RNAi沉默STAT3基因诱导BEL-7402肝癌细胞凋亡的实验研究

发布时间：2018-06-29 01:51

本文选题：STAT3 + RNA干扰　；参考：《山东大学》2015年博士论文

【摘要】：原发性肝细胞癌(Hepatocelluar Carcinoma, HCC)是严重损害全球人类生命健康的消化系统恶性肿瘤,发病率居所有恶性肿瘤中第6位,死亡率居第3位,每年死亡人数基本等于新发病数,并呈现逐年增加的趋势,专家们预计2015年——20年达到疾病平台期。肝细胞癌的发生是多步骤,多基因作用的结果。肝细胞癌的主要危险因素包括感染HBV或HCV、酒精性肝病和非酒精性血液系统疾病,并与地理区域以及种族或民族有关。HCC早期无明显症状,转移早,发现晚,恶性程度高,能够手术切除的患者较少,预后差,即使手术治疗,大多数患者术后2年复发率和5年复发率分别为50%和75%。目前,肝细胞癌的诊断和治疗手段得到了不断提升,首选方滤还是以手术为主的综合治疗,但肝细胞癌患者的预后仍不理想。因此,深入研究及了解肝细胞癌的发生发展作用机制,才能早期诊断,早期治疗,提高患者的生存期和生活质量。研究表明原发性肝细胞癌发生、发展与多种信转导通路的异常激活或者异常沉默有着密切联系。JAK/STAT3言号转导途径介导肿瘤细胞与基质或免疫细胞之间的联络,调控和诱导与肿瘤细胞增殖、分化、凋亡密切相关的基因异常表达,促进肿瘤细胞增殖、恶性转化、阻碍凋亡。JAK和STAT3是许多细胞因子受体系统的关键分子,STAT3通过多肽类配体与它们的受体相结合而被激活,同时也可以被细胞内的激酶激活,进而介导细胞激酶,生长因子和激素等信号转导至细胞核,参加细胞生长、新陈代谢、分化、生存、抑制细胞凋亡和抵抗外源性病原体。细胞因子等配体与受体结合后,gp130 (CDw13,免疫球蛋白超家族IGSF成员)趋附于受体分子LIFR,并相结合形成二聚体,JAK分别与LIFR和gp130相结合,激活相应的JAK,活化的JAK自身磷酸化,并进一步磷酸化LIFR和gp130, gp130和LIFR磷酸化位点作为STAT3的SH2区锚链位点,当然STAT3的SH2区锚链位点也可以与其他受体结合,激活其他信号转导途径,如MAPK、PI3K/Akt信号转导途径。活化的STAT3与受体分离,两个STAT3分子间通过SH2区相连接形成形成二聚体,随后穿行核孔,进入细胞核,STAT3分子与DNA相应区域结合,诱导血管生成相关基因,细胞周期调控基因及抗凋亡基因等基因表达。研究表明,如果STAT3发生异常激活就会导致细胞的异常增殖、恶性转化、调亡抑制以及免疫抑制。多中心研究显示,肝细胞癌等恶性肿瘤细胞中JAK/STAT3信号转导途径持续性激活及STAT3过表达,磷酸化STAT3在肝细胞癌等恶性肿瘤中高表达。活化的STAT3促进肝细胞癌的发生,提高肝癌细胞再生能力和侵袭性。肿瘤的生长依赖血管的生成,通过新生血管给肿瘤细胞提供丰富的养料与氧分,JAK/STAT3信号转导途径调节VEGFR-2的表达,是肿瘤血管新生和肿瘤细胞转移的主要因子。STAT3是影响肿瘤细胞免疫反应的一个重要分子,控制肿瘤细胞分泌细胞因子来调节免疫反应,造成肿瘤细胞免疫逃逸。RNAi (RNA interference)——RNA干扰,是指一系列非编码RNA,由21-23 bp长的双链RNA分子介导,诱发同源mRNA降解的转录后基因沉默(PTGS)机制。研究人员设计目标内源性mRNA,合成siRNA,核糖核酸内切酶制备siRNA或siRNA的前体(如短发夹RNA (shRNA)或长的双链RNA (dsRNA),将其倒入细胞内甚至整个器官内,高通量将基因沉默,对经典基因研究和治疗方式产生了挑战。RNAi是一种转录后基因沉默(PTGS)小分子调控RNA,包括小NA (miRNA, microRNA)和小干扰RNA (siRNA, small interfering RNA),是保护不受外部基因甚至内部基因侵扰,控制细胞生长,调控基因表达,生物体适应外界环境的重要机制,是一种比较早期的动植物,甚至微生物等生命体就已经具有的生物途径。作为转录后基因沉默(PTGS)机制的特殊性,RNAi已经成为肿瘤学基础及临床研究的一种工具。转录因子将细胞外信号转导入基因组中,是肿瘤发生、发展的关键因素,阻断转录因子是目前恶性肿瘤治疗的有效方法,针对肿瘤的靶向治疗的研究,在不同水平中断肿瘤细胞中STAT3信号通路,阻断转录因子STAT3的表达,诱导肿瘤细胞的凋亡。RNA干涉技术靶向阻断STAT3基因具有高效、特异、操作简便、低毒等特点。为研究JAK/STAT3信号转导途径对肝癌细胞凋亡的影响,本实验拟通过RNA干扰(RNA interference, RNAi)沉默BEL-7402肝癌细胞STAT3基因。该研究可能为靶向肿瘤基因治疗提供实验依据。首先构建靶向STAT3的RNA干扰载体,应用脂质体LipofectamineTM 2000将前期构建的pGC-STAT3-siRNA质粒转染至BEL-7402肝癌细胞,并检测其基因沉默效应,根据实验分为对照组、阴性质粒组、STAT3-siRNA组,分别通过分别通过流式细胞术,检测细胞凋亡比率的变化,JC-1荧光染色观察线粒体膜电势△甲m变化,并利用Western Blot方法检测Caspase 3蛋白质水平的变化。JAK/STAT3信号转导途径与肝细胞癌有关联,通过流式细胞术检测结果显示,对照组,阴性质粒组及STAT3-siRNA)贡粒组凋亡率分别为9.26±0.42%,17.53±0.98%及45.24±0.79%,经统计学分析,STAT3-siRNA组细胞凋亡率明显高于对照组和阴性质粒组(P0.05),靶向STAT3促进BEL-7402细胞凋亡。凋亡(apoptosis)是细胞在多种因素作用下,通过基因调控而发生的一系列程序化细胞死亡想象,普遍存在于生物界。细胞凋亡早期核酸酶的激活及磷酯酰丝氨酸的膜暴露晚于线粒体膜电位△Ψm下降,本实验检测各组细胞△Ψm的数值变化结果显示：实验组细胞红色荧光减弱,绿色荧光增强,线粒体膜电位△Ψm降低(58.87±1.90%,P0.05),对照组及阴性质粒组细胞绿色荧光相对较弱,红色荧光较强,线粒体膜电位△Ψm较高(90.73±1.75%,90.03±1.68%,P0.05),说明靶向STAT显著降低BEL-7402肝癌细胞线△Ψm,引起线粒体膜去极化作用。1994年caspase-3基因被Fernandez-Alnemri从数据库筛选、克隆,1996年将其编码的这种蛋白酶命名为caspase-3,目前已经获得caspase-3的X线结晶图像。caspase-3是细胞凋亡过程中最主要的终末剪切酶,在细胞凋亡中起着不可替代的作用。凋亡途径最终启动,caspase-3被降解为分子量为P17/19的acti ved-caspase-3至关重要,细胞核中的多聚ADP-核糖聚合酶(PARP,116kD)被活性caspase-3剪切,使其不能与DNA有效结合,导致Ca2+/Mg2+依赖性内切核酸酶的活性增高,进而裂解核小体间的DNA,引起细胞凋亡。Western Blotting结果显示actived-caspase3蛋白(17/19 kDa)在实验组表达明显高于对照组和阴性质粒组(0.48±0.05 vs.0.22±0.04和0.26±0.06,P0.05),Caspase 3(35kDa)表达各组无明显差异(P0.01)(图4)。提示siRNA-STAT3冗默STAT3基因可明显降低细胞线粒体△Ψm,激活caspase-3增强,促进肝癌细胞凋亡、坏死。Bcl-2家族是调节线粒体通透性的主要成分,与细胞死亡信号相连接,对细胞的死亡起决定作用。本实验通过siRNA-STAT3沉默STAT3基因,阻断JAK/STAT3信号转导途径,阻断Bcl-2基因转录,Bcl-2表达水平下降,Bcl-2同源二聚体形成受到抑制,Bax就不能与Bcl-2相结合,那么Bax就会大量在线粒体膜上形成二聚体,导致线粒体膜电势能下降,激活caspase家族一系列蛋白分子,引起凋亡级联反应,促进Bel-7402肝癌细胞凋亡。总之,通过RNAi沉默BEL-7402肝癌细胞STAT3基因,从翻译水平阻抑STAT3基因表达,来抑制JAK/STAT3信号转导途径,增加了肝癌细胞的凋亡率,表现出更强大而且稳定的促凋亡作用,为肝癌细胞的信号转导途径靶向治疗的优化提供了体外实验数据。
