let-7f-5p通过促抗凋亡蛋白表达及抑制凋亡蛋白诱导结直肠癌化疗耐药

发布时间：2018-04-30 01:06

本文选题：结直肠癌 + 化疗耐药　；参考：《兰州大学》2017年硕士论文

【摘要】：研究背景与目的结直肠癌(Colorectal cancer,CRC)是目前威胁人类生命健康最常见的恶性肿瘤之一,分别位列男性肿瘤第二位和女性肿瘤第三位,约50%以上的结直肠癌于发现时已有转移。手术切除辅助化学药物治疗是临床治疗CRC的重要方式,但多药耐药(Multidrug resisitance,MDR)严重影响了CRC的临床疗效。MDR的形成及相关机制长期以来都是结直肠癌研究中的热点和前沿问题。MicroRNAs(miRNAs)广泛参与着多种生命活动过程。在肿瘤中,miRNAs的异常表达涉及多种类型肿瘤的进展和转移。新近的研究发现,上调或下调某些特定miRNAs的表达能直接影响肿瘤对化疗药物的反应,其作为关键的调节因子,参与了多种肿瘤MDR的形成,其中就包括结直肠癌。let-7家族通过调节多种致癌信号转导,在肿瘤的发生发展中起到了广泛而重要的作用;还有报道指出,let-7/Lin28与乳腺癌细胞化疗耐药密切相关;此外let-7可通过IL-6/STAT3通路调控食管鳞状细胞癌的化疗耐药性,提示let-7家族与肿瘤耐药形成关系密切,但其在结直肠癌的化疗耐药形成中是否具有作用及其相关机制尚不明确。本研究以化疗耐药的人结直肠癌为切入点,探讨let-7f-5p对结直肠癌化疗耐药的影响及相关分子机制,旨在为临床治疗化疗耐药型结直肠癌提供新的靶点。方法1.通过TCGA数据库,分析对比化疗耐药与化疗敏感的人结直肠癌组织中let-7f-5p的表达差异,了解高表达与低表达let-7f-5p与结直肠癌化疗耐药的相互关系。2.Lipofectamine 3000转染人结直肠癌HCT116和SW480细胞系,分别构建过表达、抑制表达let-7f-5p细胞模型;利用5-氟尿嘧啶(5-fluorouracil,5-FU)处理相应细胞系,Western-blot检测过表达、抑制表达let-7f-5p后细胞系抗凋亡蛋白Bcl-2及Bcl-x L的相应改变;通过Caspase-3/-9活性检测试剂盒检测过表达、抑制表达let-7f-5p后细胞系Caspase-3/-9的活性变化。3.5-FU分别处理HCT116和SW480细胞系;过表达、抑制表达let-7f-5p,利用流式细胞术,通过Annexin V-FITC/PI染色检测细胞凋亡改变情况,通过MitoScreen JC-1检测上述细胞线粒体膜电位改变情况。4.使用TargetScan和miRanda数据库预测let-7f-5p潜在调控的靶基因;运用实时定量PCR分析HCT116和SW480细胞系中过表达或抑制表达let-7f-5p对TP53、TP53INP1、TP53INP2和Caspase-3 mRNA表达水平的影响;使用荧光素酶报告系统测定TP53、TP53INP1、TP53INP2和Caspase-3 3'UTR荧光素酶活性变化情况。结果1.let-7f-5p高表达与人结直肠癌化疗耐药相关。2.let-7f-5p通过促进抗凋亡相关蛋白表达并抑制凋亡相关蛋白表达从而诱导人结直肠癌细胞系产生化疗耐药。2.1 let-7f-5p抑制人结直肠癌细胞系耐药株的凋亡。2.2 let-7f-5p促进抗凋亡相关蛋白表达。2.3 let-7f-5p抑制凋亡相关蛋白表达。3.let-7f-5p通过直接作用于TP53、TP53INP1、TP53INP2和Caspase-3进而影响人结直肠癌细胞化疗耐受。结论以上结果证实,化疗耐药的人结直肠癌中let-7f-5p表达增高;在HCT116和SW480人结直肠癌细胞系,经5-FU处理后,过表达let-7f-5p可促进抗凋亡蛋白Bcl-2和Bcl-x L的表达,并抑制Caspase-3/-9的活性,进而引起细胞凋亡抑制,诱导肿瘤细胞化疗耐药;而let-7f-5p直接作用于TP53、TP53INP1、TP53INP2和Caspase-3并抑制其表达是可能的机制之一。本研究通过对let-7f-5p诱导人结直肠癌化疗耐药相关机制深入探讨,为临床治疗化疗耐药型结直肠癌提供了新的靶点。
[Abstract]:Background and objective Colorectal cancer (CRC) is one of the most common malignant tumors that threaten human life and health. There are second male tumors and third female tumors respectively. More than 50% of colorectal cancer has been metastases when found. Surgical excision assisted chemotherapy is an important way to treat CRC. But multidrug resistance (Multidrug resisitance, MDR) seriously affects the clinical efficacy of CRC and the formation of.MDR and the related mechanisms are a hot and frontier problem in colorectal cancer research for a long time,.MicroRNAs (miRNAs) is widely involved in a variety of life activities. In the tumor, the abnormal expression of miRNAs involves the progress and transformation of various types of tumors. Recent studies have found that up or down expression of certain specific miRNAs can directly affect the response of tumor to chemotherapeutic drugs. As a key regulatory factor, it participates in the formation of a variety of tumor MDR, including the.Let-7 family of colorectal cancer, which has been widely used in the development of cancer by regulating a variety of carcinogenic signal transduction pathways. It is also reported that let-7/Lin28 is closely related to chemoresistance of breast cancer cells; in addition, let-7 can regulate the chemotherapeutic resistance of esophageal squamous cell carcinoma through the IL-6/STAT3 pathway, suggesting that the let-7 family is closely related to the formation of tumor resistance, but whether it has a role in the formation of chemotherapeutic drug resistance in colorectal cancer