Effect And Mechanism Of SiRNA Targeting BMP-2 On Proliferation And Invasion Of Human Liver Cancer SMMC7721 Cells

Effect and mechanism of siRNA targeting BMP-2 on proliferation and invasion of human liver cancer SMMC7721 cellsPart I Expression of Bone Morphogenetic Proteins and its receptor in human liver cancer SMMC7721 cellsObjective:To examine the expression of BMP-2,3,4,5,6,7and its acceptor BMPR-IA,BMPR-IB and BMPR-Ⅱ.Method:The expression of mRNA of BMP-2,3,4,5,6,7and its acceptor BMPR-IA,BMPR-IB and BMPR-Ⅱwere measured by RT-PCR.The difference of expression of protein of BMP-2,3,4,5,6,7and its acceptor BMPR-IA,BMPR-IB and BMPR-Ⅱbetween human liver L-O2cells and liver cancer SMMC7721 cells were measured by Western blot.Results:1.RT-PCR indicated that the expression of mRNA of BMP-2 was highly and the expression of mRNA of BMP-6, BMPR-IA,BMPR-IB and BMPR-Ⅱwas Moderate(relative optical density,ROD was 0.81±0.12,0.52±0.13, 0.62±0.11,0.73±0.19,0.79±0.22,respectively).But the expression of of mRNA of BMP-3,4,5,7 were trace.2. Western blot indicated that the expression of protein of BMP-2,BMP-6 in liver cancer SMMC7721 cells was highly than in normal liver L-02 cells (P<0.01).3.Western blot indicated that the expression of protein of BMPR-IA,BMPR-IB and BMPR-Ⅱwas no significant difference between liver cancer SMMC7721 cells and normal liver L-02 cells (P>0.05).Conclusions 1.The expression of of BMP-2 was highly and the expression of BMP-6, BMPR-IA,BMPR-IB and BMPR-Ⅱwas Moderate in liver cancer SMMC7721 cells. but the expression of BMP-3,4,5,7 were trace.2.The level of expression of BMP-2,BMP-6 in liver cancer SMMC7721 cells was highly than in normal liver L-O2 cells,the level of expression of BMPR-IA,BMPR-IB and BMPR-Ⅱwas no significant difference between liver cancer SMMC7721 cells and normal liver L-02 cellsPartⅡExpression of BMP-2 and clinical significance in Primary hepatic carcinoma tissueObjective:To investigate the expression of BMP-2 in liver cancer tissue and normal liver tissue,and the correlationship between expression of BMP-2 and clinicopathologic factors of Primary hepatic carcinoma.Methods:1.30 cases of samples selected from liver cancer tissue and 10 cases of samples selected from normal liver tissue were detected respectively for the expression of BMP-2 mRNA through RT-PCR.2. The levels of mRNA and protein of BMP-2 in different clinicopathological parameters were determined with RT-PCR and Western blot and investigate the Correlationship between BMP-2 and clinicopathologic factors parameters.Results:the expression of BMP-2 was stronger in liver cancer tissue than in the normal liver tissues(P<0.01), The expressions of BMP-2 was correlated with Clinical staget,pathological differentiation and distant metastasis (P<0.01),but not with age of patients, Pathological classification,AFP and Cirrhosis(P>0.05).Conclusion:The overexpression of BMP-2 in in liver cancer tissue suggest that BMP-2 is an important feature of liver cancer,the expression of BMP-2 is closely correlated with the malignant biologic behavior of liver cancer.PartⅢThe experimental Study of siRNA targeting BMP-2 of the human liver cancer SMMC7721 cellsObjective:To observe the effect of siRNA targeting BMP-2 on the expression of BMP-2 in human liver cancer SMMC7721 cells.Methods:The molecular sequences of three siRNAs targeting BMP-2 gene were designed and synthesized. There were 6 groups in this study including groupⅠ(normal control, non-transfected cells), groupⅡ(blank control, only liposome-transfected cells), groupⅢ(negative control, non-specific siRNA-transfected cells) and groupsⅣ-Ⅵ(siRNA-A, siRNA-B and siRNA-C-transfected cells, respectively). SMMC7721 cells were instantaneously transfected using lipofectamine mrthod. The levels of mRNA and protein of BMP-2 in cells were determined with RT-PCR and Western blotting.Results:The ROD was obtained using spectrodensitometry and was 0.89±0.11, 0.93±0.12,0.92±0.14,0.65±0.08,0.32±0.07 and 0.55±0.07 in groupⅠ-Ⅵ, respectively. The mRNA expression of BMP-2 was lower in groupⅣ-Ⅵthan in groupⅠ-Ⅲ(P<0.05), especially in group BMP-2-siRNA-B the mRNA expression was the lowest (P<0.01). However, there was no statically significant difference in mRNA expression between groupⅠ-Ⅲ(P>0.05), but groupⅤhad statistically significant difference compared with groupⅣandⅥ(P<0.05). The results of Western blot are shown that The RGS of BMP-2 protein was significantly down-regulated in group BMP-2-siRNA-B (0.37±0.10). In RGS of BMP-2 protein, group BMP-2-siRNA-B has significant difference compared with groupⅣandⅥ(P <0.05), but there was no significant difference between groupⅠ-Ⅲ(P>0.05). The results of Western blot were consistent with ones of RT-PCR.Conclusion:Specific siRNA targeting BMP-2 can markedly inhibited the expression of BMP-2 in liver cancer SMMC7721 cells, especially BMP-2-siRNA-B,It Can be used for follow-up experiments.PartⅣEffect of siRNA targeting BMP-2 on the abilities of proliferation and invasion of human liver cancer SMMC7721 cellsObjective:To study the effect of siRNA targeting BMP-2 on the abilities of proliferation and invasion of human liver cancer SMMC7721 cellsMethods:In this study, there were 3 groups including group 1 (SMMC7721-nontransfection, non-transfected control cells), group 2 (SMMC7721-siControl, non-specific siRNA-transfected cells) and group 3 (SMMC7721-siBMP-2, siRNA-BMP-2-transfected cells).The abilities of proliferation, apoptosis, adhesion,migration and invasion of 3 groups were assessed by MTT assay,flow cytometry,plate adhesion model, cell scratch test and matrigel invasion assay(Transwell chamber model).Results:1.The results of MTT assay indicated that the proliferation level of SMMC7721 cells were no different at 24h(P>0.05), but obviously decreased at 48h and were Maintained for 72h after transfection with BMP-2-siRNA.Group of BMP-2-siRNA transfection induced an increase in G0/G1 Phase cells(59.30%) as compared with the group of nontransfection(32.35%) and group of siControl(31.08%), respectively, (P<0.05), Group of BMP-2-siRNA decreased in S Phase(22.39%) as compared with group of nontransfection(35.51%) and group of siControl(38.32%),respectively,(P<0.05).The apoptosis rate in group of BMP-2-siRNA (16.5%)was significantly more upgraded than group of nontransfection(1.75%) and group of siControl(1.49%) (P<0.01).2.The results of plate adhesion model assays indicated that the liver cancer SMMC7721 cells adhesion was significantly decreased in BMP-2-siRNA group (P<0.01).The results of cell scratch test indicated that The width of cell scratch was 65.8±5.3μm,36.6±4.0μm and 34.1±5.1μm in BMP-2-siRNA group, nontransfection group and siControl group, respectively. The results indicated that the ability of migration of liver cancer SMMC7721 cells was significantly decreased in BMP-2-siRNA group, and BMP-2-siRNA group had significant difference in the width of cell scratch compared with nontransfection group and siControl group (P<0.01).The results of Transwell chamber indicated that The number of cells to penetrate the membrane was 11.5±2.0,33.5±3.1 and 35.7±3.4 in BMP-2-siRNA group, nontransfection group and siControl group, respectively. The results indicated the ability of invasion of liver cancer SMMC7721 cells was significantly decreased after BMP-2-siRNA had mediated BMP-2 gene, and BMP-2-siRNA-group had significant difference in the number of invasion of cells compared with nontransfection group and siControl group (P<0.05).Conclusions:The abilities of proliferation, adhesion,migration and invasion of liver cancer SMMC7721 cells can be inhibited by siRNA targeting BMP-2 efficiently in vitro.PartⅤMechanism of siRNA targeting BMP-2 on the abilities of proliferation and invasion of human liver cancer SMMC7721 cells Objective:To study the Mechanism of siRNA targeting BMP-2 on the abilities of proliferation and invasion of human liver cancer SMMC7721 cells.Method:At 48 h after transfection, p-ERK, p-JNK and p-p38 expression of protein were measured by western blot; MMP-2,MMP-9,E-Cda and uPA expression were measured by RT-PCR,western blot and gelatinase zymography.Results:Western blot indicated that the expression of protein of p-ERK in the group of BMP-2-siRNA-transfected cells were significantly inhibited,the expression of protein of p-JNK and p-p38 in the group of BMP-2-siRNA-transfected cells were no change.RT-PCR and Western blot indicated that the expression of mRNA and protein of MMP-2,MMP-9 in the group of BMP-2-siRNA-transfected cells were significantly inhibited, gelatinase zymography indicated that similar results.The expression of E-Cad in the group of BMP-2-siRNA-transfected cells were significantly upgraded,but the expression of uPA in the group of BMP-2-siRNA-transfected cells were no change.Conclusion:Our results demonstrate that BMP-2-siRNA might decrease the abilities of proliferation and invasion of human liver cancer SMMC7721 cells which might be associated with upregulation of E-Cad and downregulation of MMP-2,MMP-9 Through the signal transduction pathway of MAPK/ERK,but seems to be disconnected with uPA and the signal transduction pathway of MAPK/JNK and MAPK/p38.