[Abstract]:Hepatocelluar Carcinoma (HCC) is the digestive system malignant tumor that seriously damages human life and health in the world. The incidence of the disease ranks sixth in all malignant tumors. The mortality rate is third. The annual death toll is basically equal to the number of new diseases, and the trend is increasing year by year. Experts expect to reach the disease in 2015 - 20 years. Stage. The occurrence of hepatocellular carcinoma is the result of multistep, multi gene action. The main risk factors for hepatocellular carcinoma include infection of HBV or HCV, alcoholic liver disease, and non-alcoholic blood system diseases, which have no obvious symptoms in the early stages of.HCC, as well as in geographical areas and ethnic or ethnic groups, early detection, high malignancy, and surgical excision. There were fewer patients and poor prognosis. The 2 year recurrence rate and 5 year recurrence rate of most patients were 50% and 75%., respectively, and the diagnosis and treatment of hepatocellular carcinoma were improved continuously. The first choice of formula filter or operation based comprehensive treatment, but the prognosis of the patients with hepatocellular carcinoma was still not ideal. Therefore, in-depth study and understanding of the prognosis of hepatocellular carcinoma patients are still not ideal. The development and development of hepatocellular carcinoma can be used for early diagnosis, early treatment, and improvement of life and quality of life. Studies have shown that the development of primary hepatocellular carcinoma is closely related to the abnormal activation or abnormal silence of a variety of signal transduction pathways that mediate tumor cells and matrix or immunization with.JAK/STAT3 transduction pathway. Communication between cells, regulating and inducing abnormal expression of genes closely related to proliferation, differentiation and apoptosis of tumor cells, promoting tumor cell proliferation, malignant transformation, blocking apoptotic.JAK and STAT3 are key molecules of many cytokine receptor systems, STAT3 is activated by the combination of peptide complexes and their receptors, and can also be used. Activated by kinases in cells, and then mediate the signal transduction of kinases, growth factors and hormones to the nucleus, and participate in cell growth, metabolism, differentiation, survival, inhibition of apoptosis and resistance to exogenous pathogens. After the binding of ligand to the receptor, gp130 (CDw13, IGSF members of the immunoglobulin superfamily) are attached to the receptor. Molecular LIFR, combined with the formation of two polymer, combined with LIFR and gp130, activates the corresponding JAK, activated JAK itself phosphorylation, and further phosphorylation of LIFR and gp130, gp130 and LIFR phosphorylation sites as STAT3 SH2 region anchor sites, and of course the binding site can also be combined with other receptors to activate other signals. Guided pathways, such as MAPK, PI3K/Akt signal transduction pathway, activated STAT3 and receptor isolation, two STAT3 molecules are formed through SH2 region to form two polymers, then pass through nuclear pores and enter the nucleus, STAT3 molecules combine with the corresponding region of DNA to induce angiogenesis related genes, cell cycle regulation gene and anti apoptotic gene expression. Studies have shown that abnormal activation of STAT3 leads to abnormal proliferation of cells, malignant transformation, suppression of apoptosis and immunosuppression. Multicenter studies show that JAK/STAT3 signal transduction pathway is persistent and STAT3 overexpression in malignant tumor cells such as hepatocellular carcinoma, and phosphorylated STAT3 is highly expressed