SIRT3基因在乳腺癌中的表达及其对乳腺癌细胞增殖能力的影响

发布时间：2018-03-08 23:21

本文选题：SIRT3　切入点：乳腺癌　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景及目的乳腺癌是世界范围内女性发病率最高的恶性肿瘤,占总癌症病例的23%和癌症死亡病例的14%。在我国,乳腺癌的发病率呈逐年上升趋势,且趋于年轻化,是对妇女健康威胁最大的疾病之一。由于缺少相应的筛查措施或治疗手段不当,我国乳腺癌的死亡率也逐渐升高。因此,深入探讨乳腺癌的发病机理十分必要。随着分子生物学机制在肿瘤学领域的更深入研究,分子靶向治疗越来越多的展现出它独有的优势。沉默信息调节因子家族(Sirtuins)被认为在调控衰老和寿命方面发挥重要作用。Sirtuins由7个成员组成,即SIRT1-SIRT7。SIRT3作为线粒体去乙酰化酶,是Sirtuins家族的重要成员。最近几年的研究发现SIRT3不仅参与了机体寿命的调节,其在肿瘤的发生、发展中亦具非常重要的作用。近期研究发现:SIRT3在肝癌细胞中的表达相对于癌旁组织显著降低,且SIRT3高表达可使总生存期(OS)与RFS延长。此外,SIRT3的高表达可以抑制细胞生长并诱导肝癌细胞的凋亡。在肺腺癌,胃癌,胰腺癌中,SIRT3表达均下调,SIRT3的过表达抑制细胞生长并诱导细胞死亡。与此结果相反,另有一些研究表明:在口腔鳞癌、食管癌、膀胱癌中SIRT3敲低显著抑制肿瘤发生,起促进肿瘤进展的作用。由此发现,SIRT3在不同类型肿瘤中的作用机制有待进一步研究。目前关于SIRT3在乳腺癌中作用的研究尚不完善,本实验以SIRT3为靶点,通过免疫组织化学测定其在乳腺癌组织中的表达情况,Western blot测定SIRT3在乳腺癌细胞的表达水平,以及RT-PCR实验测定SIRT3分子在RNA水平的表达,从而探讨SIRT3在乳腺癌疾病进展中的作用;通过将SIRT3的表达情况与患者临床病理特征进行相关性分析及生存分析,探讨SIRT3与乳腺癌预后相关性;通过研究SIRT3基因的过表达与敲低条件下分别对乳腺癌细胞增殖能力的影响,探讨SIRT3在癌细胞中的功能和性质,从而探索SIRT3是否有望成为乳腺癌治疗的潜在靶点。方法1.应用组织芯片技术,通过免疫组化法检测SITR3在乳腺癌组织及癌旁组织的表达,评价SIRT3在乳腺癌组织的表达情况,并分析SIRT3的表达与患者临床病理学特征的相关性。根据随访结果对所有病例进行单因素及多因素生存分析,分析SIRT3的表达与乳腺癌患者预后的相关性。2.应用细胞培养技术,通过Western blot检测SIRT3在乳腺癌细胞系EMT6,乳腺成纤维细胞系MEFs的蛋白表达水平。3.通过荧光定量PCR检测SIRT3 mRNA在乳腺癌细胞系EMT6,乳腺成纤维细胞系MEFs的表达情况。4.通过MTT实验观察上调及下调SIRT3基因对乳腺癌EMT6细胞增殖能力的影响。结果1、SIRT3在乳腺癌组织中的阳性表达率84.72%(122/144)低于癌旁正常组织表达率96.63%(86/89)。两组差异有统计学意义(t=7.572,P0.05)。SIRT3在乳腺癌中的表达与患者下列临床病理学特征存在相关性:年龄、淋巴结转移、TNM分期、HER-2表达(P0.05);然而,SIRT3的表达与病理分级、浸润深度、ER受体及PR受体的表达情况无关(P0.05)。单因素分析显示:乳腺癌中SIRT3表达、淋巴结转移、TNM分期、HER-2表达情况与预后相关(P0.05);多因素COX比例风险分析显示SIRT3的表达情况及TNM分期均具有独立的预后意义。2、Western blot结果显示:乳腺癌细胞中SIRT3的表达水平明显降低。定量数据显示:SIRT3在乳腺癌细胞的相对表达量(0.220±0.173)明显低于非癌性细胞(0.850±0.086),差异有统计学意义(t=7.133,P=0.002,P0.01)。3、RT-PCR结果显示:SIRT3在乳腺癌细胞的mRNA水平较非癌性细胞明显降低。SIRT3在乳腺癌细胞中的表达水平(0.520±0.069)明显低于非癌性细胞(0.886±0.034),差异有统计学意义(t=4.729,P=0.009,P0.01)。4、MTT试验结果显示:SIRT3敲低条件下细胞增殖速度增加,明显高于对照组;SIRT3过表达条件下细胞增殖速度减慢,差异有统计学意义。结论SIRT3在乳腺癌组织和乳腺癌细胞的中的表达下调,提示SIRT3可能在乳腺癌的发生发展中起抑制作用。SIRT3的表达水平对乳腺癌的预后判断具有重要意义。上调SIRT3基因可以抑制乳腺癌细胞增殖。
[Abstract]:Background and objective: breast cancer is a malignant tumor with the highest incidence of women worldwide, accounting for 23% of the total cancer cases and cancer death cases of 14%. in our country, the incidence of breast cancer increased year by year, and tend to be younger, the biggest threat to women's health is a disease due to the lack of appropriate screening. The treatment measures or improper means, of breast cancer mortality increased. Therefore, in-depth study of the pathogenesis of breast cancer is necessary. With the further study the mechanism of molecular biology in the field of oncology, molecular target therapy more and more to show its unique advantages. The silent information regulator (Sirtuins) is a family thought to play an important role in.Sirtuins is composed of 7 members in the control of aging and life, namely SIRT1-SIRT7.SIRT3 as a mitochondrial deacetylase, is an important member of the Sirtuins family. The most recent The study found that SIRT3 is not only involved in the regulation of the life of the body, the tumor also has very important role in the development. Recent research found that significantly reduced the expression of SIRT3 in hepatocellular carcinoma compared with adjacent tissues, and the high expression of SIRT3 can make the overall survival (OS) and RFS extension. In addition, the apoptosis of the high expression of SIRT3 can inhibit cell growth and induce hepatoma cells. Gastric cancer in lung adenocarcinoma, pancreatic carcinoma, the expression of SIRT3 was downregulated. Overexpression inhibited cell growth and induced cell death of SIRT3. Contrary to this, some studies show that in oral squamous cell carcinoma, esophageal cancer, bladder cancer SIRT3 knockdown significantly inhibited tumorigenesis, plays a role in promoting tumor progression. Therefore, further studies are needed on the mechanism of SIRT3 in different types of tumors. The current research on the role of SIRT3 in breast cancer is still not perfect, in this experiment SIR