内质网应激诱导miR-663过表达通过TGFB1调节肝癌细胞凋亡

发布时间：2018-06-14 00:44

本文选题：肝癌 + 内质网应激　；参考：《安徽医科大学》2016年硕士论文

【摘要】：目的:Micro RNAs(mi RNAs)在绝大部分癌症中的表达都有所失调,例如原发性肝细胞癌(hepatocellular carcinoma,HCC),但失调的确切机制尚有待研究。我们课题组在前期研究中已经发现,与正常肝细胞相比,肝癌细胞更易对内质网应激诱导的细胞凋亡(endoplasmic reticulum stress,ERS)产生抵抗,但对于mi RNAs与内质网应激诱导的凋亡抵抗(也称应激性凋亡抵抗)的相互调控机制却知之甚少。我们利用mi RNAs芯片技术,对内质网应激前后的Hep G2细胞进行mi RNAs表达谱分析,结果发现,mi R-663的表达水平发生显著上调。同时,生物信息学分析也表明了mi R-663与内质网应激和许多肿瘤的增殖、凋亡等生物学行为有着一定的相关性。本研究在此基础上,利用q RT-PCR技术进一步验证mi R-663的表达水平在内质网应激后的肝癌细胞系中的是否会上调,同时通过mi R-663模拟物(mi R-663mimics)和mi R-663阻遏物(mi R-663 inhibitors)来转染肝癌Hep G2细胞,观察mi R-663对内质网应激状态下的Hep G2细胞增殖和凋亡的影响,初步探讨mi R-663在肝癌细胞耐受内质网应激诱导的细胞凋亡中的作用。随后我们又应用了靶基因预测分析软件,进一步探索mi R-663影响肝癌细胞凋亡的下游靶基因,为阐明肝癌细胞应激性凋亡抵抗的分子机制带来新的契机,也将为肝癌药物治疗寻找新的作用靶点奠定理论基础。方法:1.以3μM的衣霉素(tunicamycin,TM)处理人肝癌Hep G2细胞株诱导内质网应激,利用mi RNAs芯片技术检测未经TM处理及TM处理后mi RNAs的表达变化,对人肝癌Hep G2细胞株内mi RNAs表达谱分析。2.以3μM的衣霉素(tunicamycin,TM)处理人肝癌细胞株(Hep G2、Be L7402和SMMC7721细胞),诱导内质网应激,利用q RT-PCR技术检测未经TM处理及TM处理24 h后,人肝癌细胞株内mi R-663表达水平的差异。3.应用Lipofectamine RNAi MAX将与mi R-663 mimics,mi R-663 inhibitors转染入Hep G2细胞中。转染24 h后,运用q RT-PCR技术测定转染后mi R-663表达水平的变化。4.应用CCK-8(Cell Counting Kit-8)法检测上调或下调mi R-663的表达水平对内质网应激状态下的Hep G2细胞增殖的影响。5.应用FCM(Flow Cytometry,流式细胞术),Annexin V-FITC/PI双染试剂盒检测下调mi R-663的表达水平对Hep G2细胞凋亡率的影响。6.应用靶基因预测软件分析的mi R-663的靶基因。7.应用qRT-PCR和ELISA技术(酶联免疫吸附试验)检测上调或下调mi R-663的表达对靶基因TGFB1表达水平的影响,内质网应激前后TGFB1表达水平的变化以及转染TGFB1 si RNA后TGFB1表达水平的变化。8.应用FCM(Flow Cytometry,流式细胞术),Annexin V-FITC/PI双染试剂盒检测下调TGFB1的表达水平对Hep G2细胞凋亡率的影响。结果:1.Mi RNAs芯片结果显示,对衣霉素处理前后的70条mi RNAs进行比较分析,mi R-663在衣霉素处理后上调最明显,尤其在衣霉素处理24 h后。2.q RT-PCR结果显示,衣霉素处理24 h后,mi R-663在Hep G2、Be L7402和SMMC7721细胞内表达均上调,其表达水平分别为未处理组的(1.93±0.16)倍、(1.88±0.24)倍和(2.15±0.21)倍。3.qRT-PCR结果显示,转染HepG2细胞24 h后,mi R-663 mimics组细胞内mi R-663的表达水平显著升高,其相对表达量约为阴性对照组的(1128.68±74.16)倍,而mi R-663 inhibitors组细胞内mi R-663的表达水平显著降低,其相对表达量约为阴性对照组的(0.27±0.13)倍。4.CCK-8结果显示,衣霉素及mi R-663 inhibitors联合处理组与衣霉素单独处理组相比,Hep G2细胞的增殖率发生了显著降低;而衣霉素及mi R-663 mimics联合处理组与衣霉素单独处理组相比,Hep G2细胞的增殖率出现了显著上升。5.FCM结果显示,衣霉素及mi R-663 inhibitors联合处理组的Hep G2细胞凋亡率显著高于衣霉素单独处理组。6.靶基因分析预测结果显示,转化生长因子β-1(transforming growth factor beta 1,TGFB1)是mi R-663的靶基因。7.qRT-PCR和ELISA结果显示,转染mi R-663 mimics使mi R-663表达水平升高时,TGFB1在m RNA和蛋白质水平的表达均降低;转染mi R-663 inhibitors使mi R-663表达水平降低时,TGFB1在m RNA和蛋白质水平的表达均升高;衣霉素处理Hep G2细胞24 h后,TGFB1在m RNA和蛋白质水平均发生了显著下调;转染TGFB1 si RNA后TGFB1在m RNA和蛋白质水平均明显下调。8.FCM结果显示,转染TGFB1 si RNA使TGFB1表达下调后,Hep G2细胞凋亡率显著低于对照组。结论:1.肝癌细胞在内质网应激状态下mi R-663表达上调。2.转染mi R-663 mimics和mi R-663 inhibitors能有效地上调和下调mi R-663的表达。3.下调mi R-663的表达能够促进内质网应激状态下肝癌细胞凋亡,抑制细胞增殖。4.肝癌细胞在内质网应激状态下TGFB1表达下调。5.上调mi R-663能抑制TGFB1m RNA和TGFB1蛋白的表达,下调mi R-663能促进TGFB1m RNA和TGFB1蛋白的表达,TGFB1可能是mi R-663的直接靶基因。6.干扰TGFB1 m RNA的表达可抑制肝癌细胞的凋亡。7.Mi R-663可能通过下调TGFB1蛋白的表达,抑制肝癌细胞凋亡。
