siRNA沉默hARD1基因对人大肠癌SW620细胞凋亡的影响研究

发布时间：2018-02-15 04:46

  本文关键词： hARD1 大肠癌 基因沉默 细胞凋亡因子　出处：《昆明医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：[目的]人停滞缺陷蛋白1(human arrest defective 1,hARD1)是酵母N-乙酷基转移酶ARD1的人类同源基因,它与NATI基因结合,生成NatA复合体,催化细胞内多种蛋白质乙酰化。大肠癌的发生发展与hARDI基因表达及细胞凋亡密切相关,但hARD1基因表达能否通过影响细胞凋亡途径进而影响大肠癌的发生发展及具体分子机制尚不明确。本实验以人大肠癌SW620细胞为研究对象,研究hARD1与细胞凋亡途径相关因子之间的关系,探讨hARD1基因被沉默后对凋亡信号通路蛋白的影响,进而揭示hARD1基因调控大肠癌细胞凋亡具体的分子机制,以期为大肠癌的早期诊断及治疗提供理论依据。[方法]1.人大肠癌SW620细胞株的复苏、培养、传代、冻存;2.实验组即hARD1沉默组,用ARD1 siRNA通过Lipofectamin2000转染试剂转染细胞株;阴性对照组即hARD1沉默阴性对照组,用NCsiRNA转染细胞株;空白对照组细胞株常规培养,不予特殊处理;3. RT-PCR检测各组细胞中hARDl的沉默效率;4.流式细胞术+PI染色检测各组大肠癌细胞的周期变化情况;5.流式细胞术和AnnexinV+PI染色检测各组大肠癌细胞的凋亡状况;6.用EdU法检测各组大肠癌细胞的增殖状况;7.荧光探针法检测各组大肠癌细胞中ROS含量;8.荧光定量PCR检测各组大肠癌细胞中细胞凋亡途径相关因子(TNF-α、Fas、caspase8、Apaf-1、caspase9; Bcl-2、Bad)的mRNA表达水平;9. Western blot检测各组大肠癌细胞中细胞凋亡途径相关因子(TNF-α、Fas、caspase8、Apaf-1、caspase9; Bcl-2、Bad)的蛋白质表达水平;[结果]1. ARD1siRNA和NCsiRNA转染人大肠癌SW620细胞后,用RT-PCR检测各组中hARD1的沉默效率:阴性对照组沉默效率为0%,实验组的沉默效率为62%;2.流式细胞术和AnnexinV+PI染色检测各组大肠癌细胞的周期及凋亡情况,结果显示:沉默人大肠癌SW620细胞hARDI基因后,三组细胞的细胞周期无明显改变;实验组细胞凋亡水平明显增加,而阴性对照组和空白对照组无明显差异;3. EdU检测细胞增殖提示:实验组与空白对照组及阴性对照组比较,实验组细胞增殖比例明显降低,阴性对照组与空白对照组比较细胞增殖比例无明显差异;4.荧光探针法检测ROS含量提示:实验组与空白对照组及阴性对照组比较,实验组ROS含量明显升高,阴性对照组与空白对照组比较ROS含量无明显差异;5.荧光定量PCR检测各凋亡因子mRNA表达水平结果提示:实验组的细胞凋亡途径相关因子(caspase9)的mRNA表达水平明显高于空白对照组和阴性对照组,而三组中细胞凋亡途径相关因子(TNF-α、caspase8、Fas、Apaf-1、Bcl-2、Bad)的mRNA表达水平无明显改变;6. Western blot检测各凋亡因子蛋白质表达水平结果提示:实验组的细胞凋亡途径相关因子(caspase8、caspase9、Fas、Apaf-1、Bcl-2)的蛋白质表达水平明显高于空白对照组和阴性对照组,而三组中细胞凋亡途径相关因子(TNF-α、Bad)的蛋白质表达水平无明显改变。[结论]1.沉默人大肠癌SW620细胞中hARD1基因后,对其细胞周期无明显影响,但能明显促进细胞凋亡和抑制细胞增殖;2.本实验证实,沉默人大肠癌SW620细胞中hARD1基因后,能够通过调节细胞凋亡途径中某些因子的转录水平和翻译水平进而促进细胞凋亡;3. hARD1基因可能作为大肠癌发生发展的潜在作用靶点,为敲除hARD1基因治疗大肠癌提供理论依据。
[Abstract]:[Objective] people Stagnation (human deficient protein 1 defective 1 arrest, hARD1) is the yeast N- B cool glycosyltransferase ARD1 homologous gene of human, it is combined with the NATI gene, generating NatA complex, catalytic intracellular protein acetylation. Colorectal cancer development and hARDI gene expression and cell apoptosis are closely related. But the expression of hARD1 gene can affect the occurrence and development of the molecular mechanism of colorectal cancer by influencing apoptosis pathway is not clear. In this experiment, human colon cancer SW620 cells as the research object, the research of hARD1 and cell apoptosis pathway related with the relationship between, to explore the influence of apoptosis signal pathway protein hARD1 gene silencing, and to reveal the molecular mechanism of hARD1 gene regulation in apoptosis of colorectal cancer cell specific, in order to early diagnosis and treatment of colorectal cancer and provide a theoretical basis. Methods]1. of human colon cancer SW620 cell line. Recovery, culture, subculture, cryopreservation; 2. experimental group hARD1 group ARD1 by siRNA silencing, Lipofectamin2000 transfection reagent transfected cell lines; negative control group: hARD1 silencing negative control group, transfected with NCsiRNA cell line; blank control group cells cultured, not special treatment; hARDl was detected by RT-PCR in 3. the silencing efficiency; cycle changes were detected in colorectal cancer cells 4. +PI staining flow cytometry; apoptosis of colorectal cancer cells were detected by flow cytometry and AnnexinV+PI 5. staining; 6. with EdU method to detect colorectal cancer cell proliferation in colorectal cancer cells; ROS levels were detected in 7. fluorescence probe method; cell apoptosis of colorectal cancer cells were detected by