MiR-377在胰腺癌增殖、凋亡中的作用及机制研究

发布时间：2018-05-27 07:02

本文选题：胰腺癌 + miRNAs　；参考：《第三军医大学》2016年博士论文

【摘要】：一、研究背景胰腺导管腺癌是全球最为致命的恶性肿瘤之一,在恶性肿瘤致死率中排名第4位,其5年生存率仅6%左右。由于胰腺癌具有明显的侵袭性,80%的胰腺癌患者在疾病确诊时就发现了早期的胰腺外转移,使得手术及药物治疗效果很差,导致很高的疾病致死率和极差的预后。究其原因,其一是胰腺导管腺癌的早期诊断存在短板,早期预警缺乏特异性及敏感性较高的分子标记,大多数患者在出现症状时,往往已出现局部侵犯和远处转移,已经失去了最佳的手术治疗时机。其二,胰腺导管腺癌是“高度异质性”疾病,致病基因多变而复杂。因此,更为敏感、特异的早期胰腺癌分子标记亟待发掘,临床需求呼吁更为深入地研究胰腺癌发生、发展的分子机制和调控通路,并可能为开发新的治疗胰腺癌的分子靶点提供思路。MicroRNA是近年来目前研究最为广泛的一种ncRNA,它在编码蛋白基因的转录后水平进行调控。它被证实在肿瘤细胞的发生、发展、增殖、分化、凋亡和侵袭等过程中有重要的调节作用。这些miRNAs具有稳定性、组织特异性、易获得性和高度保守性,可以作为很好的肿瘤疾病诊断标志物,同时这些功能性miRNAs的作用机制还远没有被人类所认识。二、研究目的本研究旨在研究miR-377在胰腺癌中的表达特征及其在胰腺癌细胞增殖和凋亡中的调控作用及其分子机制。三、材料和方法1.本研究组先运用生物信息学方法在GEO胰腺癌miRNAs芯片数据库和TCGA胰腺癌miRNAs表达谱数据库中筛选出差异表达的miRNAs。其中,miR-377在胰腺癌中表达特异性下调,并与临床预后相关。2.扩大临床样本量,通过QT-PCR技术分别检测30例胰腺导管腺癌癌组织/癌旁组织,以及胰腺癌细胞/正常胰腺导管上皮细胞中miR-377的表达水平,明确其表达差异。同时利用统计学方法分析miR-377的表达水平与患者临床参数之间的相关性。3.为了研究miR-377在胰腺癌中的功能,我们构建了异位miR-377过表达和沉默慢病毒载体,建立稳转胰腺癌细胞系以进行更为深入的功能研究。通过CCK-8实验、细胞平板克隆形成实验、Transwell迁移实验、流式细胞技术检测细胞周期、流式细胞技术检测细胞凋亡、TUNEL染色检测细胞凋亡研究miR-377对胰腺癌细胞的增殖、生长活性、迁移能力、细胞周期分布、细胞凋亡的影响。4.通过生物信息学预测工具分析miR-377潜在的靶基因,结果提示PIM-3可能是miR-377的靶控基因。通过QT_PCR、WB检测异位表达miR-377的胰腺癌细胞中PIM-3mRNA水平及蛋白水平。QT_PCR检测胰腺癌组织中miR-377与PIM-3表达量之间的相关性。WB检测胰腺癌组织与胰腺癌细胞中PIM-3蛋白水平的差异。双荧光素酶报告基因系统鉴定miR-377与PIM-3的直接结合活性。5.通过WB技术检测异位表达miR-377后,其靶蛋白PIM-3及下游通路蛋白的改变。6.通过siRNA沉默PIM-3的表达后,检测胰腺癌细胞生物学功能的改变。四、统计学方法本实验数据均用SPSS 23.0软件(Windows)进行统计,并运用Graph Pad Prism 5.0.软件进行统计图的绘制。组间计量资料的比较采用重复测量的单因素方差分析(两两比较时采用配对t检验)。运用Person相关系数分析miR-377与PIM-3 mRNA之间的相关性。Kaplan-Meier生存分析来检测miR-377与临床生存期的关系。PIM-3 mRNA的表达水平与临床参数之间的关系采用χ2检验。计量资料采用均数±标准差来表示,P0.05作为显著统计学差异的标准。五、结果1.公共数据分析结果支持miR-377在胰腺导管上皮腺癌组织中下调表达。并与临床生存预后显著相关。2.miR-377在胰腺导管腺癌癌组织和细胞株中特异性低表达,相对于正常胰腺组织和正常胰腺导管上皮细胞;且miR-377在胰腺癌中的表达水平与肿瘤大小、局部淋巴结有无转移显著相关。3.在胰腺癌细胞中过表达miR-377后可抑制细胞增殖、生长、迁移能力,阻滞胰腺癌细胞周期于G1/S转换,并促进胰腺癌细胞凋亡,减少胰腺癌细胞对于化疗药物的耐药性。4.双荧光素酶报告基因证实了miR-377的靶基因是PIM-3,并通过直接结合于它的3’-UTR区对其起到负向调控作用。QT-PCR的结果显示:miR-377的表达水平与PIM-3mRNA的表达量呈负向相关。WB结果也显示:miR-377是通过负向调控PIM-3蛋白水平,而PIM-3则通过磷酸化Ser112 Bad蛋白,抑制Bad-Bcl-XL抗凋亡通路参与胰腺癌细胞增殖、凋亡的调控。5.应用siRNA沉默PIM-3基因后,胰腺癌细胞的增殖、生长能力、克隆形成能力、迁移能力均得到抑制,并导致细胞周期阻滞、凋亡增强,而使稳定沉默miR-377表达的作用被抵消。六、结论综合实验数据可显示:miR-377在胰腺癌中特异性下调表达,并与临床不良预后相关,其表达水平与肿瘤大小、淋巴结有无转移密切相关;miR-377可抑制胰腺癌细胞增殖、生长及迁移,并引起细胞周期阻滞、凋亡增强、对化疗药物敏感性上升;miR-377通过负向调控靶基因PIM-3,抑制Bad-Bcl-XL抗凋亡通路。因此miR-377显示出一定的抑制肿瘤的特征,miR-377-Pim-3-p BAD112调控通路可能为研究胰腺癌潜在的新治疗靶点提供实验依据。
[Abstract]:First, pancreatic duct adenocarcinoma is one of the most deadly malignant tumors in the world. It ranks fourth in the mortality rate of malignant tumor, and its 5 year survival rate is only about 6%. Because of the obvious invasiveness of pancreatic cancer, 80% of the patients with pancreatic cancer found early extrapancreatic metastases at the time of diagnosis, making the surgical and drug treatment effect very effective. The reason is that the early diagnosis of pancreatic duct adenocarcinoma is short plate, early warning is short of specific and sensitive molecular markers. Most patients often have local invasion and distant metastasis when they have symptoms, and they have lost the best surgical treatment. Secondly, pancreatic ductal adenocarcinoma is a "highly heterogeneous" disease, and the pathogenic gene is changeable and complex. Therefore, it is more sensitive, specific early molecular markers of