[Abstract]:Objective: the expression of Micro RNAs (MI RNAs) in most cancers is in disorder, such as primary hepatocellular carcinoma (hepatocellular carcinoma, HCC), but the exact mechanism of the maladjustment remains to be studied. In our previous study, we have found that the hepatoma cells are more susceptible to the endoplasmic reticulum stress induced cell withering compared with normal hepatocytes. Endoplasmic reticulum stress (ERS) produces resistance, but little is known about the mutual regulation mechanism of MI RNAs and endoplasmic reticulum induced apoptosis resistance (also known as stress apoptosis resistance). We use mi RNAs chip technology to carry out mi RNAs expression profiles of Hep G2 cells before and after endoplasmic reticulum stress. At the same time, bioinformatics analysis also showed that MI R-663 and endoplasmic reticulum stress were related to the biological behavior of many tumors and apoptosis. Based on this study, the Q RT-PCR technique was used to further verify the expression of MI R-663 in the liver cancer cell lines after endoplasmic reticulum stress. The effects of MI R-663 analogue (MI R-663mimics) and MI R-663 repressor (MI R-663 inhibitors) on the proliferation and apoptosis of hepatocellular carcinoma cells under endoplasmic reticulum stress were observed by using mi R-663mimics and MI R-663 repressor (MI R-663 inhibitors). Then we used the target gene prediction analysis software to further explore the downstream target gene of MI R-663 to affect the apoptosis of hepatoma cells, which will bring new opportunity to elucidate the molecular mechanism of liver cancer cell stress resistance to apoptosis, and lay a foundation for the search for new therapeutic targets for the treatment of liver cancer. Method: 1. to 3 mu M (tunica) Mycin, TM) treated human liver cancer Hep G2 cells to induce endoplasmic reticulum stress, and used mi RNAs chip technique to detect the expression changes of MI RNAs without TM treatment and TM treatment. Endoplasmic reticulum stress, Q RT-PCR technique was used to detect the difference in the expression level of MI R-663 in human hepatoma cells without TM treatment and TM treatment 24 h.3. application Lipofectamine RNAi MAX will be transfected with MI RNAi MAX. .4. application CCK-8 (Cell Counting Kit-8) method to detect the effect of the expression level of MI R-663 on the proliferation of Hep G2 cells under the stress of endoplasmic reticulum.5. FCM (Flow, flow cytometry). The target gene.7. of MI R-663 analysis by gene prediction software applied qRT-PCR and ELISA Technology (ELISA) to detect the effect of up or down expression of MI R-663 on the level of target gene TGFB1 expression, the change of TGFB1 expression level before and after endoplasmic reticulum stress and the change of the expression level after