Study On The Function Of HRP - 3 In The Development And Progression Of Hepatocellular Carcinoma And The Function Of Transcription Factor HEY2 In

Hepatocellular carcinoma (HCC) is one of common malignant tumors in the world, and the global incidence rate has exceeded 1 million per year. China has been always the high incidence of HCC worldwide, which is about 10 times more than the U.S. and Europe. Because of its high malignancy and rapid progression, diagnosis is frequently made at an advanced stage, which makes the therapy of cytotoxic drug has poor effect, and there has been no standard effective treatment program. Therefore, it is particularly important to study HCC-causing genes and pathogenic mechanism for the multi-stage HCC diagnosis and HCC-specific targets for drug development. HRP-3 was screened by differential display PCR for high expression in HCC by our lab. Preliminary work has been found that:1) HRP-3 can promote HCC cells anchorage-independent growth; 2) over-expression of HRP-3 in hepatoma cells promote both tumor growth and tumor size in nude mice, and knockdown of endogenous HRP-3 in hepatoma cells successfully inhibited tumor growth; 3) knockdown endogenous HRP-3 can enhance the sensitivity of HCC cells to anticancer drugs. Therefore, it suggests that HRP-3 is important during HCC development and progress.On the basis of all these findings, this work is to clarify the place which has not been studied deeply. We first used real-time quantitative PCR method to detect the 102 pairs of HCC samples and found that the HRP-3 expression was significantly increased in HCC, and its high expression was significantly correlated with clinical indicators including history of hepatitis, tumor size, tumor number and prognosis. Then we designed specific peptide and prepared the HRP-3 specific polyclonal antibody; we carried Western Blot method to detected HRP-3 expression of protein level in 12 pairs HCC tissue samples and found that HRP-3 is highly expressed in HCC compared with adjacent areas, immediately the immunohistochemistry assay showed that HRP-3 was distributed both in cytoplasm and nucleus. In addition, we found a variety of liver cancer cell lines were able to secrete endogenous HRP-3 protein, and HRP-3 protein can specificly activated the MAPK/ERK signaling pathway; Although the HRP-3 protein had no effect for proliferation on adhesion, it can enhance anchorage-independent survival of HCC cells in Annoikis assay, and this effect was parcialy dependent on MAPK/ERK pathway. In vivo, we used transplantation tumor to investigate if the HRP-3 expression was related to cell proliferation and apoptosis by immunohistochemical analysis. The Ki67 and p-ERK was cell proliferation marker and TUNEL was cell apoptosis marker. We foud that high HRP-3 expression was accompanied by higher Ki67, p-ERK and lower TUNEL staining, whereas low HRP-3 levels were associated with lower Ki67、p-ERK and higher TUNEL staining. Also we found that overexpression of HRP-3 in the tumor accompanied by higher blood vessel density. Finally, we revealed that HRP-3’s distribution can transfer from nucleus to cytoplasm and form complex with RAF、 MEK、ERK. Apart from this, we explored other function of HRP-3 and found that HRP-3 regulates HCC cell survival when depletion of glouce, then increases the tolerance of energy pressure.Our research described that HRP-3 is associated with clinical parameters of liver cancer, and that HRP-3 regulate HCC anchorage-independent survival and hepatoma development through positive regulating MAPK/ERK signaling pathway. These results conformed important role of HRP-3 in regulating tumor development and progress and provide with theoretical basis for drug theropy through specific extracellular receptors or intracellular signal transduction pathway, and the HRP-3 itself can be used as diagnosis and target for hepatoma theropy.The transforming growth factor P (TGF-β) is a pleiotropic factor, exerts tumor-suppressive effects in the premalignant stage of tumor; however, in the advanced stage, TGF-β signaling turns into promoting tumor growth and invasion. Any points of disruption in TGF-β signaling will result tumor development and progression. HEY2 (hairy and Enhancer-of-split related whith YRPW motif) gene is found as a novel Hairy-related genes in mammalian. It is induced by Notch pathway and encodes protein which exercises the function of transcriptional repression. HEY2 play an important role in cardiovascular development and upregulated in osteosarcoma cells. During the study of HCC related genes, we found HEY2 could involve in TGF-β signling pathway, which prompted us to carry out reaserch for how HEY2 regulates TGF-β signaling pathway and what role is it in HCC development and progress.We found that HEY2 formed a complex with Smad3、Smad4 at transient transfection by using Co-IP and Pull-down methods; each two proteins are co-localized to the same subcellular compartments. By the dual fluorescent enzyme reporting system, we conformed that HEY2 significantly inhibited the Smad3/4-induced acctivation of the SBE reporter gene; HEY2 participated in TGF-β signaling pathway by reversing TGF-P inhibition to downstream target genes c-Myc. We further established stable Hep3B cell lines overexpressing HEY2, and determine the HEY2’s role in cell growth. The results of MTS and clone formation assays consistently indicated that overexpression of HEY2 had no effect on cell nomal growth, but can enhance the growth ability against TGF-β which can inhibit cell growth. Finally, we studied the HEY2 significance on physiological level, we used the Western Blot and immunohistochemistry to detect the HEY2 expression in human hepatocellular carcinoma and adjacent, the results show that HEY2 increasingly expressed in HCC. To sum up the results, HEY2 probably played a role in liver cancer occurrence and development by inhibiting the TGF-P signaling pathway.This study suggested a novel mode for function of HEY2, and it may be involved in the occurrence and development of liver cancer.