in malignant tumors such as hepatocellular carcinoma. STAT3 promotes the development of hepatocellular carcinoma and improves the regeneration ability and invasiveness of hepatoma cells. The growth of the tumor depends on the formation of blood vessels and provides rich nutrients and oxygen in the tumor cells through the neovascularization. The JAK/STAT3 signal transduction pathway regulates the expression of VEGFR-2, which is the main factor of tumor blood tube neoplasm and tumor cell metastasis,.STAT3 An important molecule that affects the immune response of a tumor cell, controls the secretion of cytokines by the tumor cells to regulate the immune response and causes the immune escape of the tumor cells,.RNAi (RNA interference) - RNA interference. It refers to a series of non coded RNA, mediated by 21-23 BP long double stranded RNA molecules, and induces the post transcriptional gene silencing (PTGS) of the degradation of homologous mRNA. Mechanism. Researchers designed target endogenous mRNA, synthesized siRNA, ribonucleic acid endonuclease to prepare precursors of siRNA or siRNA (such as short hairpin RNA (shRNA) or long double stranded RNA (dsRNA), and pour it into cells and even the whole organ. High throughput can silence the gene, and the challenge of classical gene research and treatment is a transcription. Gene silencing (PTGS) small molecules regulate RNA, including small NA (miRNA, microRNA) and small interference RNA (siRNA, small interfering RNA). It is an important mechanism to protect the growth of cells, regulate gene expression, and adapt to the environment of the outside world. It is a kind of early animal and plant, or even microorganism, and so on. The biological pathway that the life body has already has. As a special mechanism of post transcriptional gene silencing (PTGS), RNAi has become a tool for the basis of oncology and clinical research. The transcription factors transfer the extracellular signal into the genome, which is the key factor for the development of tumor, and blocking the transcription factors is effective for the treatment of malignant tumors. Methods, aiming at the target therapy of tumor, the STAT3 signaling pathway in tumor cells was interrupted at different levels and the expression of transcription factor STAT3 was blocked. The apoptosis.RNA interference technique of tumor cells was induced to target the STAT3 gene with high efficiency, specificity, simple operation, low toxicity and so on. To study the JAK/STAT3 signal transduction pathway for hepatoma cells The effect of apoptosis, this experiment is to silence the STAT3 gene of BEL-7402 hepatoma cells by RNA interference (RNA interference, RNAi). This study may provide experimental basis for targeting tumor gene therapy. Firstly, construct RNA interference vector of targeted STAT3, and transfect the early constructed pGC-STAT3-siRNA plasmid to BEL-7402 by liposome LipofectamineTM 2000. The gene silencing effect of hepatoma cells was detected. According to the experiment, the cells were divided into control group, negative plasmid group and STAT3-siRNA group. By flow cytometry, the changes of apoptosis ratio were detected by flow cytometry, JC-1 fluorescence staining was used to observe the changes of mitochondrial membrane potential delta m, and the Western Blot method was used to detect the change of Caspase 3 protein level.J The AK/STAT3 signal transduction pathway was associated with hepatocellular carcinoma. By flow cytometry, the apoptosis rate of the control group, negative plasmid group and STAT3-siRNA) was 9.26 + 0.42%, 17.53 + 0.98% and 45.24 + 0.79% respectively. By statistical