TGFB1 Si RNA. Ytometry, flow cytometry), Annexin V-FITC/PI double staining kit was used to detect the effect of down regulation of TGFB1 expression on the apoptosis rate of Hep G2 cells. Results: the results of 1.Mi RNAs chip showed that the 70 mi RNAs before and after the treatment of ycomycin were compared and analyzed, MI R-663 was up to the most obvious after being treated with ycomycin, especially in the treatment of ycomycin treatment 24. T-PCR results showed that the expression of MI R-663 increased in Hep G2, Be L7402 and SMMC7721 cells after 24 h treatment, and the expression level was (1.93 + 0.16) times, (1.88 + 0.24) times (1.88 + 0.24 times) and (2.15 + 0.21) times.3.qRT-PCR, respectively. The relative expression was about (1128.68 + 74.16) times of the negative control group, while the expression level of MI R-663 in the MI R-663 inhibitors group decreased significantly, and the relative expression amount was about (0.27 + 0.13) times.4.CCK-8 results in the negative control group, which showed that the combined treatment group of ycomycin and Mi R-663 inhibitors was compared with the single treatment group of ycomycin, Hep G. The proliferation rate of 2 cells decreased significantly, while the proliferation rate of Hep G2 cells increased significantly compared with the single treatment group of ycomycin and MI R-663 mimics, which showed that the Hep G2 cell withering rate of the combined treatment group of ycomycin and MI R-663 inhibitors was significantly higher than that of ycomycin alone treated group.6. target group. The result of analysis and prediction showed that transforming growth factor beta -1 (transforming growth factor beta 1, TGFB1) was the result of.7.qRT-PCR and ELISA of the target gene of MI R-663. The expression of TGFB1 at the level of M RNA and protein increased. After 24 h of Hep G2 cells treated with mycophencin, TGFB1 decreased significantly at the RNA and protein levels of M. The expression of MI R-663 in 1. hepatoma cells under endoplasmic reticulum stress up regulation of.2. transfected mi R-663 mimics and MI R-663 inhibitors can effectively regulate the expression of MI R-663, the expression of.3. down regulation can promote the apoptosis of hepatoma cells under the stress of endoplasmic reticulum and inhibit the proliferation of hepatoma cells The down regulation of TGFB1 expression under the stress of endoplasmic reticulum up regulation of.5. up regulation of MI R-663 can inhibit the expression of TGFB1m RNA and TGFB1 protein, and the downregulation of MI R-663 can promote TGFB1m RNA and the expression of TGFB1 protein. The expression of white inhibits the apoptosis of hepatoma cells.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

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