| Hepatocellular carcinoma (HCC), one of the most common cancers worldwide, ranks as the fifth most common malignancy in men and the seventh in women. It is among the top three causes of cancer death in the world. There are at least700,000new cases of HCC per year around the world. In China alone360,000new cases of this tumour occur each year representing more than50%of the world total. Etiological factors for HCC are well-defined that infection with hepatitis B or C virus, heavy alcohol intake, and aflatoxin intake are the important risk factors for HCC. Currenly, surgical resection is the mainstay of treatment for HCC. Curative surgical resection rate is increased greatly and5-year survival rate after surgical therapy is also improved to50%-60%during the past decades, because of the successive advancement in operation mode for HCC. However, compared with other malignant digestive-tract diseases, long-term survival rate of patients with HCC is far less satisfactory due to the high postsurgical recurrence and metastasis rate. Furthermore, the factors that mediate HCC invasion and recurrence are complicated and muti-factorial. Hence, it is urgent to identify crucial carcinogenic pathways and find out new therapeutic targets for HCC.Recent evidence highlighted that tumor microenvironment (TME) provided the indispensable cues to the maintenance of the cancer stem cells and to enhance the seeding of cancer cells at metastatic sites. Nonetheless, TME is spatially and temporally heterogeneous, suggesting that the effects of different components of TME on cancer cells are distincted. TME is composed of endothelial cells and their precursors, fibroblasts, carcinoma-associated fibroblasts (CAF), myofibroblasts, inflammatory and immune cells, extracellular components including extracellular matrix (ECM), as well as soluble factors. Most recently, several studies have described that CAFs are a key determinant in malignant progression of cancer via regulating EMT, and that many cytokines and chemokines are the crucial incentives for EMT in TME. However, the role of normal tumor-host cells in tumorigenesis and progression is largely unknown.In the first part of this study, we used two cell culture models for simulating the interactions of tumor cells with their microenvironment, in order to identify the exact roles of diverse compositions of TME on cancer cells. Based on the results of the first part, we further uncoverd the mechanisms of MRC-5regulating HCC cells invasion, migration, proliferation and apoptosis.Part I Induction of EMT-like procession and MET in vitroObjective:Epithelial-mesenchymal transition (EMT) is believed to play an important role in metastasis of cancer. The reverse of EMT, mesenchymal-epithelial transition (MET) is also related to tumor metastasis. It is agreed that tumor microenvironment (TME) may be the inducer of EMT/MET. The purpose of this study was to investigate whether cancer cells can be induced to undergo EMT/MET in vitro. Methods:We used two cell culture models to simulate the effect of TM on cancer cells. Co-culture model:Bel-7402(HCC cell line) were co-cultured with HL-7702(normal liver cell line) or RF/6A (Retinal vascular endothelial cell) in double-layer six well plates. Conditioned medium model:Bel-7402were cultured in the conditioned medium of MRC-5, HL-7702, RF/6A. The invasion and migration ability of HCC cells was determined by transwell assay. The expression profiles of epithelial marker, mesenchymal marker, EMT-related TFs and MMPs were evaluated by western blot. The expressiong of F-actin, mesenchymal marker and membrane expressions of E-cadherin/p-catenin complex, a-catenin and y-catenin were further verfied by confocal laser scanning microscope. RT-PCR was used to detect laminins and integrins mRNA expression levels in Bel-7402, Bel-7402co-cultured with RF/6A, Bel-7402cultured in MRC-5-CM, Bel-7402cultured in HL-7702-CM, Bel-7402cultured in RF/6A-CM, Bel-7402before and after transfection with miR-200a mimics.Results:Bel-7402was