T3 as the target, through the determination of its expression in breast cancer tissue by immunohistochemistry, Western blot determination of SIRT3 expression levels in breast cancer cells, and the expression of RT-PCR assay of SIRT3 molecules in the RNA level, so as to explore the progress in breast cancer for SIRT3; the expression of SIRT3 and the clinicopathologic features of the patients with correlation analysis and survival analysis, the relationship between SIRT3 and the prognosis of breast cancer; through the study of SIRT3 gene overexpression and knockdown conditions effect on cell proliferation of breast cancer, to explore the nature and function of SIRT3 in cancer cells, and to explore whether SIRT3 is expected to become a potential target for the treatment of breast cancer methods 1.. The application of tissue microarray technology, by immunohistochemistry to investigate the expression detection of SITR3 in breast carcinoma tissue and the expression situation in breast cancer tissue and the evaluation of SIRT3 Correlation analysis between the expression and the clinical pathological features of SIRT3. Survival analysis was performed by single factor and multiple factors on all cases according to the follow-up results, analysis the correlation between.2. cell SIRT3 expression and prognosis of breast cancer by Western blot culture technique, the detection of SIRT3 in breast cancer cell line EMT6, breast fibroblast cell line MEFs the protein expression level of.3. was detected by SIRT3 mRNA fluorescence quantitative PCR in breast cancer cell lines EMT6, breast fibroblast cell lines expressing MEFs.4. MTT up and down through the experimental observation of the effect of SIRT3 gene on cell proliferation of EMT6 breast cancer. Results 1. The positive expression of SIRT3 in breast cancer was 84.72% (122/144) lower than the adjacent normal tissues was 96.63% (86/89). There was significant difference between two groups (t=7.572, P0.05).SIRT3 expression and clinical pathology in breast cancer patients following the special Correlation between syndrome: age, lymph node metastasis, TNM staging, the expression of HER-2 (P0.05); however, the expression of SIRT3 and pathological grade, depth of invasion, independent of the expression of ER receptor and PR receptor (P0.05). Single factor analysis showed that the expression of SIRT3 in breast cancer, lymph node metastasis, TNM staging, HER-2 the expression is correlated with the prognosis (P0.05); multivariate COX proportional hazard analysis showed that the expression of TNM and SIRT3 stage were independent prognostic factors of.2 Western blot, the results showed that the expression level of SIRT3 in breast cancer cells decreased significantly. The quantitative data showed that SIRT3 in the relative expression of breast cancer cells (0.220. 0.173) was significantly lower than that in non cancerous cells (0.850 + 0.086), the difference was statistically significant (t=7.133, P=0.002, P0.01).3, RT-PCR results showed that SIRT3 in breast cancer cell mRNA levels than non cancerous cells decreases expression of.SIRT3 in breast cancer cells Ping (0.520 + 0.069) was significantly lower than that in non cancerous cells (0.886 + 0.034), the difference was statistically significant (t=4.729, P=0.009, P0.01).4, MTT test results showed that the proliferation rate of SIRT3 cells on the condition of low increase, significantly higher than the control group; SIRT3 overexpression slowed down the cell proliferation condition was statistically significant difference. Conclusion expression of SIRT3 in breast cancer and breast cancer in the cell, suggesting that SIRT3 may be in the development of breast cancer prognosis from the expression level of the inhibitory effect of.SIRT3 on breast cancer. It is important to determine the upregulation of SIRT3 gene can inhibit the proliferation of breast cancer cells.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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