Effects Of Bone Morphogenetic Protein-2 On Proliferation And Apoptosis Of Human Hepatoma HepG2 Cells

BACKGROUND:Primary hepatocarcinoma(PHC)is the third most prevalent malignant diseases in China.In all malignant tumors, death rate caused by PHC ranks the 2^ndin males,the 4^thin females.While, primary hepatocellular carcinoma(HCC)constitutes 91.5%of PHC in our country.Unfortunately,etiology and mechanism of this malignancy are still not clearly elucidated.Many factors and assumptions have been propounded to solve the puzzling problem,then proliferation and apoptosis of tumor cells have been received more and more attention.Bone morphogenetic protein-2(BMP-2),a member of TGF-βsuperfamily,acts important regulational parts in growth,differentiation and apoptosis of cells and formation of multiple tissues and organs. Studies have confirmed that BMP-2 promotes cell differentiation and apoptosis,inhibit cell proliferation.Expectation of this study is to elucidate:what the role of BMP-2 in the development of hepatocellular carcinoma is,what the effect induced by BMP-2 on cell proliferation and apoptosis in hepatocellular carcinoma is,whether BMP-2 play a role through its receptor BMPR(bone morphogenetic protein receptor)in hepatocellular carcinoma or not,what the post-receptor signal transduction pathway is when BMP-2 transfering its signals in hepatocellular carcinoma,aiming to provide theoretical basis for further exploration of effects exerted by BMP-2 on heptocellular carcinoma,and new data or clue for prevention and cure on hepatocellular carcinoma.First,chapterâ… ,we investigated the expression of BMP-2 and its receptors in HepG2 cells,and the relationship between BMP-2 intervention and HepG-2 cellular proliferation and apoptosis.RNA interference(RNAi),a newly developed experimental technology,can be used to highly effectively block specific genes,which has been widely adopted in research work related to functional genenomes,genetic therapy to tumors and other fields.Chapterâ…¡,by adopting siRNA to interfere inhibition of expression of bone morphogenetic protein receptor-â…¡(BMPR-â…¡),we try to find whether BMP-2 exerts effects by the way of BMPR-â…¡on HepG-2 cellular proliferation and apoptosis.There have not been reported about the post-receptor signal transduction pathway of BMP-2/BMPR in hepatocellular carcinoma,expecting to elucidate it,chapterâ…¢,we investigated the post-receptor signal transduction pathway of BMP-2 inducing proliferation and apoptosis on human hepatoma HepG-2 cells.Chapter one The Effects of Bone Morphogenetic Protein-2 (BMP-2)on Proliferation and Apoptosis in Human Hepatoma HepG2 CellsObjective:To characterize the effects of BMP-2 on proliferation and apoptosis of human heaptoma HepG2 cells. Methods:(1)The expression of BMP-2,BMPR-IA,BMPR-IB and BMPR-â…¡protein were measured by Western blot.(2)Cell survival was determined by MTr assay.(3)Cell proliferation was assessed by [³H]TdR incorporateion.(4)Cell growth curve was assessed by trypan blue resisting assay.(5)Cell apoptosis was assessed by ELISA and flow cytometry.Results:(1)Western blot analysis showed that human hepatoma HepG-2 cells expressed BMP-2 and its receptor BMPR-IA,BMPR-IB, BMPR-â…¡.Expression of BMP-2 protein attained from human hepatoma HepG-2 cells was significantly degraded comparing with normal hepatic cells,and P＜0.01.Expression of BMP-2 receptor BMPR-IA,BMPR-IB, BMPR-â…¡in HepG-2 cells and normal hepatic cells had no difference, P＞0.05.(2)Result detecting cell vitality by MTT analysis showed that values attained from 10ng/ml group and 100ng/ml group were significantly decreased when compared with control,P＜0.05.(3)When assessing effects of different concentration of BMP-2 on HepG cell proliferation by[³H]TdR incorporation method,1ng/ml group and 10ng/ml group were significantly depressed when compared with control, P＜0.05,100ng/ml group were more significantly depressed when compared with control,P＜0.01.(4)Trypan blue resisting assay demonstrated that BMP-2 exerted significant effects on HepG2 cell growth.After treated with 100ng/ml BMP-2,cell numbers decreased markedly.What's more,with treated time elongation,such effects became more significantly,and when compared with control,P＜0.05.(5) Adopting flow cytometry to assess effects of BMP-2 on apoptosis of HepG-2 cells,percentage of apoptosis in 100ng/ml group was significantly heightened,When compared with control group,p＜0.05.