Hepatocellular Precancerous Injuries Induce Mesenchymal Of Bmscs And Tumor Promotion

Background:With the developing of cancer research, the tumor microenvironment has aroused widespread concern by governments and academia fields in recent years. People gradually realized that tumor is heterogeneity, and only a small part of cells which we called cancer stem cells (CSCs) are the source of tumor formation and development. Moreover, there was a close relationship between CSCs and the tumor microenvironment. It was found that normal microenvironment can inhibit tumor development, and abnormal microenvironment can promote tumor development. This prompts that mutation might not be the unique factor in regulating the tumor formation, microenvironment might also be involved. Although a variety of cells in the tumor microenvironment play a certain role in the tumor development, the role of tumor associated fibroblasts (TAFs) might be more important. Bone marrow mesenchymal stem cells (BMSCs) are among the key sources of TAFs. Liver fibrosis is a wound healing response in liver, also a necessary intermediate of hepatocirrhosis. There is a close relationship between hepatic fibrosis and hepatocellular carcinoma, but the intrinsic relationship between them is unclear. We speculated that liver fibrosis might be a potential factor in hepatocellular carcinoma, but the role of the matrix before tumor had rarely been reported.Objective:We aimed to investigate the effect of gene expression in liver cells after short-time exposure to exogenous carcinogen, to explore the role of secreted factors of the damaged liver cells on malignant transformation of the microenvironment, and to clarify the influence of transformed microenvironment in tumorigenicity and tumor migration.Methods:1.Primary culture and appraisal the rat mesenchymal stem cellsWe used the whole bone marrow adherent method to culture the rat bone marrow mesenchymal stem cells (BMSCs) which were identified by detecting its multi-directional differentiation potential and cell surface markers.2.The roles of exogenous carcinogens on BRL cell1) After exposed to Aflatoxin B1(AFB1), cells survival rate of BRL wasassessed by Methyl thiazolyl tetrazolium (MTT) assay. Through drawing growth curve,the differences in proliferation ability were evaluated before and after exposed to AFB1.2) The chromosome damage of BRL cells exposed to AFB1was analysis by Karyotype.3) MTT, SCGE and Hoechst33258stain were detect the cumulative cytotoxicity for DEN on human liver stem cells.4) Real-time quantitative PCR was used to detect the levels of gene expressions change after the BRL cell exposed to AFB1.3.The microenvironment variation and their effects on the ability of the tumor information.1) To establish the Transwell co-cultured model and simulate the influence of damage liver cells on BMSCs.2) Immunohistochemical was used to compare the level of α-SMA expression on BMSCs before and after induced.3) Soft agar test, scratch test and invasion test were used to assess the effect of microenvironmental variation on tumor information.Results:1. The toxicity effect of DEN in fetal hepatic stem cellsThe survival rate of fetal hepatic stem cell was significantly lower than that of control group (P<0.05) at the dose of900,1350and1800μg/ml after48h treatment. When human hepatic stem cells were exposed to DEN for24h, cellular apoptosis occurred with karyopyknosis and deep staining. Break in DNA strands was found at the concentrations of900and1350μg/ml of DEN; the content of DNA in tail [(18.44±4.99)%, (17.33±3.29)%, respectively] was higher than the control [(0.015±0.004)%](P<0.01), while the comet length was decreased at the higher concentrations of DEN (1800and2250μg/ml)2. The toxicity effect of AFB1in BRL cellsThe survival rate of BRL cells was43.59% at the dose of3mmol/L AFB1by MTT assay. It was significantly lower than the control (P<0.05). The value of IC50was2.96mmol/L3. Successfully cultured rat BMSCsThe cells showed spindle-shaped or spindle-shaped, and the cell shape was similarly to fibroblast cells. Detection of surface markers on BMSCs showed that positive rate of CD29, CD44, CD45was92.5%,65%,3.5%respectively. In the conditioned medium, BMSCs could differentiate into osteoblast or fat cells.4. Karyotype analysisChromosomal abnormalities including polyploidy, gap, and breakage were observed in BRL after exposure to AFB1for1Oh.5. Real-time quantitative PCR detected the mRNA levelsThe expressions of TGF-β, PDGF-A, PDGF-B, PDGF-C and PDGF-D mRNAs were increased in varied degrees in AFB1-exposed BRL compared with untreated BRL cells.6. The effect of damaged BRL in BMSCsWe co-cultured AFB1-exposed BRL and BMSCs by using Tranwell chamber, and detected upregulation of a-SMA expression in BMSCs.7. To investigate the effect of malignant transformed BMSCs on HepG2cell lineTumorigenicity, tumor migration and invasion abilities of HepG2were enhanced in malignant transformed BMSCs by using soft-agar cloning assay, scratch test and invasive test.Conclusions1. DEN could induce directly DNA damage of human hepatic stem cells in vitro, so it might have some potential genetic toxicity;2. Pretreatment with AFB1increased the TGF-β and PDGF mRNA levels of BRL cells, and the cytokines of secreted by injured BRL cells up-regulated the expression of α-SMA in BMSCs.3. Precancerous injuries of BRL cells could cause the microenvironment change, which can enhance the ability of tumor formation, migration and invasion.