP and cleaved caspase-3after over-expression of rHSG, in addition, effect of over-expressed rHSG on PI3K/Akt and MAPK signaling pathways induced by insulin-like growth factor (IGF-1).Result1. It was observed that in C6cells infected with Adv-rHSG-GFP for24h, the proteinexpression of rHSG and green fluorescent protein were increasing with MOI; After infectionwith Adv-rHSG-GFP(MOI=100), green fluorescent protein could be observed after12h andincreased as time went on within24h, while no change from24-36h and decreased after48h(P<0.01); rHSG protein expression level raised within the range of12-48h and maximizedat48h(P﹤0.01).2. It is observed that in C6cells of group infected with Adv-rHSG-GFP, the proteinexpression level of rHSG was more than group of uninfected and group infected withAdv-GFP(P<0.01); the results from flow cytometer indicated that the cell cycle was arrestedin G0/G1pHase, and MTT, cytometry and plate cloning formation showed that rat glioma cellproliferation was markedly inhibited; the expression of p27Kip1and p21Cip1were increasedwhile the expression of PCNA was decreased.3. It is observed that in C6cells of group infected with Adv-rHSG-GFP, rHSG proteincould be expressed at a high level; Hoechst33342/PI double staining and comet assay showedthat rHSG induced C6cell apoptosis and DNA damage; the results of western blot indicatedthat, after over-expression of rHSG, the expression of Full-length PARP was obviouslyincreased at time points of24,72and96h(P<0.1) while decreased at48h(P<0.1); theexpression of cleaved PARP and cleaved caspase-3were obviously increased(P<0.1) andrHSG had significant suppression on IGF-1-inducing activation of Akt and Erk1/2protein,wherein the suppression effect is stronger in p-Erk1/2than in Akt.Conclusion1. The recombinant adenovirus vectors containing rHSG can infect C6cells effectively,and the highest infection efficiency can be obtained at MOI=100and24h.2. It can be concluded that the over-expression of rHSG can suppress the cellproliferation of rat glioma cells and the suppression of rHSG may be functioned byparticipating in cell cycle regulation.3. It can be concluded that the over-expression of rHSG can promote apoptosis of ratglioma cells and the induction of rHSG may be functioned by participating in PI3K/Akt and MAPK signaling pathways, mainly of which is MAPK.