Investigation Of The Proliferation And Migration Mechanism Mediated By Oncoprotein HBXIP In Cancer

Hepatitis B X-interacting protein (HBXIP), a19kDa protein, was originally identified by its interaction with the C terminal of hepatitis B virus (HBV) X protein (HBx). It was reported that HBXIP plays important roles in many cellular process, such as cell proliferation, apoptosis, mitosis, centrosome dynamics and cell cycle progression. HBXIP is also a component of mTORC1pathway in response to amino acid. Our group has reported that HBXIP promoted breast cancer cell proliferation and migration, and was overexpressed in breast cancer tisssues. However, the mechanism is still unclear. Skp2, involving in DNA replication, cell cycle, apoptosis, invasion and metastasis, is necessary to enter S phase, and overexpress in many tumors. In the present study, we first report that HBXIP is a novel oncogenic cofactor of Skp2to result in proliferation and migration in cancer. The main contents are showed as follows:Part One:Effect of HBXIP on proliferation of breast cancer cellsTo investigate the relationship of HBXIP and Skp2expression in clinical specimens, we detected the expression of HBXIP and Skp2protein by immunohistochemical (IHC) staining in tissue microarrays, the stain results suggests that Skp2is closely relevant to HBXIP in breast cancer tissues. Moreover, we found that the mRNA levels of HBXIP was positively correlated with those of Skp2in30clinical fresh breast cancer tissue samples detected by real-time PCR, which is consistant with the above conclusion. Luciferase reporter gene assays showed that HBXIP could regulate the activity of Skp2promoter. And HBXIP was able to regulate the mRNA and protein levels of Skp2in breast cancer cell lines by western blot and RT-PCR. ChIP assay demons’trated that HBXIP may occupy the-640/-443region of Skp2promoter including E2F1binding site. Co-IP and EMS A assay show that purified protein of HBXIP and Skp2could combine to each other. Furthermore, luciferase reporter gene assays showed that HBXIP failed to active the Skp2promoter when the E2F1site was mutated. In function, HBXIP promoted the proliferation of breast cancer cells via Skp2examined by MTT, colony formation assay and flow cytometry in vitro and Skp2is necessary for the proliferation of breast cancer cell enhanced by HBXIP in vivo.Part Two:Effect of HBXIP on migration of ovarian cancer cellsTo investigate the relationship of HBXIP and Skp2expression in ovarian cancer specimens, we detected the expression of HBXIP and Skp2protein by immunohistochemical (IHC) staining in tissue microarrays, the stain results suggests that Skp2is closely relevant to HBXIP in ovarian cancer tissues. Additionally, HBXIP up-regulated the mRNA and protein levels of Skp2in two kinds of ovarian cancer cell lines, SKOV3and CAOV3cell lines, in a dose dependent manner. Luciferase reporter gene assays showed that HBXIP could up-regulate the activity of Skp2promoter in ovarian cancer cells. ChIP assay showed that HBXIP could occupy the-640/-443region of Skp2promoter in which include a Spl binding site. Luciferase reporter gene assays showed that HBXIP failed to stimulate the activity of Skp2promoter when the binding site of Sp1in Skp2promoter was mutated in ovarian cancer cells. In function, wound healing assay showed HBXIP promoted the migration of ovarian cancer cells via Skp2in vitro.We investigated the role of HBXIP and Skp2in tumors progression in the aspects of molecular, cellular, animal and clinical specimens. Our results showed that HBXIP was a novel oncoprotein, and it promoted proliferation and migration of cancer cells via many pathways. Our investigation provided new evidence for the mechanism of HBXIP on tumorigenesis, and HBXIP may serve as a target for the therapy of cancer.