Characterization Of A Dual-color Fluorescence Tracing Model Of Hepatocellular Carcinoma And The Experimental Researches Of MiR-146b-5p Effect The Proliferation Andmigration Of Hepatocellular Carcinoma Cells

Part I Characterization of a dual-color fluorescence tracing model of hepatocellular carcinomaObjective: To establish a dual-color fluorescence tracing orthotopic transplantation model of hepatocellular carcinoma, and analyze its characterization of tumor biological.Methods: Stable high red fluorescent protein(RFP)-expressing cells were injected into the liver lobe of enhanced green fluorescent protein(EGFP)-expressing nude mice.The growth and metastasis of tumor were visualized dynamically by the whole-body in vivo fluorescence imaging system in real time. When tumor-bearing mice appeared distressed, they were sacrificed and an autopsy was conducted. HCC tissues were removed from tumor-bearing mice, and frozen slices were made. The organizational structure of transplanted tumors was observed under the fluorescence microscope. Tumor nodules and ascites from tumor-bearing mice were subculturing. Immunohistochemistry was used to evaluate the expression of tumor-related protein.Results: A dual-color fluorescence tracing orthotopic transplantation tumor model of HCC was successfully established. It can mimic the characterization of tumor biological such as invasion, metastasis, malignant ascites. The growth and metastasis of tumor were visualized by the whole-body in vivo fluorescence imaging system. Under the fluorescence microscope, tumor cells with red fluorescence and host cells with green fluorescence were intertwined in the reconstruction region of tumor tissue. Cell fusion between tumor and host cells could be observed clearly. Tumor-related protein PDGFRÉ‘, p STAT3, IRAK1,MMP9, N-RAS was expressed in HCC tissues.Conclusion: The dual-color fluorescence tracing orthotopic transplantation model of HCC is stable, reliable and easy to repeat. Using this, mutual interactions between hepatoma cells and host tissues could be observed directly to further elucidate the development of tumor microenvironment.Part II The experimental researches of mi R-146b-5p effect the proliferation and migration of hepatocellular carcinoma cellsObjective: To detect the expression level of the mi R-146b-5p in hepatocellular carcinoma cells and study its effects on proliferation and migration of hepatocellular carcinoma cells, then exploring its mechanism of the role.Methods: Detect the express level of mi R-146b-5p by means of real-time RT-PCR in hepatocellular carcinoma cell lines and hepatic cells. Then, build overexpression of mi R-146b-5p group, negative control group, and the blank control group; compared the differences of three groups of cells in the proliferation and migration using the method of CCK-8 test, plate clone-formation test, transwell technique, wound healing test. We searched the putative targets of mi R-146b-5p using prediction program and then using the method of western blot to validated the target-genes.Results: The expression level of mi R-146b-5p is generally low in hepatocellular carcinoma cells. The cell proliferation ability and migration ability of overexpression of mi R-146b-5p group was lower than that in group of blank control group and negative control group. And the protein expression level of IRAK1 and p STAT3 were negatively regulated bymi R-146b-5p.Conclusion: mi R-146b-5p inhibition of the proliferation and migration of hepatocellular carcinoma cells, probably by targeting IRAK1 and p STAT3.