| Hepatocellular carcinoma(HCC) is one of the most common malignant tumors in the world and the incidence of HCC continues to rising. Epidemiological and experimental studies showed that viral hepatitis had particular relationships with the incidence of HCC, and hepatitis B was the most closely relationships with HCC. About 90% patients of HCC in China carried hepatitis B virus(HBV), coupled with high degree of malignancy and poor prognosis of HCC, we were faced with the increasingly serious situation of HCC. So far, conventional surgery and liver transplantation was still the best treatment of HCC. However, because of insidious onset of HCC and the lack of specific clinical manifestations, most patients visited at a late stage and missed the best timing of surgery with extremely poor prognosis. Therefore, in order to explore the mechanism of development, invasion and metastasis of HCC and improve long-term prognosis, the studies of HCC associated with molecules has become a research hotspot.HACE1 is located on chromosome 6q21 which is one site of more frequently mutated in malignant tumors. HACE1 gene was firstly discovered with closely related to the occurrence of Wilms’ tumor. Futhermore, more evidence had demonstrated that lower expression or mutations of HACE1 associated with a variety of human malignant tumors, including of breast cancer, colorectal cancer and lymphomas. However, there have been few available data concerning the precise function of HACE1 gene in HCC. Therefore, the experiment focused on the biological characteristics of HACE1 gene in HCC cells, explore its effect on the occurrence, development and invasion, metastasis and its relationships with clinical pathological features and prognosis of HCC.Part I The expression of HACE1 gene in HCC cell lines and hepatocellular carcinomaObjective: To investigate the expression pattern in HCC cell lines such as Huh7, MHCC97 H, HCCLM3, MHCC97 L, HepG2, SMCC7721 and normal liver cell line L02 and study the expression pattern in HCC tissues and corresponding adjacent tissues, in order to explore its relationships with HCC.Methods: We detected the expression of mRNA and protein of HACE1 gene in HCC cell lines, normal liver cell line L02 and 40 pairs of HCC and their corresponding adjacent tissues using RT-PCR and western blot.Results: The expression of HACE1 gene in different HCC cell lines such as Huh7, MHCC97 H, HCCLM3, MHCC97 L, HepG2, SMCC7721 was significantly lower than the normal liver cell line L02. Among them, the highest expression of HACE1 gene was detected in Huh7 cells, and the lowest was SMCC7721 cells in all HCC cell lines. Therefore, to verify the role of HACE1 in HCC cell lines, we upregulated by transfection of pcDNA3.1-HACE1 into SMCC7721 cells and Huh7 cells was transfected with siRNA targeting HACE1 for downregulation. The expression of HACE1 gene in HCC tissues was significantly lower than paired adjacent non-tumor tissues at both the mRNA and protein levels.Conclusion: HACE1 is an important tumor suppressor gene, and the mutation of HACE1 in cancer tissues plays an vital role in the occurrence of HCC. PartII The relationships on HCC proliferation and migration by regulation of HACE1 gene expressionObjective: Explore the role of HACE1 gene on proliferation and migration in Huh7 and SMCC7721 HCC cell lines.Methods: we knocked down HACE1 gene in human HCC Huh7 cells by transient transfected with siRNA of HACE1 or negative control(NC) siRNA and overexpressed HACE1 in HCC SMCC7721 cells through transfected with pcDNA3.1-HACE1 or mock plasmid, respectively. Build stable transfected Huh7 cell line of HACE1 siRNA and SMCC7721 cell line of pcDNA3.1- HACE1. Knockdown and overexpression efficiency determined by RT?PCR and western blot analysis. Cell Counting Κit-8(CCK-8), transwell and wound healing assays were performed to investigate the effects on cellular proliferation and migration after overexpression and knockdown of HACE1.Results: The results showed that stable transfected Huh7 cell line of HACE1 siRNA and SMCC7721 cell line of pcDNA3.1- HACE1 was established successfully. The results indicated that downregulation of HACE1 expression significantly promoted the proliferation and migration of the Huh7 cells by CCK8, Transwell and Wound healing assays(P<0.01), and upregulation of HACE1 expression obviously inhibited the proliferation and migration of the SMCC7721 cells by CCK8, Transwell and Wound healing assays(P<0.05).Conclusion: HACE1 gene is involved in the development, invasion and metastasis of HCC.Part III Correlation between the expression differences of HACE1 gene andclinicopathological features and prognosis in HCCObjective: To investigate correlation between the expression differences of HACE1 gene in HCC and clinicopathological features and prognosis of HCC.Methods: Immunohistochemistry(IHC) was performed to detect the expression of HACE1 gene of 102 cases of HCC tissues, in order to investigate correlation between the expression differences of HACE1 gene and clinicopathological features and prognosis in HCC.Results: The results found that 75 samples(73.52%) had low HACE1 expression, 27 samples(26.48%) had high HACE1 expression in all 102 patients, and low expression of HACE1 gene of HCC patients accompanied by high malignant tumor. According to the level of HACE1 expression in the tumor tissues, we demonstrated that low levels of HACE1 expression was significantly associated with serum AFP level(P<0.001), tumor differentiation(P<0.05) and vascular invasion(P<0.05). The overall survival for five years was significantly decreased in patients with low HACE1 expression compared to patients with high HACE1 expression(P<0.05). In addition, univariate and multivariate Cox regression analysis demonstrated that AFP level, tumor differentiation and HACE1 level were significantly associated with overall survival, and which were significant prognostic factors for HCC patients.Conclusion: The expression differences of HACE1 gene in tissues of HCC is closely related to the malignant degree and prognosis of HCC. |
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