本文選題：食管癌 + miR-25-3p　； 參考：《石河子大學(xué)》2017年碩士論文

【摘要】：目的:根據(jù)課題組前期的研究,我們通過(guò)細(xì)胞學(xué)實(shí)驗(yàn)證明提高miR-25-3p在食管鱗癌(esophageal squamous cell carcinoma,ESCC)細(xì)胞系中的表達(dá)量后,細(xì)胞侵襲和增殖能力增強(qiáng),說(shuō)明miR-25-3p在ESCC的發(fā)生發(fā)展中可能具有重要的功能性調(diào)控作用。本研究希望通過(guò)生物信息學(xué)等技術(shù)篩選驗(yàn)證miR-25-3p調(diào)控機(jī)制,為進(jìn)一步了解miR-25-3p在食管癌中的作用提供理論基礎(chǔ)。方法:第一部分:在GEO數(shù)據(jù)庫(kù),搜索框輸入檢索關(guān)鍵詞"esophageal squamous cell carcinoma",從檢索結(jié)果中進(jìn)行數(shù)據(jù)挖掘。將數(shù)據(jù)導(dǎo)入基于R語(yǔ)言設(shè)計(jì)的GEO2R芯片數(shù)據(jù)分析工具,得到ESCC細(xì)胞中差異性表達(dá)的m RNA集合;另一方面,使用Target Scan7.0靶基因預(yù)測(cè)軟件預(yù)測(cè)miR-25-3p所有可能的具有高度保守性的靶基因列表,將上述得到的兩列基因取交集,得到具有ESCC組織特異性高的miR-25-3p可能靶基因。將靶基因上傳至DAVID基因功能注釋軟件進(jìn)行富集分析,細(xì)胞功能富集選擇GO數(shù)據(jù)庫(kù),通路富集選擇KEGG數(shù)據(jù)庫(kù)。通過(guò)文獻(xiàn)及結(jié)果分析篩選所得靶基因。第二部分:脂質(zhì)體包裹合成miRNA轉(zhuǎn)染ESCC細(xì)胞系EC109,分為三組(1.增強(qiáng)組(miR-25-3p表達(dá)量增加),轉(zhuǎn)染miR-25-3p agomir;2.抑制組(miR-25-3p表達(dá)量降低),轉(zhuǎn)染miR-25-3p antagomir;3.對(duì)照組,轉(zhuǎn)染空白序列)。熒光實(shí)時(shí)定量PCR(q RT-PCR)技術(shù)檢測(cè)三組細(xì)胞NOTCH1表達(dá)量的變化。熒光素酶報(bào)告基因檢測(cè)系統(tǒng)驗(yàn)證miR-25-3p與NOTCH1之間在3′非編碼區(qū)(Untranslated Regions,UTR)的靶向位點(diǎn)。第三部分(miR-25-3p靶向NOTCH1對(duì)細(xì)胞增殖與侵襲功能的作用研究)課題組前期已經(jīng)對(duì)miR-25-3p對(duì)EC109細(xì)胞增殖和侵襲能力的研究進(jìn)行了研究,本次通過(guò)轉(zhuǎn)染NOTCH1 si RNA沉默NOTCH1,模擬miR-25-3p調(diào)控NOTCH1,觀察細(xì)胞功能,分為兩組(1.沉默組(抑制NOTCH1基因表達(dá)),轉(zhuǎn)染NOTCH1 si RNA;2.對(duì)照組,轉(zhuǎn)染si-scrambled)。Transwell侵襲實(shí)驗(yàn)檢測(cè)轉(zhuǎn)染后細(xì)胞侵襲能力,CCK-8法檢測(cè)細(xì)胞轉(zhuǎn)染后細(xì)胞增殖能力。結(jié)果:通過(guò)Target Scan 7.0預(yù)測(cè)與miR-25-3p有高度保守結(jié)合位點(diǎn)的靶基因,共1037個(gè)。GEO2R中設(shè)置組別,KYSE30和KYSE180高移動(dòng)能力細(xì)胞亞系設(shè)置為KYSE D組,親本細(xì)胞設(shè)置為KYSE U組。通過(guò)GEO2R的一致性檢驗(yàn),8個(gè)樣本間一致性較好,得到差異基因24491個(gè),去掉miRNA及未錄入Gene symbol數(shù)據(jù)庫(kù)的基因ID,最終得到19491個(gè)差異基因。與上述預(yù)測(cè)的靶基因取交集,得到miR-25-3p可能在ESCC中參與調(diào)控的基因623個(gè)。KEGG篩選條件:P值0.05且基因數(shù)2個(gè),基因主要富集于Fox O、c AMP、細(xì)胞骨架等信號(hào)通路;GO篩選條件:P值0.05且基因數(shù)2個(gè),GO中與侵襲、增殖相關(guān)基因NOTCH1、PTEN、TGFB2等。結(jié)合既往文獻(xiàn)、生物信息學(xué)功能分析,以及miR-25-3p與靶基因之間的負(fù)性調(diào)控機(jī)制,選擇NOTCH1作為miR-25-3p的靶基因進(jìn)行驗(yàn)證及細(xì)胞功能性實(shí)驗(yàn)。轉(zhuǎn)染miR-25-3p后q RT-PCR檢測(cè)結(jié)果顯示增強(qiáng)組miR-25-3p的表達(dá)水平明顯高于對(duì)照組(p0.05),抑制劑組miR-25-3p的表達(dá)明顯低于對(duì)照組(p0.05),miR-25-3p在ESCC細(xì)胞系EC109中的高表達(dá)及低表達(dá)細(xì)胞模型構(gòu)建成功。q RT-PCR檢測(cè)轉(zhuǎn)染后NOTCH1在各組的表達(dá)情況,增強(qiáng)組NOTCH1表達(dá)較對(duì)照組顯著降低(p0.05),抑制組NOTCH1表達(dá)較對(duì)照組升高(p0.05)。熒光素酶報(bào)告基因檢測(cè)系統(tǒng)顯示miR-25-3p靶向NOTCH1 3′UTR互補(bǔ)位點(diǎn),是熒光素酶活性較對(duì)照組明顯降低(p0.05),說(shuō)明miR-25-3p與NOTCH1之間具有靶向調(diào)控關(guān)系。EC109細(xì)胞轉(zhuǎn)染si-NOTCH1后,沉默組NOTCH1表達(dá)量較對(duì)照組明顯降低,構(gòu)建NOTCH1沉默細(xì)胞模型成功,Transwell侵襲實(shí)驗(yàn)結(jié)果顯示沉默組細(xì)胞侵襲能力較對(duì)照組明顯增強(qiáng)(p0.05),CCK-8增殖實(shí)驗(yàn)結(jié)果顯示沉默組細(xì)胞增殖能力較對(duì)照組明顯增強(qiáng),與miR-25-3p高表達(dá)細(xì)胞模型細(xì)胞功能學(xué)結(jié)果一致。結(jié)論:miR-25-3p可以通過(guò)靶向NOTCH1影響ESCC細(xì)胞系EC109的增殖和侵襲能力。
[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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 李武;賀慶芝;孫詩(shī)博;潘小元;曾懷才;;has-miR-16靶基因預(yù)測(cè)及生物信息學(xué)分析[J];中華疾病控制雜志;2016年02期