pancreatic cancer need to be unearthed urgently. Clinical requirements call for more in-depth study of the pathogenesis of pancreatic cancer, the molecular mechanisms and regulatory pathways of development, and the possibility of developing new molecules for the treatment of pancreatic cancer. .MicroRNA is the most widely studied ncRNA in recent years, which regulates the post transcriptional level of the encoded protein gene. It has been proved to play an important role in the pathogenesis, development, proliferation, differentiation, apoptosis and invasion of the tumor cells. These miRNAs are stable, tissue specific and easy to obtain. And highly conserved, it can be used as a good marker for diagnosis of tumor disease, and the functional mechanisms of these functional miRNAs are still far from being recognized by humans. Two. The purpose of this study was to study the expression of miR-377 in pancreatic cancer and its regulatory role in the proliferation and apoptosis of pancreatic cancer cells and its molecular mechanism. Three Materials and methods 1. study group first used bioinformatics methods to select the differentially expressed miRNAs. in the miRNAs chip database of GEO pancreatic cancer and the miRNAs expression database of TCGA pancreatic cancer. The specific down-regulation of miR-377 in pancreatic cancer was expressed, and the clinical samples were enlarged by.2. in the clinical prognosis, and 30 were detected by QT-PCR technology respectively. The expression level of miR-377 in pancreatic ductal carcinoma tissue / paracancerous tissue and pancreatic carcinoma cell / normal pancreatic duct epithelial cells, and the correlation between the expression level of miR-377 and the clinical parameters of the patients was analyzed by statistical methods.3. in order to study the function of miR-377 in pancreatic cancer, we construct it. The ectopic miR-377 overexpression and the silencing of the lentivirus vector to establish a stable pancreatic cancer cell line for further functional study. Through the CCK-8 experiment, the cell plate clone formation experiment, the Transwell migration experiment, the flow cytometry test cell cycle, the flow cytometry to detect the cell apoptosis, and the TUNEL staining to detect the apoptosis of the cells, MI The effects of R-377 on the proliferation, growth activity, migration, cell cycle distribution and apoptosis of pancreatic cancer cells.4. can be used to analyze the potential target genes of miR-377 through bioinformatics prediction tools. The results suggest that PIM-3 may be the target gene of miR-377. WB detection of PIM-3mRNA levels and eggs in pancreatic cancer cells with ectopic expression of miR-377 through QT_PCR Correlation between miR-377 and PIM-3 expression in pancreatic cancer tissues by white level.QT_PCR.WB detection of PIM-3 protein levels in pancreatic cancer tissues and pancreatic cancer cells. The double luciferase reporter gene system identified the direct binding activity of miR-377 and PIM-3 by the WB technique to detect the expression of miR-377 by WB technique, and the target protein was PIM-3 and lower. The changes in the pathway protein.6. were used to detect the changes in the biological function of pancreatic cancer cells after