Effects Of SiRNA-Targeting BMPR-Ⅱ On The Invasion And Proliferation Of Human Liver Cancer Cells And Its Signaling Pathway Mechanism About VEGF-C

Background and purpose:BMPs play a vital role in neoplasms’ invasion and proliferation,but it is still notclear about the relative receptor(BMPR) and its inductive effects on HCC’s invasionand proliferation.Our preliminary study showed that BMP-2could promoteproliferation and invasion of hepatoma carcinoma cells.The mechanism may becaused by the up-regulated expression of MMP-7,MMP-9and the down-regulatedexpression level of E-cadherin through MAPK/ERKsignal pathway.Recent researchrevealed that BMPR-II is the key regulatory factor in the BMP signal pathway.So weinfered that BMPR-II might play an important role in HCC’s invasion andproliferation.On the basis of the precendent researches,we aimed to firstly researchthe function of BMPR-II and further explore the mechanism of its relative signalpathway,ultilizing the technolody of small RNA interference,at the new point ofBMPR-II,which may futher expound the molecular mechanism of the proliferationand invasion mediated by BMP and do a favor to search for new molecular targetsand new thougt in clinic therapy.Purpose:To observe the effects of small interfering RNA (siRNA)-targeting bonemorphogen-etic protein receptor II(BMPR-II) on the invasion and proliferation ofhuman liver cancer cells and its signaling pathway mechanism about VEGF-C.Experimental Design:The mRNA and protein levels of BMPR-II in cells of HepG-2,SMMC7721andHep3B were determined with reverse transcription-PCR and westernblotting,screening the highest experssion of BMPR-II in them.ThreesiRNAs-targeting BMPR-II gene were synthesized. There were six groups includinggroup A (non-transfected cells), group B (only liposome-transfected cells), group C(non-specific siRNA-transfected cells) and groups D-F (siRNA-a,siRNA-b andsiRNA-c-targeting BMPR-II transfected cells,respec-tively). Liver cancer cells wereinstantaneously transfected using lipofectamine method. The mRNA and protein levels of BMPR-II in cells were determined with reverse transcription-PCR andwestern blotting.The abilities of proliferation and invasion of transfected cells wereassessed using MTT assay and transwell assay, respectively.The changes of cellapoptosis and cell cycle were were assessed using flow cytometry methods.Toobserve the changes of relative protein’s expression,when each signaling pathway ofMAPKs was blocked before,after and blocked with siRNA-BMPR-II,using specificinhibitors of MAPKs signaling pathway,including SB203580(P38inhibitor),PD98059(ERK inhibitor) and SP6000(JNK inhibitor).Results:Western blot and RT-PCR indicated that HepG2(1.00±0.04,1.01±0.06P<0.01)was the highest expression of BMPR-II in the three liver cancerlines.Expression of BMPR-II at the level of mRNA and protein in groups D-F weresignificantly inhibited, especially in group F(0.20±0.01,0.39±0.02， P<0.01).MTTassay and transwell assay revealed that the number of cell growth and celltransmembrane in siBMPR-II group(48.27%±0.76%,25.20±1.60,P<0.05) weresignificantly lower than Normal control group and negative control group aftertransfection48hours.Flow cytometric analysis showed that cell apoptosis insiBMPR-II group(37.03±0.56,P<0.01)was the highest than Normal control group andnegative control group,S phase of cell cycle was significantly blocked aftertransfection48hours(50.63±13.09P<0.01).Western blot indicated that the level ofp-P38(0.45±0.05P<0.01),p-ERK(0.35±0.03P<0.01) and were significantlydecreased after silencing BMPR-II,the level of p-ERK was not significantlychanged,and the level of VEGF-C(0.33±0.05P<0.01) was significantlydownregulated.The level of VEGF-C in PD9805group and SB203580group weresignificantly downre-gulated (P<0.01),but the SB203580group was not significantlychanged.The level of VEGF-C in PD98059+siBMPR-II group and SB203580+siBMPR-II group were significantly downregulated (P<0.01),but the SB203580group was not significantly changed.In them,The level of VEGF-C in SB203580group was downregulated much than PD9805group as well as SB203580+siBMPR-II group vs PD98059+siBMPR-II group.Conclusions: HepG2was the highest expression of BMPR-II in the three liver cancerlines.siRNA-targeting BMPR-II can markedly inhibited the expression of BMPR-II inliver cancer HepG2cells, and decrease the abilities of proliferation andinvasion,promote the abilities of cell apoptosis and block S phase of cell cycle of livercancer cells, especially siRNA-a-BMPR-II group.The inhibitory effects ofsiRNA-c-targeting BMPR-II on the abilities of oncological bioactivity of human livercancer HepG2cells may be caused by the downregulation of VEGF-C throughMAPK/P38and MAPK/ERK pathway, whereas is not related to MAPK/JNKpathway.especially,induced the phosphorylation of p38mitogen-activated proteinkinase (MAPK) conpared to the ERK MAP kinases selectively.