The Mechanisms Of HMBOX1, A Nuclear Transcription Factor, Regulating Human Hepatoma Cell Biological Characterizations

Object:Hepatocellular carcinoma(HCC) refers to the malignant tumor occured in liver, including the primary and the secondary. China is the region of the highest incidence of primary HCC, the number of patients died of HCC in China accounts for more than a half of the world. Traditional treatments for HCC include surgery, radiofrequency ablation and chemotherapy. As a new treatment method, biological treatment emerged in recent years provides a new choice for the patients unable or unwilling to accept the traditional treatments.The fundamental reasons why HCC is difficult to be cured completely are recurrence and metastasis. The result of research shows that tumor stem cells may play a key role in this process. To date, CD133is considered as a biomarker of tumor stem cells, and CD133positive cells show much more malignant phenotypes. Therefore, differentiation therapy on tumor stem cells will surely be of great theoretical value and social significance.HMBOX1, a novel gene, was isolated from human pancreatic cDNA library in2006, belonging to the Homeobox gene family. Recently, HMBOX1b was identified as a splicing variant of HMBOX1. Studies show that HMBOX1is a potential nuclear transcription factor with transcriptional inhibitory activity. A few studies of HMBOX1focus on its regulatory activities on NK cell function and differentiation of bone-marrow stromal cells into vascular endothelial cells; however, the molecular mechanism remains unknown. HNF-1Alpha, the other member of HNF class, plays an anti-tumor activity. However, the study relating to hepatocellular carcinoma and HMBOX1has not been reported.In this study, we constructed HMBOX1/HMBOX1b expression plasmids and investigated the role of HMBOX1on biological characteristics of hepatoma cell line HepG2, hoping to find a new target for biological treatment. Methods:1. Immunohistochemistry was performed to detect the HMBOX1expression levels of clinical specimens;2. PCR and Western Blot were used to detect the mRNA or protein levels of HMBOX1in a variety of hepatoma cell lines;3. Construction of HMBOX1/HMBOX1b EGFP fusion eukaryotic expression plasmids;4. Immunofluorescence was performed to ensure the sub-cellular localization of HMBOX1/HMBOX1b;5. CCK-8and FACS were used to analysis proliferation and cell cycle;6. qPCR was performed to detect the "sternness" and pro-inflammatory genes expression on HepG2cells over-expressed HMBOX1;7. MTT was used to analysis the sensitivity of HMBOX1over-expressed HepG2cells to NK cell cytolysis and FACS was used to detect the levels of cytotoxicity associated molecules;8. Microarray was performed to analysis the signaling pathways, biological process and molecular functions HMBOX1involved in.Results:1. A positive correlation between the HMBOX1expression level and the degree of differentiation;2. The expression of HMBOX1in hepatoma cell lines was lower than that in hepatocytes;3. pEGFP-N1-HMBOX1and pEGFP-N1-HMBOX1b plasmids were successfully constructed;4. The subcellular distribution of HMBOX1was unique, and the subcellular distribution of HMBOX1b was different from HMBOX1;5. Over-expression of HMBOX1didn’t influence the proliferation ability and cell cycle of HepG2cells; 6. Over-expression of HMBOX1down-regulated the "sternness" and pro inflammatory genes in HepG2cells;7. The cytotoxicity of NKL against HMBOX1over-expressed HepG2cells was increased;8. HMBOX1may be involved in a number of tumor-associated and immune-associated signaling pathways.Conclusion:HMBOX1, as transcription inhibitory factor, plays roles in regulating the functions of NK cells and promoting bone marrow stromal cells differentiate into vascular endothelial cells. However, the studies on HMBOX1in hepatocellular carcinoma have not been reported. We firstly found a positive correlation between the HMBOX1expression level and the differentiation degree of clinical specimens, as well as the expression of HMBOX1in hepatoma cell lines was lower than that of normal liver cells. We speculated that HMBOX1may be involved in oncogenesis or development of HCC.We did research on subcellular localization of HMBOX1/HMBOX1b with the successfully constructed EGFP fusion eukaryotic expression plasmids. We found that the subcellular distribution of HMBOX1was unique, and HMBOX1b was different from HMBOX1.In HMBOX1over-expressed HepG2cells,"Sternness" and proinflammatory cytokines were decreased. We also found that the cytotoxicity of NKL against HMBOX1over-expressed HepG2cells was increased. Finally, by microarray we found that HMBOX1may be involved in regulating tumor-associated and immune-associated signaling pathways, and also involved in organ development and cell differentiation.In our study, we discovered a transcription factor involved in oncogenesis or development of HCC, but the specific regulating mechanisms need to be further investigated. This study provided a new target for prevent and cure HCC.