2 代航;康冰;左的于;左國(guó)慶;;MicroRNA-30a-5p對(duì)肝癌細(xì)胞株SMMC-7721增殖凋亡及侵襲轉(zhuǎn)移的影響[J];中華肝臟病雜志;2014年12期

3 Fa-Jun Xie;Yi-Ping Zhang;Qiu-Qing Zheng;Hong-Chuan Jin;Fa-Liang Wang;Ming Chen;Lan Shao;De-Hong Zou;Xin-Min Yu;Wei-Min Mao;;Helicobacter pylori infection and esophageal cancer risk:An updated meta-analysis[J];World Journal of Gastroenterology;2013年36期

4 董明明;雋兆東;蘇俊武;管英俊;于麗;冷召廷;朱鳳祥;;Notch4表達(dá)與胸中段食管鱗癌侵襲、轉(zhuǎn)移及預(yù)后的關(guān)系[J];中華胸心血管外科雜志;2013年08期

5 朱龍萍;金麗艷;蔣日月;汪茜茜;蔣健;毛朝明;陳德玉;;食管鱗癌腫瘤微環(huán)境中miRNA與TGF-β1的相關(guān)性分析[J];細(xì)胞與分子免疫學(xué)雜志;2013年05期

6 ;Circulating microRNAs: Novel biomarkers for esophageal cancer[J];World Journal of Gastroenterology;2010年19期

7 張永利;張可杰;閔祥輝;鹿全意;劉文勵(lì);;Notch1通路活化抑制EC109細(xì)胞的增殖及機(jī)制探討[J];中國(guó)癌癥雜志;2009年08期

，

本文編號(hào)：2022718