| Hepotocelluar carcinoma (HCC) is the fifth most commonly diagnosed cancer worldwide. In 2012, an estimated 780,000 new liver cancer cases have occurred throughout the world, and about half of the cases were accounted for by China. The incidence of male HCC were higher than that among females in China, indicating there were significant differences between the sexes in the susceptibilities of correlated HCC risk factors. Therefore, the seeking of important tumor markers and interventional targets has recently been a research focus during the genesis and development of male HCC.Testis-specific protein Y-encoded 1 (TSPY1) is a member of the TSPY/TSPY-L/SET/NAP-1 (TTSN) protein superfamily, which has a highly conserved SET/NAP domain. TSPY1 can interact with multiple transcription factors which were involved in many important cellular processes such as gene expression, chromatin organization, protein synthesis and cell cycle progression.Studies have confirmed that TSPY1 was aberrantly expressed in the epithelium of gonadoblastoma, testicular germ cell tumor and prostatic cancer, suggesting it might play a key role in tumor genesis and development.Our workgroup had previously found TSPY1 was the differentially expressed protein in male HCC by using quantitative proteomic technique combined with iTRAQ and mass spectrometry, Realtime-PCR and Western Blot techniques. On this basis, the present study would adopt Realtime-PCR and Western Blot techniques to analyze the expressions of TSPY1 mRNA and protein in Y chromosome positive hepatocellular carcinoma cells. Lentiviral vector transfection and RNA interference techniques were applied to observe the alterations of proliferation, invasion and metastasis of hepatocellular carcinoma cell line MHCC97-H with TSPY1 silencing. Furthermore, hypoxia inducible factor-la (HIF-la) was selected as the study subject to investigate whether TSPY1 affected the invasion ability of hepatocellular carcinoma cells by regulating HIF-1? expression or not. It will provide new idea for the further treatment and prevention on male HCC with TSPY1 as a target point.PART ONE THE EXPRESSION ANALYSIS OF TSPY1 IN Y CHROMOSOME POSITIVE HEPATOCELLULAR CARCINOMA CELLSThe human TSPY1 was mainly expressed in spermatogonia,spermatocyte and partial sperm cells of embryonic and adult testis. We had previously found that TSPY1 was specifically over-expressed in male HCC tissues, suggesting it might be correlated with the development and progression of male HCC. The purpose of this part was to analyze the expressions of TSPY 1 mRNA and protein in the Y chromosome positive hepatocellular carcinoma cells,including SMMC-7721, HuH-7, Hep G2, MHCC97-L, MHCC97-H and HCCLM3, by using Realtime-PCR and Western Blot techniques. The relative expression of TSPY 1 mRNA in hepatocellular carcinoma cells was detected via Realtime-PCR by adopting ?-actin as the reference gene and the Delta-delta Ct method. The relative expression of TSPY1 protein was analyzed via Western Blot by using GAPDH protein as the internal control and the Bio-rad Quantity One software. The results of Realtime-PCR showed that there were high TSPY1 mRNA levels in the high-metastatic potential hepatocellular carcinoma MHCC97-H and HCCLM3 cells, while low TSPY1 mRNA levels in the non-and low-metastatic potential hepatocellular carcinoma SMMC-7721 and Hep G2 cells, respectively. Furthermore, the results of Western Blot were consistent with that of Realtime-PCR. Thus, we concluded that TSPY1 was associated with the metastatic potential of Y chromosome positive hepatocellular carcinoma cells,and TSPY1 might play an important role in the proliferation, invasion and metastasis of HCC cells.PART TWO THE EFFECTS OF TSPY1 ON THE BIOLOGICAL FUNCTIONS OF HEPATOCELLULAR CARCINOMA CELL LINE MHCC97-HThe protein encoded by TSPY1 belongs to the TSPY/TSPY-L/SET/NAP-1(TTSN) protein superfamily. TSPY1 could affect many important cellular processes such as gene expression, nucleosome assembly, chromatin remodeling, protein synthesis and cell cycle progression through the interaction with multiple transcription factors, thereby participating in the genesis and progression of malignancy. In this part, RNA interference technique was used to establish the hepatoma cell line MHCC97-H with TSPY1 silencing, and the expressions of TSPY1 mRNA and protein were determined by Realtime-PCR and Western Blot methods. Furthermore, CCK8 cell proliferation assay and Transwell chamber invasion experiment were applied to investigate the alterations of proliferation and invasion of the hepatoma cell line MHCC97-H with TSPY 1 silencing, respectively. The results of Realtime-PCR and Western Blot showed that the expressions of TSPY1 mRNA and protein weresignificantly down-regulated by ientiviral transfection, which indicating we had successfully established the hepatoma cell line MHCC97-H with TSPY1 silencing. The result of CCK8 cell proliferation assay showed that the proliferation capacity of hepatocellular carcinoma MHCC97-H cells with TSPY1 silencing was significantly lower than the cells of control group. The result of Transwell chamber invasion assay showed the number of hepatocellular carcinoma MHCC97-H cells with TSPY1 silencing that passing through the chamber was less than the control group. In conclusion, we had successfully established the hepatoma cell line MHCC97-H with TSPY1 silencing, and confirmed that silencing TSPY1 gene expression could significantly inhibit the proliferation, invasion and metastasis of HCC cells.PART THREE REGULATION EFFECT OF TSPY1 ON THE EXPRESSION OF HIF-1AInvasion and metastasis are the primary factors causing poor prognosis of patients with HCC. However, the molecular mechanisms underlying these biological behaviors have not been completely elucidated. Research had proven that hypoxia microenvironment could not only enhance the invasion and metastasis ability of tumor cells but also promote the proliferation of tumor cells,neovascularization and multidrug resistance. Hypoxia inducible factor-1 (HIF-1),one of the key regulatory factors implicated in the regulation of cellular response to hypoxia microenvironment, generally exists in mammal and human body.HIF-1 was composed of HIF-1? and HIF-1?, and HIF-1? was considered as the specific regulatory subunit. In this part, we used Realtime-PCR and Western Blot techniques to detect the expressions of HIF-la mRNA and protein based in the hepatocellular carcinoma MHCC97-H cells with TSPY1 silencing. The results indicated that the expressions of-HIF-1? mRNA and protein inhepatocellular carcinoma MHCC97-H cells with TSPY1 silencing were significantly lower than that in control group. We therefore reached the conclusion that TSPY1 might promote the metastasis of male HCC by regulating HIF-1? expression.CONCLUSIONS1. TSPY1 was associated with the metastatic potential of Y chromosome positive hepatocellular carcinoma cell lines.2. We had successfully established the hepatocellular carcinoma MHCC97-H cells with TSPY1 silencing, and found that silencing TSPY1 gene expression could significantly inhibit the proliferation, invasion and metastasis of HCC cells.3. TSPY1 might influence the invasion and metastasis of male HCC by regulating HIF-1? expression, and thus it was probably a potential target for the prevention and treatment of male HCC. |
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