本文選題：SIRT3　切入點(diǎn)：乳腺癌　出處：《鄭州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景及目的乳腺癌是世界范圍內(nèi)女性發(fā)病率最高的惡性腫瘤,占總癌癥病例的23%和癌癥死亡病例的14%。在我國,乳腺癌的發(fā)病率呈逐年上升趨勢,且趨于年輕化,是對婦女健康威脅最大的疾病之一。由于缺少相應(yīng)的篩查措施或治療手段不當(dāng),我國乳腺癌的死亡率也逐漸升高。因此,深入探討乳腺癌的發(fā)病機(jī)理十分必要。隨著分子生物學(xué)機(jī)制在腫瘤學(xué)領(lǐng)域的更深入研究,分子靶向治療越來越多的展現(xiàn)出它獨(dú)有的優(yōu)勢。沉默信息調(diào)節(jié)因子家族(Sirtuins)被認(rèn)為在調(diào)控衰老和壽命方面發(fā)揮重要作用。Sirtuins由7個(gè)成員組成,即SIRT1-SIRT7。SIRT3作為線粒體去乙酰化酶,是Sirtuins家族的重要成員。最近幾年的研究發(fā)現(xiàn)SIRT3不僅參與了機(jī)體壽命的調(diào)節(jié),其在腫瘤的發(fā)生、發(fā)展中亦具非常重要的作用。近期研究發(fā)現(xiàn):SIRT3在肝癌細(xì)胞中的表達(dá)相對于癌旁組織顯著降低,且SIRT3高表達(dá)可使總生存期(OS)與RFS延長。此外,SIRT3的高表達(dá)可以抑制細(xì)胞生長并誘導(dǎo)肝癌細(xì)胞的凋亡。在肺腺癌,胃癌,胰腺癌中,SIRT3表達(dá)均下調(diào),SIRT3的過表達(dá)抑制細(xì)胞生長并誘導(dǎo)細(xì)胞死亡。與此結(jié)果相反,另有一些研究表明:在口腔鱗癌、食管癌、膀胱癌中SIRT3敲低顯著抑制腫瘤發(fā)生,起促進(jìn)腫瘤進(jìn)展的作用。由此發(fā)現(xiàn),SIRT3在不同類型腫瘤中的作用機(jī)制有待進(jìn)一步研究。目前關(guān)于SIRT3在乳腺癌中作用的研究尚不完善,本實(shí)驗(yàn)以SIRT3為靶點(diǎn),通過免疫組織化學(xué)測定其在乳腺癌組織中的表達(dá)情況,Western blot測定SIRT3在乳腺癌細(xì)胞的表達(dá)水平,以及RT-PCR實(shí)驗(yàn)測定SIRT3分子在RNA水平的表達(dá),從而探討SIRT3在乳腺癌疾病進(jìn)展中的作用;通過將SIRT3的表達(dá)情況與患者臨床病理特征進(jìn)行相關(guān)性分析及生存分析,探討SIRT3與乳腺癌預(yù)后相關(guān)性;通過研究SIRT3基因的過表達(dá)與敲低條件下分別對乳腺癌細(xì)胞增殖能力的影響,探討SIRT3在癌細(xì)胞中的功能和性質(zhì),從而探索SIRT3是否有望成為乳腺癌治療的潛在靶點(diǎn)。方法1.應(yīng)用組織芯片技術(shù),通過免疫組化法檢測SITR3在乳腺癌組織及癌旁組織的表達(dá),評價(jià)SIRT3在乳腺癌組織的表達(dá)情況,并分析SIRT3的表達(dá)與患者臨床病理學(xué)特征的相關(guān)性。根據(jù)隨訪結(jié)果對所有病例進(jìn)行單因素及多因素生存分析,分析SIRT3的表達(dá)與乳腺癌患者預(yù)后的相關(guān)性。2.應(yīng)用細(xì)胞培養(yǎng)技術(shù),通過Western blot檢測SIRT3在乳腺癌細(xì)胞系EMT6,乳腺成纖維細(xì)胞系MEFs的蛋白表達(dá)水平。3.通過熒光定量PCR檢測SIRT3 mRNA在乳腺癌細(xì)胞系EMT6,乳腺成纖維細(xì)胞系MEFs的表達(dá)情況。4.通過MTT實(shí)驗(yàn)觀察上調(diào)及下調(diào)SIRT3基因?qū)θ橄侔〦MT6細(xì)胞增殖能力的影響。結(jié)果1、SIRT3在乳腺癌組織中的陽性表達(dá)率84.72%(122/144)低于癌旁正常組織表達(dá)率96.63%(86/89)。兩組差異有統(tǒng)計(jì)學(xué)意義(t=7.572,P0.05)。SIRT3在乳腺癌中的表達(dá)與患者下列臨床病理學(xué)特征存在相關(guān)性:年齡、淋巴結(jié)轉(zhuǎn)移、TNM分期、HER-2表達(dá)(P0.05);然而,SIRT3的表達(dá)與病理分級、浸潤深度、ER受體及PR受體的表達(dá)情況無關(guān)(P0.05)。單因素分析顯示:乳腺癌中SIRT3表達(dá)、淋巴結(jié)轉(zhuǎn)移、TNM分期、HER-2表達(dá)情況與預(yù)后相關(guān)(P0.05);多因素COX比例風(fēng)險(xiǎn)分析顯示SIRT3的表達(dá)情況及TNM分期均具有獨(dú)立的預(yù)后意義。2、Western blot結(jié)果顯示:乳腺癌細(xì)胞中SIRT3的表達(dá)水平明顯降低。定量數(shù)據(jù)顯示:SIRT3在乳腺癌細(xì)胞的相對表達(dá)量(0.220±0.173)明顯低于非癌性細(xì)胞(0.850±0.086),差異有統(tǒng)計(jì)學(xué)意義(t=7.133,P=0.002,P0.01)。3、RT-PCR結(jié)果顯示:SIRT3在乳腺癌細(xì)胞的mRNA水平較非癌性細(xì)胞明顯降低。SIRT3在乳腺癌細(xì)胞中的表達(dá)水平(0.520±0.069)明顯低于非癌性細(xì)胞(0.886±0.034),差異有統(tǒng)計(jì)學(xué)意義(t=4.729,P=0.009,P0.01)。4、MTT試驗(yàn)結(jié)果顯示:SIRT3敲低條件下細(xì)胞增殖速度增加,明顯高于對照組;SIRT3過表達(dá)條件下細(xì)胞增殖速度減慢,差異有統(tǒng)計(jì)學(xué)意義。結(jié)論SIRT3在乳腺癌組織和乳腺癌細(xì)胞的中的表達(dá)下調(diào),提示SIRT3可能在乳腺癌的發(fā)生發(fā)展中起抑制作用。SIRT3的表達(dá)水平對乳腺癌的預(yù)后判斷具有重要意義。上調(diào)SIRT3基因可以抑制乳腺癌細(xì)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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.9

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1 杜文英;乳腺癌分子亞型的臨床與病理特點(diǎn)[D];鄭州大學(xué);2011年

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5 葛廣哲;樹，

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