Effect Of Mesenchymal Stem Cell On Mechanobiological Behavior Of Hepatoma Cells And Its Involved Molecular Mechanism

MSCs possess critical properties, exhibit self-renewal ability and multi-lineagedifferentiation potential which can differentiate to osteoblasts, chondrocytes, adipocytesand myocyte under various culture conditions. As MSCs can be recruitted to theenvironment of inflammation and the sites of tissue injury, these cells play a significantrole in repair and regeneration of tissue damage through a combined effect ofanti-inflammatory, paracrine, proliferation and differentiation. The use of MSCs forgene delivery and cell therapy has been regarded as an important tool. Recently, MSCshave become the most widely used stem cell type which in tissue repair andregeneration.The malignant tumor was caused by the disorder of cell cycle or the unfetter celldivide and growth, it threatens human health seriously, however, there is still a hugechallenge against the prevention and treatment of this disease. Inflammatorymicroenvironment was one of the most important characteristics of tumor tissues.Considering the fact that the normal tissue accompanied by malignant change also couldbe treated as a special kind of tissue damage. So tumor tissue can recruit MSCs toinvolve in remodeling the microenvironment of tumor, which further significantlyinfluenced the biological behavior of tumor cells.In the area of tumor studies, the interactive dialogue and influences betweentumor cells and MSCs will be a hot topic in the future. However, the mechanism of thisphenomenon is not completely clear. So our research mainly focuses on the changes ofbiological behavior of rat hepatoma cells (CBRH-7919) in a microenvironment ofco-culturing with MSCs and discusses the possible signaling pathway. The main contentof this research and results are as follows:1. Isolation and culture of cellPercoll density gradient centrifugation and adherent culture was used to separateMSCs. The cells were maintained in L-DMEM supplemented with10%fetal bovineserum. The separated MSCs showed morphological uniform and swirl distribution.Using flow cytometry to analyze the expression of cell surface antigen, the resultsshowed that CD44and CD90positive expression of cell surface antigen, but CD34negative expression. Rat hepatoma cell line CBRH-7919cells were accorded to the conventionalmethod culture which maintained in L-DMEM supplemented with10%fetal bovineserum.2. Effect of conditioned medium from mesenchymal stem cells on CBRH-7919cellsmigrationUsing a transwell assay, we determined the effects of MSC-CM on the migrationof CBRH-7919cells. The results showed that a significant increase of migration ofCBRH-7919cells in the presence of MSC-CM compared to control groups.Further studies showed that, the mRNA expression of CXCR4was increased byMSC-CM (p<0.05), western blot confirmed that the up-regulation of CXCR4inresponse to MSC-CM (p<0.01). Immunofluorescent staining showed that an increaseexpression of F-actin from cytoskeleton by MSC-CM, and AFM showed that theYoung’s modulus of the MSC-CM-treated cells was significantly decreased. AMD3100,the inhibitor of CXCR4, could significantly inhibit MSC-CM-induced theoverexpression of CXCR4in CBRH-7919cells, the F-actin expression of cytoskeletonand the Young’s modulus of cells returned to control level. And it also could inhibit theabilities of the migration of cells that MSC-CM promoted. The results demonstrated thatMSC-CM could promote cells migration by inducing the expression of CXCR4inCBRH-7919cells, remodeling cytoskeleton, and decreasing cell stiffness.3. Effect of Mesenchymal stem cells on CBRH-7919cells proliferationUsing a transwell system, we co-cultured MSCs and CBRH-7919, and then theproliferation of CBRH-7919cells was examined by MTT assay. A significant increaseof proliferation of CBRH-7919cells was observed in the presence of MSCs. There wason significantly increased after co-culture one day. However, there was a significantlyincreased (p<0.01) after four days. Moreover, the proliferation of experimental groupwas increased1.6times by control group (p<0.05). The RT-PCR results showed that itcould enhanced the expression of Wnt5a gene and β-catenin gene in CBRH-7919cellswhen co-cultured with MSCs. Western blot further confirmed the up-regulation ofβ-catenin in response to MSCs (p<0.01). The results demonstrated that MSCs couldpromote CBRH-7919cells proliferation, and Wnt5a/β-catenin pathways play a crucialrole in this process.Taken together, our results showed that MSCs microenvironment promotes tumorcells migration by lessening cell stiffness and increasing F-actin formation throughup-regulation of CXCR4expression. At the same time, MSCs microenvironment also promotes tumor cells proliferation through Wnt5a and β-catenin signal pathways. Theseresults suggesting that MSCs could been recruit into tumor sites and participate inremodeled tumor microenvironment, promoted tumor cells migration and proliferation,which further suggesting the security issues of MSCs in cancer research, clinicalapplication and biotechnology.