miR-25-3p靶向NOTCH1对EC109细胞侵袭和增殖的影响

发布时间：2018-06-15 16:43

本文选题：食管癌 + miR-25-3p　；参考：《石河子大学》2017年硕士论文

【摘要】：目的:根据课题组前期的研究,我们通过细胞学实验证明提高miR-25-3p在食管鳞癌(esophageal squamous cell carcinoma,ESCC)细胞系中的表达量后,细胞侵袭和增殖能力增强,说明miR-25-3p在ESCC的发生发展中可能具有重要的功能性调控作用。本研究希望通过生物信息学等技术筛选验证miR-25-3p调控机制,为进一步了解miR-25-3p在食管癌中的作用提供理论基础。方法:第一部分:在GEO数据库,搜索框输入检索关键词"esophageal squamous cell carcinoma",从检索结果中进行数据挖掘。将数据导入基于R语言设计的GEO2R芯片数据分析工具,得到ESCC细胞中差异性表达的m RNA集合;另一方面,使用Target Scan7.0靶基因预测软件预测miR-25-3p所有可能的具有高度保守性的靶基因列表,将上述得到的两列基因取交集,得到具有ESCC组织特异性高的miR-25-3p可能靶基因。将靶基因上传至DAVID基因功能注释软件进行富集分析,细胞功能富集选择GO数据库,通路富集选择KEGG数据库。通过文献及结果分析筛选所得靶基因。第二部分:脂质体包裹合成miRNA转染ESCC细胞系EC109,分为三组(1.增强组(miR-25-3p表达量增加),转染miR-25-3p agomir;2.抑制组(miR-25-3p表达量降低),转染miR-25-3p antagomir;3.对照组,转染空白序列)。荧光实时定量PCR(q RT-PCR)技术检测三组细胞NOTCH1表达量的变化。荧光素酶报告基因检测系统验证miR-25-3p与NOTCH1之间在3′非编码区(Untranslated Regions,UTR)的靶向位点。第三部分(miR-25-3p靶向NOTCH1对细胞增殖与侵袭功能的作用研究)课题组前期已经对miR-25-3p对EC109细胞增殖和侵袭能力的研究进行了研究,本次通过转染NOTCH1 si RNA沉默NOTCH1,模拟miR-25-3p调控NOTCH1,观察细胞功能,分为两组(1.沉默组(抑制NOTCH1基因表达),转染NOTCH1 si RNA;2.对照组,转染si-scrambled)。Transwell侵袭实验检测转染后细胞侵袭能力,CCK-8法检测细胞转染后细胞增殖能力。结果:通过Target Scan 7.0预测与miR-25-3p有高度保守结合位点的靶基因,共1037个。GEO2R中设置组别,KYSE30和KYSE180高移动能力细胞亚系设置为KYSE D组,亲本细胞设置为KYSE U组。通过GEO2R的一致性检验,8个样本间一致性较好,得到差异基因24491个,去掉miRNA及未录入Gene symbol数据库的基因ID,最终得到19491个差异基因。与上述预测的靶基因取交集,得到miR-25-3p可能在ESCC中参与调控的基因623个。KEGG筛选条件:P值0.05且基因数2个,基因主要富集于Fox O、c AMP、细胞骨架等信号通路;GO筛选条件:P值0.05且基因数2个,GO中与侵袭、增殖相关基因NOTCH1、PTEN、TGFB2等。结合既往文献、生物信息学功能分析,以及miR-25-3p与靶基因之间的负性调控机制,选择NOTCH1作为miR-25-3p的靶基因进行验证及细胞功能性实验。转染miR-25-3p后q RT-PCR检测结果显示增强组miR-25-3p的表达水平明显高于对照组(p0.05),抑制剂组miR-25-3p的表达明显低于对照组(p0.05),miR-25-3p在ESCC细胞系EC109中的高表达及低表达细胞模型构建成功。q RT-PCR检测转染后NOTCH1在各组的表达情况,增强组NOTCH1表达较对照组显著降低(p0.05),抑制组NOTCH1表达较对照组升高(p0.05)。荧光素酶报告基因检测系统显示miR-25-3p靶向NOTCH1 3′UTR互补位点,是荧光素酶活性较对照组明显降低(p0.05),说明miR-25-3p与NOTCH1之间具有靶向调控关系。EC109细胞转染si-NOTCH1后,沉默组NOTCH1表达量较对照组明显降低,构建NOTCH1沉默细胞模型成功,Transwell侵袭实验结果显示沉默组细胞侵袭能力较对照组明显增强(p0.05),CCK-8增殖实验结果显示沉默组细胞增殖能力较对照组明显增强,与miR-25-3p高表达细胞模型细胞功能学结果一致。结论:miR-25-3p可以通过靶向NOTCH1影响ESCC细胞系EC109的增殖和侵袭能力。
[Abstract]:Objective: according to the previous study of the group, we have proved that the enhancement of cell invasion and proliferation ability of miR-25-3p in esophageal squamous cell carcinoma (ESCC) cell line has been proved by cytological experiments. This shows that miR-25-3p may have important functional regulation in the development of ESCC. The miR-25-3p regulation mechanism is screened by bioinformatics and other techniques to provide a theoretical basis for further understanding the role of miR-25-3p in esophageal cancer. Method: the first part: in the GEO database, the search box is used to retrieve the keyword "esophageal squamous cell carcinoma", and the data mining is carried out from the retrieval results. The data is introduced into R based on R. The GEO2R chip data analysis tool for language design is used to obtain the m RNA set of differential expression in ESCC cells. On the other hand, the Target Scan7.0 target gene prediction software is used to predict all possible highly conserved target gene list of miR-25-3p, and the above two columns are intersected to obtain miR- with high ESCC tissue specificity. 25-3p may be the target gene. The target gene was uploaded to the DAVID gene functional annotation software for enrichment analysis, cell function enrichment selected the GO database, and the pathway was enriched to select the KEGG database. The target gene was screened by literature and result analysis. The second part: liposome encapsulated and synthesized miRNA transfer ESCC cell line EC109, divided into three groups (1. groups (miR). -25-3p expression increased), transfection of miR-25-3p agomir, 2. inhibition group (miR-25-3p expression reduction), transfection of miR-25-3p antagomir, 3. control group, transfected blank sequence. Fluorescence real-time quantitative PCR (Q RT-PCR) technique was used to detect the changes in the expression of NOTCH1 in three groups of cells. The fluorescein reporter gene detection system verified that miR-25-3p and NOTCH1 were in 3 ' The target loci of the coding region (Untranslated Regions, UTR). The third part (Research on the effect of miR-25-3p targeting NOTCH1 on cell proliferation and invasion) research group has studied the proliferation and invasion ability of miR-25-3p to EC109 cells in the earlier period. This time the NOTCH1 Si RNA was transfected to the NOTCH1 NOTCH1, analog miR-25-3p regulation and regulation, The cell function was divided into two groups (1. silent groups (inhibition of NOTCH1 gene expression), transfection of NOTCH1 Si RNA, 2. control group, si-scrambled.Transwell invasion test to detect the invasion ability of the transfected cells, and CCK-8 method to detect cell proliferation after transfection. Results: a highly conservative binding site was predicted by Target Scan 7 and miR-25-3p. The target gene was set in a total of 1037.GEO2R groups, and the sublines of KYSE30 and KYSE180 high mobility cells were set to KYSE D group, and the parent cells were set to KYSE U group. Through the consistency test of GEO2R, the consistency of the 8 samples was good, and 24491 different genes were obtained. The gene ID was removed from miRNA and the Gene symbol database was removed. Finally, 19491 differences were obtained. Allogeneic. With the target gene predicted above, 623.KEGG screening conditions for genes involved in the regulation of ESCC were obtained: the P value was 0.05 and the number of genes was 2, and the genes were mainly enriched in Fox O, C AMP, cytoskeleton and so on; GO screening conditions: P value 0.05 and the number of genes 2, GO and invasion, proliferation related genes NOTCH1 On the basis of previous literature, functional analysis of bioinformatics, and the negative regulatory mechanism between miR-25-3p and target genes, NOTCH1 was selected as the target gene for miR-25-3p and cell functional experiments. After transfection of miR-25-3p, Q RT-PCR detection results showed that the expression level of miR-25-3p in the enhanced group was significantly higher than that of the control group (P0.05), and the inhibitors were significantly higher than those of the control group (P0.05). The expression of miR-25-3p was significantly lower than that of the control group (P0.05). The high expression of miR-25-3p in the ESCC cell line EC109 and the construction of the low expression cell model were successfully constructed by.Q RT-PCR to detect the expression of NOTCH1 in each group. The expression of NOTCH1 in the enhanced group was significantly lower than that in the control group (P0.05), and the NOTCH1 expression in the inhibition group was higher than that of the control group (P0.05). The reporter gene detection system showed that miR-25-3p target NOTCH1 3 'UTR complementation site, the luciferase activity was significantly lower than the control group (P0.05), indicating the relationship between miR-25-3p and NOTCH1 with the targeting regulation of.EC109 cells transfected with si-NOTCH1, the expression of NOTCH1 in the silent group was significantly lower than that in the control group, and the construction of NOTCH1 silencing cell model was successful, Tra The invasion test of nswell showed that the cell invasiveness of the silent group was significantly enhanced (P0.05). The CCK-8 proliferation test showed that the cell proliferation ability of the silent group was significantly enhanced than the control group, and it was consistent with the functional results of the miR-25-3p high expression cell model cells. Conclusion: miR-25-3p can affect the ESCC cell line EC109 by targeting NOTCH1. Proliferation and invasiveness.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

【参考文献】