【作者】 王新红；

【导师】 孟祥伟；

【作者基本信息】 吉林大学 ， 内科学， 2010， 博士

【摘要】 原发性肝癌是世界上最常见的十种恶性肿瘤之一,我国又是原发性肝癌的高发区。一旦发现多为中晚期,失去了手术机会,现有的治疗方法效果亦不理想。因此,长久以来肝癌的研究一直为基础和临床科研人员所关注。β-catenin是细胞质中一种重要的多功能蛋白,处于Wnt信号通路的活化中心,与形成肿瘤的多种因素密切相关,参与肿瘤发生、发展过程的多个环节。因此,本研究欲应用siRNA干扰β-catenin信号传导通路,以观察其对肝癌细胞生长、细胞周期、细胞凋亡相关因子、血管生成因子、端粒酶及其它信号通路的影响及siRNA能否用于肝癌的治疗。首先筛选能够有效沉默β-catenin基因表达的siRNA,然后用有效的siRNA转染入肝癌HepG2细胞,在不同的时间点用RT-PCR及western blot法检测β-catenin的mRNA及蛋白质表达,MTT法检测肝癌细胞的生长,72h和96h用流式细胞仪检测细胞周期。最后用western blot法检测细胞周期、细胞凋亡、血管生成因子、端粒酶及其它信号通路部分相关蛋白质的表达,用RT-PCR法检测VEGF-A、VEGF-C的mRNA表达。从而可知siRNA可以沉默β-catenin基因及蛋白质的表达,并使细胞周期阻滞于G0/G1期。一次转染可在一定时间内抑制肝癌细胞的生长,在肝癌的治疗中起一定的作用,但不能治愈肝癌。肝癌细胞中β-catenin信号传导通路可以调节肝癌的细胞生长、细胞周期、细胞凋亡、血管生成因子、端粒酶及其它信号通路等,从而抑制β-catenin信号通路在肝癌的治疗中起一定的作用。我们需要进一步深入研究,以了解肝癌的发生、发展过程,从而寻找更为有效的治疗肝癌的方法。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of ten most common malignant tumors in the world and our country is its high incidence. HCC has the hidden genesis, the rapid development and the high malignance. Once it is found, it is in the late stage and the patient loses the chance of operation. The current treatment is also not satisfactory. Therefore, the study of HCC is always concerned by the basic and clinical researches for a long time.β-catenin, as a multifunctional protein in cytoplasm, is close to many factors that form tumors and participates in a lot of steps of tumor genesie and development. The current researches indicate that there are the abnormal expressions of cell cycle, apoptosis, angiogenesis, telomerase, signaling pathway, and so on, in HCC, but the relation betweenβ-catenin and these factors is not clear.Based on the above findings, in HepG2 cells, we silenceβ-catenin gene and observe the change of cell growth, cell cycle, apoptosis, angiogenesis, telomerase and signaling pathway, thereby studying the relation betweenβ-catenin and these factors and validating theβ-catenin role on HCC genesis and development and the siRNA role on HCC gene therapy. These will offer the basis of theory for HCC pathogenesis and therapy so that people may better study and treat HCC.(1) Bolting effective siRNA againstβ-cateninIn our studies, after the siRNA marked by fluorescence was transfected into the HepG2 cells, the transfection efficiency was detected. The different siRNA againstβ-catenin was teansfected into the HepG2 cells,β-catenin protein expression was detected by western blot at 48 and 72h. We drew a conclusion that the first siRNA againstβ-catenin was the most effective. After the first siRNA againstβ-catenin was transfected into HepG2 cells, the mRNA and protein expressions ofβ-catenin were detected respectively by RT-PCR and western blot.β-catenin mRNA expression was most obviously inhibited at 24 h and increased gradually at 48, 72 and 96 h after the transfection.β-catenin protein expression decreased gradually at 24, 48 and 72 h and slightly increased at 96 hours after the transfection. These proved that the first siRNA againstβ-catenin could specifically silenceβ-catenin expression again. And we studied its time-varying pattern, thereby selecting 72 and 96h as the point time of future experiments. These lay a solid foundation for future experiments.(2) The influence of silencingβ-catenin on HCC cell growthIn our studies, after the siRNA againstβ-catenin was transfected into the HepG2 cells, we observed the cell shape and measured HCC cell growth by MTT assay. After the transfection, the cell growth was inhibited at 24, 48 and 72 h and the weakened inhibition was manifested by slightly increased rate of cell growth at 96 h. Therefore, Only one time transient transfection may temporarily inhibit HCC cell growth, which palys a certain role in the therapy of HCC, but may not cure HCC. We will further study if multiple transfections or stable transfection would work as a powerful tool to cure HCC in the future. Our results also suggest that other signaling pathways may have some functions to compensate the inhibition ofβ-catenin signaling pathway. It needs to be further study in the future.(3) The influence of silencingβ-catenin on HCC cell cycleIn our studies, after the siRNA againstβ-catenin was transfected into the HepG2 cells, HCC cell cycle was detected by flow cytometry and the protein expression of cyclin A and cyclin E was detected by western blot. After the transfection, the cell cycle was blocked in G0/G1 phase at 72 h and was slightly returned and proceeded to M phase at 96 h. HCC cells began to multiplied again at 96 hours. The result was similar to that of MTT. After the transfection, the protein expression of cyclin A and cyclin E increased at 72 h and decreased at 96 h, so we may know that they are related toβ-catenin signaling pathway and are also related to other factors, andβ-catenin may regulate cell growth through affecting cell cycle.