| Liver cancer is one of the most common malignant tumor in the world, with China alone accounting for more than50%of the total. Liver cancer rate is the highest in Guangxi. Among primary liver cancers occurring worldwide, Hepatocellular carcinoma (HCC) represents the major histologic type. Hepatocarcinogenesis is a complex process with multi-gene and multi-factor participation.Sp3is a member of the Sp transcription factor family, which expressed in all immature mammalian cells. Sp3takes part in many functions of cells, including proliferation, apoptosis and differentiation. Overexpression of Sp3can be found in many tumors. The roles of Sp3in tumors are different which can act as an activator or a repressor of transcription. The roles in HCC is still not clear.Role of Wnt/β-catenin signaling in HCC was discovered over a decade ago. Aberrant expression of (3-catenin is a central component of Wnt/β-catenin signaling. β-catenin and E-cadherin form a functional unit that maintains cellular cohesion and tissue architecture. β-catenin also can ativate the transcription of oncogene,mcluding cyclin D1and c-myc.Our preliminary studies indicate that Sp3is a tumor activator gene of transcription in HCC and has a crosstalk with P-catenin.To further confirm our preliminary findings, the role of Sp3and its relationship with β-catenin in HCC will be investigated in cells and xenograft of athymic mice as well as in human HCC tissues. Results from this project will deepen our understanding of the mechanisms of HCC development and may potentially be translated into new therapeutic or diagnostic approaches for prevention and treatment of HCC.We studied the biological effects and mechanism in HepG2cells knockdown of Sp3by RNA interference in vitro and in vivo by using cellular and molecular biology techniques. Observed the growth of xenograft of athymic mice and explored the mechanism. Detected the expression of Sp3and β-catenin in HCC and paratumorous tissues, using immunohistochemistry (IHC) and demonatrate the correlation between the expression of Sp3, β-catenin protein and clinicopathologic features.Including the following three parts:Part1Silencing of Sp3by RNAi influences biological behavior of human hepatocellular carcinoma HepG2cellsObjectiveSp3-shRNA lentiviral vector was used to transfected to human hepatocellular carcinoma HepG2cells for knockdown the Sp3gene in vivo. Observed the influence of biological behavior of Sp3-shRNA in HepG2cells, and preliminarily revealed the mechanism.MethodsSp3-shRNA and NC-shRNA were transfected into HepG2cells with lentiviral vector. The stable transfectants were selected:Lv-shRNA-Sp3and Lv-shRNA-NC. The expression of Sp3were evaluated by Real-time PCR, Western blot and immuno fluorescence staining. The effects of Sp3-shRNA on growth, proliferation, cell cycle, apoptosis, migration and invasion of HepG2were assessed by MTT, flow cytometric, transmission electron microscopy analysis and transwell.Using Real-time PCR to detect the expression of caspase-3and caspase-9mRNA, and detecting the expression of pro-caspase-3and pro-caspase-9protein in three groups cells.Results1, The levels of Sp3mRNA and protein was lower in Lv-shRNA-Sp3group cells (F=41.570,P<0.001; F=5.409,P=0.045);2, MTT showed that the growth of Lv-shRNA-Sp3group cells was slow-growing(P=0.05). Flowcytometry showed that cell proliferation was significantly inhibited, cell cycle progression was blocked in G1(F=18.946,P=0.003) and apoptosis rate was higher (F=53.647, P=0.000) in Lv-shRNA-Sp3group cells. Transmission electron microscopy analysis showed that the ultrastructural changes of the cells was destroyed. Transwell assay showed that the migration and invasion of Lv-shRNA-Sp3group cells were significantly inhibited comparison with Lv-shRNA-NC and HepG2cells (F=92.912, P=0.000; F=45.282, P=0.000)3, The expression of caspase-3mRNA, caspase-9mRNA in Lv-shRNA-Sp3group cells were significantly higher than other groups (F=19.297, P=0.002; F=34.016,P=0.001). And the expression of Pro-Caspase-3protein and Pro-Caspase-9protein were significantly lower than Lv-shRNA-NC and HepG2cells (F=5.409,P=0.045; F=5.798,P=0.005).Conclusions1, The stable transfectants were selected:Lv-shRNA-Sp3and Lv-shRNA-Sp3group cells.2, These results demonstrate that Sp3gene may influences biological behavior of human hepatocellular carcinoma HepG2cells in vitro. Silincing of Sp3would inhibit the proliferation, imigration and invasion of HepG2cells, and induce the apoptosis. Sp3gene up-regulate the development of HCC.3, The higher rate apoptosis has relationship with activation of caspase-3and caspase-9in Lv-shRNA-Sp3group cells. Part2Effects of HepG2cells stably knockdown Sp3on growth of xenogeneic graft in nude miceObjectiveConstruction of xenogeneic graft in nude mice with three group cells to observed the influence of Sp3-shRNA in nude mice, and preliminarily revealed the mechanism.MethodsThe Lv-shRNA-Sp3, Lv-shRNA-NC and HepG2cells were injected into nude mice. The xenografts were observed by tumor formation rate and The growth of tumor. Observed the pathological morphology of cells in three groups. Analyzed for Sp3mRNA and protein expression by Real-time PCR, Weatern blot and IHC. Detected the rates of apoptosis in three groups using TUNEL staining.Results1, The tumor formation rate of Lv-shRNA-Sp3group was lower than Lv-shRNA-NC and HepG2groups (60%vs100%,100%); The growth of tumor was slower than others(P<0.05), the volume of Lv-shRNA-Sp3group was (189.02±36.7) mm3,which was significantly smaller than in Lv-shRNA-NC and HepG2groups (F=8.728, P=0.005).2, The effects of invasion was weaker in Lv-shRNA-Sp3group cells.3, The tumor with Lv-shRNA-Sp3had marked inhibition of expression of Sp3mRNA and protein (F=5.765, P=0.04; F=7.387,P=0.006)。