fluorescence quantitative PCR in 8. related factors (Fas, caspase8, TNF- alpha, Apaf-1, caspase9; Bcl-2, Bad) the expression level of mRNA; 9. Western blot were detected in colorectal carcinoma The apoptotic pathway in cell associated factor (TNF- alpha, Fas, caspase8, Apaf-1, caspase9; Bcl-2, Bad) the protein expression of]1. and NCsiRNA ARD1siRNA; the transfection of human colon cancer SW620 cells, using hARD1 RT-PCR to detect the silencing efficiency: negative control group silencing efficiency was 0%, the experimental group silencing efficiency 62%; 2. flow cytometry and AnnexinV+PI staining cycle and apoptosis of colorectal cancer cells were detected, the results showed that the silencing of human colon cancer SW620 cells after hARDI gene, cell cycle of cells in three groups had no obvious change; the level of apoptosis in experimental group increased significantly, while the negative control group and blank control group had no significant difference cell proliferation assay; 3. EdU note: the experimental group and blank control group and negative control group, cell proliferation in experimental group decreased significantly, negative control group and blank control group cell proliferation ratio of ignorance It is suggested that the detection of significant difference; the content of ROS 4. fluorescence probe method: experimental group and blank control group and negative control group, the experimental group was significantly increased ROS content, negative control group and blank control group ROS content had no significant difference; 5. fluorescence quantitative PCR detection of apoptosis factor mRNA expression level showed that cell apoptosis in experimental group the related factor (caspase9) expression level of mRNA was significantly higher than the control group and negative control group, while the apoptosis pathway related factor in the three groups (TNF- alpha, caspase8, Fas, Apaf-1, Bcl-2, Bad) mRNA expression level had no obvious change; 6. Western blot to detect the protein expression of apoptosis factor results the experimental group: cell apoptosis related factors (caspase8, caspase9, Fas, Apaf-1, Bcl-2) protein expression level was significantly higher than that of blank control group and negative control group, while the apoptosis pathway related in three groups Factor (TNF- alpha, Bad) protein expression level had no obvious change in hARD1. Conclusion]1. silencing of human colorectal cancer cell line SW620 in genes that had no significant effect on the cell cycle, but it can significantly promote cell apoptosis and inhibit cell proliferation; 2. confirmed this experiment, hARD1 human colon cancer cells SW620 gene silence, through some factors regulating cell apoptosis pathway in the level of transcription and translation and promote cell apoptosis; potential target gene hARD1 3. may occur as the development of colorectal cancer, to provide theoretical basis for the knockout of hARD1 gene therapy for colorectal cancer.

【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34

【参考文献】

相关期刊论文 前4条

1 张渝;刘展;方圆;阳佳;张明亮;;SiRNA抑制CDK6表达阻滞大肠癌细胞增殖和细胞周期进展[J];实用医学杂志;2015年12期

2 白松;邵佳发;王维琦;钟孝斌;赵翔宇;;hARD1在人大肠腺癌中的表达及与肿瘤分化的相关性[J];世界华人消化杂志;2011年15期

3 白松;周媛媛;田风;赵翔宇;钟孝斌;;ARD1在人大肠腺癌细胞SW620、LS-174T和HCT-8中的表达[J];南方医科大学学报;2011年05期

4 Kyung-Eun Lee;Young-Seoub Hong;Byoung-Gwon Kim;Na-Young Kim;Kyoung-Mu Lee;Jong-Young Kwak;Mee-Sook Roh;;p73 G4C14 to A4T14 polymorphism is associated with colorectal cancer risk and survival[J];World Journal of Gastroenterology;2010年35期

，

本文编号：1512457