The Effects And Molecular Mechanism Of Bone Marrow-derived Mesenchymal Stem Cells On Proliferation And Metastasis Of Human Hepatocellular Carcinoma In Vitro And In Vivo

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors which is the third leading cause of cancer death in the world, and the second in China. The clinical features of HCC are fast-growing, easy to migrate, and the poor prognosis. Studies have shown that tumor growth and metastasis is not an isolated process,but based on the interaction between tumor cells and the surrounding microenvironment and the body environment.Mesenchymal stem cells (MSCs) have long been known to contribute to the maintenance and regeneration of connective tissues, and as a component of the tumor microenvironment.MSCs become a research hotspot in recent years. MSCs are derived from the mesoderm of a class which has a high degree of self-renewal capacity and differentiation potential of non-hematopoietic stem cells, mainly present in the connective tissue, organ interstitium and bone marrow. According to current reports of view, MSCs support or suppress tumor growth is a discrepancy. Li Hong of our lab reported that the migration and invasion ability of fused cells were enhanced after the fusion between MSCs and low-metastatic hepatocellular carcinoma cell line HepG2, and cell fusion can promote HepG2cells metastasis in vivo. In this study, our aim is to investigate the effects and molecular mechanism of rat bone marrow-derived MSCs on the proliferation and migration of human hepatocellular carcinoma cell in vitro and vivo and it was expected to explore the role of MSCs in HCC development and help to identify new therapeutic target for anti-tumor growth and metastasis.Objectives:1. Investigate the effects of rat bone marrow-derived MSCs on the proliferation and migration of human hepatocellular carcinoma cell in vitro and vivo.2. Exploration of the molecular mechanism.Methods:1. Identification and cultivation of MSCs. Bone marrow was isolated under sterile conditions from femurs and tibias. Briefly, bones were isolated and marrow was flushed out with L-DMEM. Cells were propagated in the medium at37℃in a5%CO2humidified incubator. Non-adherent cells were removed after4and24hrs and every2-3days thereafter by gently washing with medium. Cultures were grown to80-90%confluency before passing. Morphology of the cells was observed by microscope. The expression of CD34, CD45, CD90, and CD105were detected by flow cytometry. Collect conditioned media (MSC-CM).2. Investigate the effects of MSCs on the proliferation of human hepatocellular carcinoma cell. The cell growth was measured by MTT assay, colony formation and soft agar colony formation assay. Cell cycle progressions were analyzed by flow cytometry. Western blot were used to detect the expression of cyclin D1.3. Investigate the effects of MSCs on the migration of human hepatocellular carcinoma cell. Scratch test and transwell chamber was used to detect infiltration and migration of human hepatocellular carcinoma cells after treated with MSC-CM. Established the liver cancer metastatic model. Detect of TGF-β content in MSC-CM by ELISA kit.EMT markers (E-cadherin and Vimentin) and EMT regulatory factors (Snail and Twist1) expression were detected by Western blot; The mRNA levels were evaluated by RT-PCR; MMP2and MMP9changes were detected by Gelatin zymography.Results:1. The cells isolated and cultured from bone marrow were MSCs, which were identified by the surface markers including CD105+, CD90+, CD34-, CD45- and by flow cytometry. Growth curve showed that the1st and4th generation of MSCs has much stronger growth ability than the8th generation.2. Mesenchymal stem cells promote the proliferation and colony formation abilityof cell line HepG2indirectly by MSC-CM (P<0.05). Cell number in Go/G1phase was decreased and that in S and G2/M phase was increased(P<0.05). MSCs promote the expression of protein Cyclin D1in HepG2(P<0.05).3. The results of scratch test, infiltration test and migration test indicated that the SMMC-7721cells were more migratory and invasive after treated with MSC-CM. The results of the liver cancer metastatic model showed the average number of liver metastasis in Mix group (4.42±1.39) was higher than SMMC-7721group (2.42±0.97); Rate of lung metastasis was85.71%in Mix group, and28.57%in SMMC-7721group. The concentrations of TGF-β1in5th generation of MSC-CM are0.508±0.027pg/ml. After treated with MSC-CM, results of western blot detection indicated that low expression of E-cadherin and high expression of Vimentin in SMMC-7721cells. Further testing showed the expression of EMT regulatory factors Twist1and Snail were also highly expressed The same results was obtained by RT-PCR. Gelatin zymography results confirmed that MMP2and MMP9were highly expressed.Conclusions:1. MSCs can promote the proliferation and migration of human hepatocellular carcinoma cells in vitro and vivo.2. Under the affect of MSCs, human hepatocellular carcinoma cells experienced EMT and highly expressed MMP2and MMP9. That may be the mechanism of enhancement of metastatic ability in vivo.