analysis, the cell withering rate of the STAT3-siRNA group was significantly higher than that of the control group and the negative plasmid group (P0.05). STAT3 promotes apoptosis of BEL-7402 cells. Apoptosis (apoptosis) is a series of programmed cell death imaginations that occur through gene regulation and regulation by a variety of factors. The activation of nuclease in the early stage of apoptosis and the membrane exposure of phosphonyl serine later than the mitochondrial membrane potential delta m decreased. The results of the numerical change of the group cell delta m showed that the red fluorescence of the experimental group was weakened, the green fluorescence enhanced, the mitochondrial membrane potential delta m decreased (58.87 + 1.90%, P0.05), the green fluorescence of the control group and the negative plasmid group was relatively weak, the red fluorescence was stronger and the mitochondrial membrane potential delta m was higher (90.73 + 1.75%, 90.03 + 1.68%, P0.05), indicating the target. STAT significantly reduced BEL-7402 hepatoma cell line delta m, causing mitochondrial membrane depolarization, caspase-3 gene was screened by Fernandez-Alnemri from database and cloned by Fernandez-Alnemri in 1996. In 1996, the protease was named caspase-3. At present, the X-ray crystallized.Caspase-3 of Caspase-3 is the most important in the process of cell apoptosis. Terminal shear enzyme plays an irreplaceable role in cell apoptosis. Apoptosis pathway eventually starts, and caspase-3 is degraded to acti ved-caspase-3 with molecular weight P17/19, and the poly ADP- ribose polymerase (PARP, 116kD) in the nucleus is cut by active Caspase-3, so that it can not be effectively combined with DNA, resulting in Ca2+/Mg2+ dependence. The activity of nuclease increased and then cleavage of DNA between the nucleosomes, causing apoptosis.Western Blotting results showed that the expression of actived-caspase3 protein (17/19 kDa) in the experimental group was significantly higher than that of the control group and negative plasmid group (0.48 + 0.05 vs.0.22 + 0.04 and 0.26 + 0.06, P0.05), Caspase 3 (35kDa) expressed no significant difference (P0.01) (Fig. 4). The siRNA-STAT3 redundant STAT3 gene can obviously reduce the mitochondrial delta m, activate caspase-3 and promote the apoptosis of the hepatoma cells. The necrotic.Bcl-2 family is the main component of the mitochondrial permeability, which is connected with the cell death signal, and plays a decisive role in the cell death. This experiment blocked the JAK/STAT by siRNA-STAT3 silencing the STAT3 gene and blocked JAK/STAT. 3 signal transduction pathway, blocking Bcl-2 gene transcription, Bcl-2 expression level decline, Bcl-2 homologous two polymer formation is inhibited, Bax can not be combined with Bcl-2, then Bax will form a large number of polymers on the mitochondrial membrane, leading to the decrease of the mitochondrial membrane potential energy, activating a series of protein molecules in the caspase family, causing apoptosis cascade reaction and promoting B. El-7402 hepatoma cell apoptosis. In a word, RNAi silencing the STAT3 gene of BEL-7402 hepatoma cells, inhibiting the expression of STAT3 gene from the translation level, inhibiting the JAK/STAT3 signal transduction pathway, increasing the apoptosis rate of the hepatoma cells, showing a stronger and stable apoptosis promoting effect, and optimizing the target therapy for the signal transduction pathway of the hepatoma cells. The experimental data were provided in vitro.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7

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