and its phase. The system is not clear. In this study, the effect of let-7f-5p on chemotherapy resistance of colorectal cancer and its molecular mechanism were discussed with chemotherapy resistant human colorectal cancer, and the aim of this study was to provide new targets for the clinical treatment of chemotherapy resistant colorectal cancer. Method 1. the TCGA database was used to analyze and compare the colorectal cancer sensitive and chemotherapy sensitive colorectal cancer. The relationship between the expression of let-7f-5p in cancer tissue and the relationship between high expression and low expression of let-7f-5p and chemotherapy resistance of colorectal cancer.2.Lipofectamine 3000 transfected HCT116 and SW480 cell lines of human colorectal cancer, respectively, to construct overexpression, inhibit the expression of let-7f-5p cell model, and use 5- fluorouracil (5-fluorouracil, 5-FU) to deal with the corresponding cell lines. Western-blot detected over expression, inhibited the corresponding changes in the anti apoptotic protein Bcl-2 and Bcl-x L in the cell lines after the expression of let-7f-5p, and detected the expression by the Caspase-3/-9 activity detection kit and inhibited the activity of Caspase-3/-9 in the cell line after the expression of let-7f-5p, and.3.5-FU treated HCT116 and SW480 cell lines respectively; overexpressed and inhibited the expression let-7f-5p, The changes of apoptosis were detected by flow cytometry and Annexin V-FITC/PI staining. The changes of mitochondrial membrane potential were detected by MitoScreen JC-1..4. was used to predict the potential target genes of let-7f-5p using TargetScan and miRanda databases. The real-time quantitative PCR analysis of HCT116 and SW480 cell lines was used for overexpression or inhibition. The effect of let-7f-5p on the expression of TP53, TP53INP1, TP53INP2 and Caspase-3 mRNA; the changes in the activity of TP53, TP53INP1, TP53INP2 and Caspase-3 3'UTR luciferase by the luciferase reporter system. Inhibition of apoptosis related proteins to induce the expression of apoptosis related proteins in human colorectal cancer cell lines to induce chemotherapeutic drug resistance.2.1 let-7f-5p to inhibit apoptosis of.2.2 let-7f-5p in human colorectal cancer cell lines and promote anti apoptosis related protein expression of.2.3 let-7f-5p to inhibit apoptosis related protein expression.3.let-7f-5p by direct action on TP53, TP53INP1, TP53INP2. And Caspase-3 further affect the chemotherapy tolerance of human colorectal cancer cells. Conclusion the above results confirm that the expression of let-7f-5p in colorectal cancer is higher in chemoresistant human colorectal cancer; in HCT116 and SW480 human colorectal cancer cell lines, after 5-FU treatment, overexpression of let-7f-5p can promote the expression of anti apoptotic protein Bcl-2 and Bcl-x L, and inhibit the activity of Caspase-3/-9. The effect of let-7f-5p on TP53, TP53INP1, TP53INP2, Caspase-3 and inhibition of its expression is one of the possible mechanisms. This study provides an in-depth study of the mechanism of chemotherapeutic drug resistance in human colon cancer induced by let-7f-5p and provides a clinical treatment for the clinical treatment of chemotherapy resistant colorectal cancer. New targets.

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34

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