Thyroid Hormone/Thyroid Hormone Receptor ? Signaling Promotes Tumorigenicity Through Inducing Cancer Stem-like Cell Self-renewal In Hepatocellular Carcinoma

OBJECTIVES:1)To prove the self-renewal regulatory role of thyroid hormone(Thyroid hormone,TH)for Liver cancer stem cells(Liver cancer stem cells,LCSCs).2)To clear and definite the regulation and control function of the expression of thyroid hormone receptor alpha(Thyroid hormone receptor alpha,TR?)for the clone formation ability of CSQT-2 and MHCC97-H liver cancer cells.3)To clear and definite the role the TH/TR?signaling pathway plays in regulating the growth of liver cancer cells in vivo.4)To explore the correlation between the TH/TR?signaling pathway and the expression of regulatory factors in known cancer stem cells(cancer stem cells,CSCs).METHODS:1)We would establish the low adsorption model of tumor cells by using CSQT-2 cells firstly,in order to detect the ability of cell self-renewal,and screen and identify the hormones/Nuclear receptor(NR)signaling pathways that may affect LCSCs by using 24 kinds of different hormones/Nuclear receptor agonists or antagonists that are currently known to stimulate liver cancer cells.2)Through Realtime qPCR,we plan to detect the difference in expression of TR?in different clinical paired specimens(normal liver tissue,HCC and portal venous thrombosis).3)We would interfere TR?and TR?(Thyroid hormone receptor beta,TR?)by establishing shRNA lentivirus specific for TR?and TR?,in order to clear and definite whether TR?is specific to the regulation effect of the TH/TR?signaling pathway on the self-renewal of liver cancer cells.4)By means of antibody incubation and flow cytometry detection and analysis,we could clear and definite whether specific interference with TR?that can effectively inhibit the induction of CD90~+cells in CSQT-2 liver cancer cells by TH/TR?signaling pathway.5)By virtue of IC50 detection,we could clear and definite whether the interference of TR?could significantly increase the sensitivity of hepatocellular carcinoma cells to epirubicin and 5-fluorouracil,to confirm the fact that TR?plays a role in the chemotherapy resistance of hepatocellular carcinoma regulated by the TH/TR?signaling pathway.6)Through the classical serial dilution assay with different number of liver cancer cells in NOD/SCID mice,we could clear and definite whether the ratio of LCSCs in liver cancer cells and the proliferation of liver cancer cells in vivo would be affected after TR?interfered.7)By means of Realtime-qPCR and chromatin immunoprecipitation(ChIP),we continue to explore the molecular mechanism that the TH/TR?signaling pathway promotes the self-renewal capacity of Hepatocellular carcinoma(HCC).RESULTS:In the study,we report that the TH/TR?signaling pathway promotes cell self-renewal in HCC cells.Moreover,the TH/TR?signaling pathway also increases the percentage of CD90~+HCC cells and promotes drug resistance of HCC cells and TH mainly through its receptor(Thyroid receptor,TR)plays a role in the process.In addition,we found that it can significantly inhibit the function of TH/TR?signaling pathway on liver cancer cells self-renewal capacity in vitro,the quantity of CD90+liver cancer cells,drug resistance and the tumors growth in vivo in virtue of shRNA interference that was specific to TR?in the functional study.By analyzing primary human HCCs samples,we found that TR?transcript level is significantly elevated in primary liver cancer and portal vein tumor thrombus(portal vein tumor thrombus,PVTT),compared to that of corresponding normal liver tissue.Knocking down TR?not only inhibits HCC self-renewal in vitro but also suppresses HCC tumor growth in vivo.Interestingly,we also found that the TH/TR?signaling pathway in liver cancer cells has a synergistic effect with the NF-?B signaling pathway:TR?and p65 cooperatively drive the expression of BMI1 by co-binding to the promoter region of BMI1 gene,and the NF-?B signaling pathway plays a role in the process of liver cancer cells self-renewaling,which is induced by the TH/TR?signaling pathway.CONCLUSION:In summary,our study uncovers a novel function that the TH/TR?signaling pathway plays a important role in regulating LCSCs.And it can specifically increase the proportion of CD90~+liver cancer cells and promote the self-renewal capacity of liver cancer cells.Moreover,it would increase the tolerance of liver cancer cells to chemotherapy drugs.From the clinical point of view,it is of potential application value to identify the environmental or genetic factors that affect the stem cell-like characteristic of the tumor,and these findings might be useful for developing novel therapies to HCC by targeting the TH/TR? signaling pathway.