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

【摘要】：目的:根據(jù)課題組前期的研究,我們通過細胞學實驗證明提高miR-25-3p在食管鱗癌(esophageal squamous cell carcinoma,ESCC)細胞系中的表達量后,細胞侵襲和增殖能力增強,說明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。本研究希望通過生物信息學等技術(shù)篩選驗證miR-25-3p調(diào)控機制,為進一步了解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,搜索框輸入檢索關(guān)鍵詞"esophageal squamous cell carcinoma",從檢索結(jié)果中進行數(shù)據(jù)挖掘。將數(shù)據(jù)導入基于R語言設(shè)計的GEO2R芯片數(shù)據(jù)分析工具,得到ESCC細胞中差異性表達的m RNA集合;另一方面,使用Target Scan7.0靶基因預(yù)測軟件預(yù)測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行富集分析,細胞功能富集選擇GO數(shù)據(jù)庫,通路富集選擇KEGG數(shù)據(jù)庫。通過文獻及結(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細胞系EC109,分為三組(1.增強組(miR-25-3p表達量增加),轉(zhuǎn)染miR-25-3p agomir;2.抑制組(miR-25-3p表達量降低),轉(zhuǎn)染miR-25-3p antagomir;3.對照組,轉(zhuǎn)染空白序列)。熒光實時定量PCR(q RT-PCR)技術(shù)檢測三組細胞NOTCH1表達量的變化。熒光素酶報告基因檢測系統(tǒng)驗證miR-25-3p與NOTCH1之間在3′非編碼區(qū)(Untranslated Regions,UTR)的靶向位點。第三部分(miR-25-3p靶向NOTCH1對細胞增殖與侵襲功能的作用研究)課題組前期已經(jīng)對miR-25-3p對EC109細胞增殖和侵襲能力的研究進行了研究,本次通過轉(zhuǎn)染NOTCH1 si RNA沉默NOTCH1,模擬miR-25-3p調(diào)控NOTCH1,觀察細胞功能,分為兩組(1.沉默組(抑制NOTCH1基因表達),轉(zhuǎn)染NOTCH1 si RNA;2.對照組,轉(zhuǎn)染si-scrambled)。Transwell侵襲實驗檢測轉(zhuǎn)染后細胞侵襲能力,CCK-8法檢測細胞轉(zhuǎn)染后細胞增殖能力。結(jié)果:通過Target Scan 7.0預(yù)測與miR-25-3p有高度保守結(jié)合位點的靶基因,共1037個。GEO2R中設(shè)置組別,KYSE30和KYSE180高移動能力細胞亞系設(shè)置為KYSE D組,親本細胞設(shè)置為KYSE U組。通過GEO2R的一致性檢驗,8個樣本間一致性較好,得到差異基因24491個,去掉miRNA及未錄入Gene symbol數(shù)據(jù)庫的基因ID,最終得到19491個差異基因。與上述預(yù)測的靶基因取交集,得到miR-25-3p可能在ESCC中參與調(diào)控的基因623個。KEGG篩選條件:P值0.05且基因數(shù)2個,基因主要富集于Fox O、c AMP、細胞骨架等信號通路;GO篩選條件:P值0.05且基因數(shù)2個,GO中與侵襲、增殖相關(guān)基因NOTCH1、PTEN、TGFB2等。結(jié)合既往文獻、生物信息學功能分析,以及miR-25-3p與靶基因之間的負性調(diào)控機制,選擇NOTCH1作為miR-25-3p的靶基因進行驗證及細胞功能性實驗。轉(zhuǎn)染miR-25-3p后q RT-PCR檢測結(jié)果顯示增強組miR-25-3p的表達水平明顯高于對照組(p0.05),抑制劑組miR-25-3p的表達明顯低于對照組(p0.05),miR-25-3p在ESCC細胞系EC109中的高表達及低表達細胞模型構(gòu)建成功。q RT-PCR檢測轉(zhuǎn)染后NOTCH1在各組的表達情況,增強組NOTCH1表達較對照組顯著降低(p0.05),抑制組NOTCH1表達較對照組升高(p0.05)。熒光素酶報告基因檢測系統(tǒng)顯示miR-25-3p靶向NOTCH1 3′UTR互補位點,是熒光素酶活性較對照組明顯降低(p0.05),說明miR-25-3p與NOTCH1之間具有靶向調(diào)控關(guān)系。EC109細胞轉(zhuǎn)染si-NOTCH1后,沉默組NOTCH1表達量較對照組明顯降低,構(gòu)建NOTCH1沉默細胞模型成功,Transwell侵襲實驗結(jié)果顯示沉默組細胞侵襲能力較對照組明顯增強(p0.05),CCK-8增殖實驗結(jié)果顯示沉默組細胞增殖能力較對照組明顯增強,與miR-25-3p高表達細胞模型細胞功能學結(jié)果一致。結(jié)論:miR-25-3p可以通過靶向NOTCH1影響ESCC細胞系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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.1

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