4, The apoptosis rate was higher in Lv-shRNA-Sp3group than in two other groups (F=27.297,P=0.001)。Conclusions1, Construction of xenogeneic graft in nude mice with three group cells.2, Sp3gene may influences biological behavior of human hepatocellular carcinoma HepG2cells in vivo. And Sp3gene up-regulate the development of HCC. Part3Expression of Sp3and β-catenin in hepatocellular carcinoma and their relationshipObjectiveDetected the expression of Sp3, P-catenin protein and mRNA in HCC and paratumorous tissues, and demonatrated the correlation between the expression of Sp3, P-catenin and clinicopathologic features. Investigated the relationship between Sp3and P-catenin in cells, xenogeneic graft of nude mice and HCC.Methods1,83cases of HCC and paratumorous tissues had been collected and all were diagnosed by the Department of Pathology of the first affiliated hospital of Guangxi medical university. The expression of Sp3and P-catenin protein and mRNA were detected in all the tissues by immunohistochemistry and RT-PCR. And then investigated the relationship between the expression of Sp3, P-catenin and clinicopathologic features.2, Detected the expression of Sp3, P-catenin in xenogeneic graft of nude mice and cells by Real-time PCR, Western blot and IHC to investigate the relationship between Sp3and P-catenin.Results1, Immunohistochemistry staining revealed the positive rate of Sp3protein in HCC was significantly higher than their paratumorous tissues (χ2=53.597, P=0.000); and the positive rate of Sp3protein in cirrhosis paratumorous tissues was significantly higher than in the nomorl paratumorous tissues (χ2=4.218, P=0.037);2, Immunohistochemistry staining revealed the positive rate of P-catenin protein in HCC was significantly higher than their paratumorous tissues(χ=60.049, P=0.000);3, Analysed the Sp3and pathologic parameters, the expression of Sp3protein was significantly associated with the size of tumor (χ2=12.261, P=0.000)and the degree of HCC differentiation (χ2=6.363, P=0.010), but not significantly associated with the other clinicopathological features (P>0.05);4, Analysed the β-catenin and pathologic features, the expression of P-catenin protein was significantly associated with the size of tumor (χ2=7.525, P=0.005), the degree of HCC differentiation (χ2=11.270, P=0.001) and the portal vein tumor thrombus and the presence of metastasis (χ2=4.910, P=0.028), but not significantly associated with the other clinicopathological features (P>0.05).5, The results of RT-PCR show that the expression of Sp3mRNA in HCC was significantly higher than their paratumorous tissues (t=6.118, P<0.0001);6, The results of RT-PCR show that the expression of β-catenin mRNA in HCC was significantly higher than their paratumorous tissues;7, Analysed the Sp3mRNA and pathologic parameters, the expression of Sp3mRNA was significantly associated with the size of tumor (t--2.549, P=0.015) and the degree of HCC differentiation (t=-4.238, P=0.000), but not significantly associated with the other clinicopathological features (P>0.05);8, Analysed the β-catenin mRNA and pathologic parameters, the expression of β-catenin mRNA was significantly associated with the size of tumor (t=-2.941, P=0.005) and the degree of HCC differentiation (t=-3.512, P=0.001), but not significantly associated with the other clinicopathological features (P>0.05);9, The3-year overall survival rate was45.8%; 10, Univariate analysis releaved that the patients with high expression of Sp3protein had poor survival than the low expression (P=0.010). And the patients with high expression of β-catenin protein had poor survival than the low expression (P=0.001) too.11, Multivariate analysis showed that expression of Sp3and β-catenin protein were both the independent predictor of survival (HR=2.586, P=0.006; HR=2.289, P=0.009).12, By Spearman correlation analysis, the expression of Sp3and β-catenin protein was positively correlation (r=0.298, P=0.006);13, By Spearman correlation analysis, the expression of Sp3and β-catenin mRNA was positively correlation (r=0.835, P<0.001);14, The expression of Sp3、β-catenin mRNA in Lv-shRNA-Sp3group were higher than Lv-shRNA-NC and HepG2groups (F=41.570, P<0.001; F-18.089, P=0.003); and the expression of Sp3、β-catenin protein in Lv-shRNA-Sp3group were higher than two other groups too (F=5.409, P=0.045; F=9.925, P=0.005).15, In xenografts of nude mice, the expression of Sp3、β-catenin mRNA in Lv-shRNA-Sp3group were higher than Lv-shRNA-NC and HepG2groups (F=5.765, P=0.04; F=17.246, P=0.003); and the expression of Sp3、 β-catenin protein in Lv-shRNA-Sp3group were higher than two other groups too (P<0.05).Conclusions1, The level of Sp3and P-catenin expression in HCC were fairly high, which both have the relationship with the size of tumor and the poor degree of HCC differentiation. This releaves that Sp3and β-catenin play the important role in hepatocarcinogenesis. 2, High expression of Sp3and β-catenin in HCC may have value as the adverse prognostic factor.3, The expression of Sp3wsa significantly associated with β-catenin protein in HCC tissues.4, Silencing of Sp3can inhibit the expression of β-catenin in HepG2cells and in xenografts of nude mice. |
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