(4) The influence of silencingβ-catenin on HCC apoptosisIn our studies, after the siRNA againstβ-catenin was transfected into the HepG2 cells, the protein expression of apoptosis-related factors (caspase-3, XIAP, Grp78 and HSP27) was detected by western blot. The protein expression of phosphorylated (p)-caspase-3 and Grp78 increased at 72h and decreased at 96h. The protein expression of caspase-3 and XIAP decreased at 72h and increased at 96 h. The HSP27 protein expression showed no change following transfection. These indicate that Wnt/β-catenin signaling pathway can regulate the expression of these proteins (caspase-3,XIAP,Grp78) and the latter is downstream target proteins of the former. Wnt/β-catenin isn’t related to the HSP27 protein expression. In short, the Wnt/β-catenin signaling pathway contributes to HCC apoptosis, proliferation, and differentiation through regulation of the expression of angiogenesis factors.(5) The influence of silencingβ-catenin on HCC angiogenesisIn our studies, after the siRNA againstβ-catenin was transfected into the HepG2 cells, the protein expression of angiogenesis factors (MMP-2, MMP-9, VEGF-A, VEGF-C and bFGF) was detected by western blot and the mRNA expression of VEGF-A and VEGF-C was detected by RT-PCR. The protein expression of MMP-2, MMP-9, VEGF-A, VEGF-C and bFGF decreased at 72h and increased at 96 h. The mRNA expression of VEGF-A and VEGF-C is similar to the protein expression. These indicate that Wnt/β-catenin signaling pathway can regulate the expression of these proteins and the latter is downstream target proteins of the former. In short, the Wnt/β-catenin signaling pathway contributes to HCC angiogenesis, infiltration and metastasis through regulation of the expression of angiogenesis factors.(6) The influence of silencingβ-catenin on HCC telomerase and other signaling pathwayIn our studies, after the siRNA againstβ-catenin was transfected into the HepG2 cells, the protein expression of telomerase (TERT) and other signaling pathways (MAPK family, Akt1, Smad3, STAT3 and GSK-3β) was detected by western blot. ERK1/2 (p44/42MAPK), JNK/SAPK, p38MAPK, and Akt1 protein levels showed no change following transfection, while their phosphorylated protein levels showed changes. After the transfection, p-ERK1/2 (p-p42MAPK) protein expression increased gradually at 72 and 96h; expression of p-JNK/SAPK protein 54kD form decreased while levels of the 46kD form gradually increased at 72 and 96h; no p-p38MAPK protein was observed in HepG2 cells; p-Akt1 protein expression decreased at 72h and increased again slightly at 96h. The protein expression of TERT decreased at 72h and increased at 96 h. The protein expression of Smad3 increased at 72h and decreased at 96h. The STAT3 protein expression showed no change following transfection. GSK-3βand p-GSK-3βprotein expression increased gradually at 72 and 96 h. Thus,β-catenin signaling pathway may affect Ras/MAPK and PI3K/Akt signaling pathways through regulating the phosphorylation state of ERK1/2, JNK/SAPK and Akt1 protein in HepG2 cells. In addition, the Wnt/β-catenin signaling pathway may participate in HCC genesie and development through regulating the protein expression of GSK-3β, p-GSK-3β, Smad3, TERT. These signaling pathways activate each other, thereby form an intricate network.In summary, siRNA may silence the mRNA and protein expression ofβ-catenin and block the cell cycle in G0/G1 phase. Only one time transient transfection may temporarily inhibit HCC cell growth, which palys a certain role in the therapy of HCC, but may not cure HCC.β-catenin signaling pathway is related to the protein expression of cyclin A, cyclin E, caspase-3, XIAP, Grp78, MMP-2, MMP-9, VEGF-A, VEGF-C, bFGF, TERT and Smad3, while it is not related to HSP27 and STAT3 protein expression in HCC.β-catenin signaling pathway may affect Ras/MAPK and PI3K/Akt signaling pathways through regulating the phosphorylation state of ERK1/2, JNK/SAPK and Akt1 protein in HepG2 cells. These pathways andβ-catenin pathway might regulate some downstream factors in common, thereby compensating for the inhibitory effect ofβ-catenin and affecting tumor cell growth and others factors.In short,β-catenin signaling pathway may contribute to affect HCC cell growth, cell cycle, apoptosis, angiogenesis, telomerase and other signaling pathway through regulating these factors, so silencingβ-catenin may play a certain role on the therapy of HCC. This pathway also might be involved in HCC genesis and development through regulating these factors. Only make the processes and interrelations of the signaling pathway clear, we can understand tumor genesis, development, prognosis and prevention and control cell growth, differentiation and apoptosis to achieve to cure the tumor through interfering the signaling pathway.更多还原