(6) Apoptosis detected by ELISA analysis demonstrated that,treated with different concentrations of BMP-2 for 24 hours duration,cell apoptosis rates attained from 1/ng/ml BMP-2 group was significantly upgraded when compared with control,and P＜0.05,10ng/ml,100ng/ml,200ng/ml BMP-2 group were more significantly upgraded,P＜0.01.While,treated with BMP-2 on the same concentration of 100ng/ml,apoptosis rates in 6 hours group went significantly higher when compared with control group, P＜0.05,furthermore,12,24,48 hours group more significantly higher than control group,P＜0.01.Conclusion:(1)BMP-2,BMPR-IA,BMPR-IB and BMPR-â…¡are expressed in HepG2 cells,while BMP-2 expression rate is significantly less than that from normal liver cell control,BMPR expression rate has no difference in two type cells.We supposed that decrease of BMP-2 expression in hepatocellular carcinoma maybe the cause of development of hepatocellular carcinoma,perhaps,BMP-2 plays a role through its receptor BMPR in hepatocellular carcinoma and normal hepatic cell.(2) BMP-2 dos-dependent and time-dependent inhibites HepG2 cells proliferation and promotes HepG2 cells apoptosis,with treated time elongation or dose increasing,effect induced by BMP-2 on hepatocellular carcinoma becomes more markedly.We concluded that BMP-2 can regulate the development of hepatocellular carcinoma by inhibiting cell proliferation and promoting cell apoptosis.Chapter two The Role of Bone Morphogenetic Protein Receptor(BMPR)on BMP-2's Effects on Proliferation and Apoptosis in Human Hepatoma HepG2 CellsObjective:The present study adopted small interfering RNA(siRNA) method to inhibit expression of BMPR-â…¡of HepG2 cell,aiming to observe whether BMP-2 exert proliferation and apoptosis of HepG2 cell through BMPR.Methods:(1)SiRNA interference was used to down-regulate the expression of BMPR-â…¡in HepG2 cells.(2)Expression of BMPR-â…¡protein in cultured HepG2 cells were detected by Western blot analysis.(3)Cell proliferation was assessed by[³H]TdR incorporateion. (4)Cell apoptosis was assessed by ELISA.Results:(1)Western blot analysis showed that HepG2 cell transinfected by BMPR-â…¡-siRNA expressed no BMPR-â…¡proteins.(2)By [³H]TdR incorporation,incorporation quantities attained from BMP(+)control-siRNA group was significantly more decreased when compared with BMP(-)control-siRNA group,P＜0.01.Incorporation quantities attained from BMP(+)BMPR-â…¡-siRNA group was significantly more increased when compared with BMP(+)control-siRNA group, P＜0.01.(3)With ELISA method,HepG-2 apoptosis rates in BMP(+)control-siRNA group was significantly more upgraded than BMP(-)control-siRNA group,P＜0.01.HepG-2 apoptosis rates in BMP(+)BMPR-â…¡-siRNA group was significantly decreased When compared with BMP(+)control-siRNA group,P＜0.01.Conclusion:(1)HepG2 cells transinfected by BMPR-â…¡-siRNA showed inhibition of expression of BMPR-â…¡proteins;(2)HepG2 cells transinfected by BMPR-â…¡-siRNA down-regulated expression of BMPR-â…¡, further inhibited proliferation and apoptosis initiated by BMP-2 through BMPR.Chapter three The Signaling Pathway of the Effects of Bone Morphogenetic Protein-2(BMP-2)on Proliferation and Apoptosis in Human Hepatoma HepG2 CellsObjective:The present study was undertaken to investigate the post-receptor signal transduction pathway of the BMP-2 on Proliferation and Apoptosis in Human Hepatoma HepG2 Cells.Methods:(1)Expression of p-JNK,JNK,p-p38,p38,p-ERK1/2, ERK1/2,Akt and p-Akt in cultured HepG2 cells were detected by Western blot analysis.(2)HepG2 cells cultured with BMP-2 combing JNK specific inhibitor SP600125 or BMP-2 combing RNAi by BMPR-â…¡-siRNA,JNK phosphorylation was analyzed by western blot.(3) HepG2 cells interfered by BMP-2 combining MAPK signal transduction blocking agent SP600125,SB203580,PD98059,PI-3 signal transduction blocking agent LY294002,then,Cell proliferation was assessed by [³H]TdR incorporation,Cell apoptosis was assessed by ELISA,in order to study the post-receptor signaling pathway.Results:(1)BMP-2 time-dependent induced the expression of p-JNK in HepG2 cells.This effect was blocked by suppression of BMPR-â…¡with siRNA.(2)SP600125,but not PD98059,SB203580,or LY294002 abolished the effects of BMP-2 on HepG2 cells proliferation and apoptosis.Conclusion:(1)BMP-2 can time-dependent induce JNK phosphorylation through its receptor BMPR-â…¡.(2)BMP-2 acts on the proliferation and apoptosis through the BMPR/JNK signaling pathway in HepG2 cells,independence with PI-3 K/Akt pathway and other MAPK pathway including ERK MAPK,p38 MAPK pathway.