co-cultured with HL-7702{Bel-7402-(HL-7702)} for90days and with RF/6A {Bel-7402-(RF/6A)} for90days. The invasion and migration ability of Bel-7402-(HL-7702) was determined by transwell assay at day44,60and74. Compared with control, we showed that Bel-7402-(HL-7702) was weakened to invade and migrate (P<0.05). Meanwhile, we determined whether Bel-7402-(RF/6A) rendered altered ability of invasion and migration using transwell assay at day44and day60. We also found Bel-7402-(RF/6A) was declined to invade and migrate, too (P<0.05). Epithelial marker E-cadherin, a-catenin and γ-catenin in co-cultured Bel-7402were up-regulated. Immunofluorescence evaluation also demonstrated that the membrane expression of E-cadherin, a-catenin and β-catenin was increased in co-cultured Bel-7402. What’s more, compared with the no co-culture Bel-7402, F-actin-rich protrusions in co-cultured Bel-7402were decreased. In light of this, we speculated that co-cultured Bel-7402underwent MET. In contrast, MRC-5-CM induced" Bel-7402to undergo EMT-like procession. Bel-7402cultured in MRC-5-CM was named Bel-7402-(MRC-5)-CM. We found that Bel-7402-(MRC-5)-CM were beyond recognition at day14. Cells became elongated. In addition, F-actin-rich protrusions were also observed obviously. The invasion and migration ability of Bel-7402-(MRC-5)-CM were increased significantly (P<0.05). The morphology and the invasion and migration ability of Bel-7402-(HL-7702)-CM or Bel-7402-(RF/6A)-CM in respond to control did not change significantly at day14. RT-PCR results indicated that laminin Al, A2and integrinA4, Al1, AL, AM, AV, B6, B7, B8were up-regulated in Bel-7402-(MRC-5)-CM, and that integrinB4was down-regulated. Conversely, laminin A1, A2and integrinA4, A11, AL, AM, AV, B6, B7, B8were down-regulated in Bel-7402-(RF/6A) and in Bel-7402transfected with miR-200a mimics. IntegrinB4was up-regulated in Bel-7402-(HL-7702)-CM, Bel-7402-(RF/6A)-CM, Bel-7402-(RF/6A) and in Bel-7402transfected with miR-200a mimics.Conclusion:We demonstrate that HL-7702and RF/6A can induce Bel-7402to undergo MET, and that MRC-5can induce Bel-7402to undergo EMT-like precession. Tumor microenvironment is the inducer of EMT/MET by regulation E-cadherin/catenin complex, laminins and integrins. Part Ⅱ MRC-5fibroblast conditioned medium mediates multiple pathways regulating invasion, migration, proliferation and apoptosis in Hepatocellular carcinomaObjective:Role of MRC-5fibroblast conditioned medium (MRC-5-CM) in breast cancer cell motility has been well established. However, its role in hepatocellular carcinoma (HCC) is less clear. The aim of our study was to investigate the effect of MRC-5-CM on HCC and to explore the underlying mechanisms.Methods:Using a combination of techniques, such as transwell assay, western blot, immunofluorescence, flow cytometry, conditioned medium culture model, the role of MRC-5-CM in HCC was dissected out.. Results: MRC-5-CM induced Bel-7402and MHCC-LM3to undergo non-classical epithelial-mesenchymal trasition (EMT) but not via up-regulating typical EMT-related transcription factors (Snail/Twistl/ZEB-1/ZEB2). The induction of non-classical EMT was via quintessential redistribution of α, β,γ-catenin, P120catenin and E-cadherin. MRC-5-CM activated α6, β3, β4,β7-integrin/FAK/Src signaling pathway and increased actived form of MMP2. The flow cytometry results showed that MRC-5-CM induced G1phase arrest in Bel-7402with reduction of cells in S phase. In contrast, MRC-5-CM induced S phase arrest in MHCC-LM3with reduction of cells-in G2/M phase. The results also exhibited that MRC-5-CM inhibited apoptosis in Bel-7402while induced apoptosis in MHCC-LM3.Conclusion:MRC-5-CM promoted cancer cells invasiveness through initiating non-classical EMT, activating Integrin/FAK/Src signaling pathway and increasing actived form of MMP2; MRC-5-CM exerted distinct roles in cancer cells viability by typical cell cycle and apoptosis associated genes and other unknown mechanosensors... |
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