siRNA silencing the expression of PIM-3. Four, the statistical methods were statistically used by the SPSS 23 software (Windows), and the Graph Pad Prism 5.0. software was used to draw the statistical map. The comparison of the data between groups was compared with the single cause of the repeated measurement. Analysis of prime variance (22 compared with paired t test). Person correlation coefficient was used to analyze the correlation between miR-377 and PIM-3 mRNA to determine the relationship between miR-377 and the clinical survival time. The relationship between the expression level of.PIM-3 mRNA and the clinical parameters was tested by the chi 2 test. The results indicated that P0.05 was a significant statistical difference. Five, results 1. the results of public data analysis supported the downregulation of miR-377 in pancreatic ductal epithelial adenocarcinoma. It was significantly related to the survival prognosis of.2.miR-377 in the specific low expression of.2.miR-377 in pancreatic ductal adenocarcinoma tissue and cell lines, relative to normal pancreatic tissue and normal pancreas. The expression level of miR-377 in pancreatic cancer is significantly related to the size of the tumor and the size of the tumor and the metastasis of the local lymph nodes..3. can inhibit the proliferation, growth and migration of miR-377 in pancreatic cancer cells, block the cell cycle of pancreatic cancer in G1/S conversion, and promote the apoptosis of pancreatic cancer cells and reduce the pancreatic cancer cells. The drug resistance.4. double luciferase reporter gene confirmed that the target gene of miR-377 is PIM-3, and the result of the negative regulation of.QT-PCR through its 3 '-UTR region shows that the expression level of miR-377 and the expression of PIM-3mRNA are negatively related.WB results also show that miR-377 is through the negative regulation of PIM-3. Protein level, and PIM-3 by phosphorylated Ser112 Bad protein, inhibit Bad-Bcl-XL anti apoptosis pathway to participate in the proliferation of pancreatic cancer cells. Apoptosis regulation,.5. application siRNA silent PIM-3 gene, pancreatic cancer cell proliferation, growth, clone formation ability, migration ability to inhibit, and lead to cell cycle block, apoptosis enhancement, and cause The effect of stable silence miR-377 expression was offset. Six, conclusion the comprehensive experimental data can show that miR-377 is specific down expression in pancreatic cancer and is related to poor clinical prognosis. The expression level is closely related to the size of the tumor and the metastasis of lymph nodes. MiR-377 can inhibit the proliferation, growth and migration of pancreatic adenocarcinoma cells and cause cell cycle. Blocking, increasing apoptosis and increasing sensitivity to chemotherapeutic drugs; miR-377 through the negative regulation of target gene PIM-3, inhibiting the anti apoptosis pathway of Bad-Bcl-XL. Therefore, miR-377 shows certain characteristics of tumor inhibition, and miR-377-Pim-3-p BAD112 regulation pathway may provide experimental basis for the study of potential new therapeutic targets for pancreatic cancer.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.9

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