A Study On Tumor Invasion And Metastasis Promoted By BMP-4-induced Epithelial-mesenchymal Transition In Hepatocellular Carcinoma

Background:Hepatocellular carcinoma is the one of the most prevalent cancer and the third most frequent cause of cancer-related death. Since tumorigenesis is a multiple-stage procedure, some underlying molecular mechanisms remain unclear. Bone morphogenetic protein (BMP) belongs to the transforming growth factor β superfamily. BMP has broad functions including the regulation of many types of normal tissue pattering and epithelial-mesenchymal interaction in the developmental process. One of the BMP family members, BMP-4is essential for development and is involved in a variety of biological programs, one of which is tumor development. In addition a recent study revealed that BMP-4induces epithelial to mesenchymal transition (EMT), which is a crucial step for cancer invasion and metastasis in ovarian cancer cells. It appeals to find the relationship between BMP-4and EMT in more common cancer diseases, one of which is the hepatocellular carcinoma.Materials&Methods:In this study, various assays were adopted to test the influence of BMP-4on cell growth and proliferation, cell cycle and apoptosis, invasion, metastasis and EMT. Such are MTT assay, colony formation assay, transwell migration assay, flow cytometry, RT-PCR and real-time quantitative PCR, and immunofluorescence.Results:BMP-4had no influence on proliferation and colony formation of L02, but stimulated colony formation of HepG2(135.333±11.015V.S.46.667±11.719, P=0.001). Its specific antagonist Noggin significantly reduced colony formation of HCCLM3(135.000±7.000V.S.45.000±13.454, P=0.001). As to apoptosis, BMP-4seemed to play a protective role. Furthermore, after treatment of BMP-4, mRNA expression levels of MMP-2and MMP-9in L02and HepG2increased (the former was2.481±0.819V.S.0.795±0.215and2.817±0.763V.S.0.908±0.258separately, the P value was0.026and0.015separately; the latter was4.242±0.862V.S.0.644±0.236and3.716±1.334V.S.0.541±0.169separately, the P value was0.002and0.015separately). Cell lines also presented morphology change and improved invasion and metastasis ability, which implied that BMP-4induced EMT in HCC cell lines.Conclusions:It reveals that BMP-4(≥50ng/mL) plays positive role in HepG2proliferation, while different concentrations of BMP-4have no significant differences on proliferation of L02and HCCLM3. BMP-4can promote wound healing and cell migration. It can induce S phase arrest in HepG2and HCCLM3, but G2/M phase in L02to reduce apoptosis. After BMP-4treatment, mRNA expression level of MMP-2and MMP-9in L02 and HepG2improves, as well as cell invasion ability. BMP-4can also induce epithelial-mesenchymal transition in human hepatocellular carcinoma cell lines.