本文選題：神經(jīng)膠質(zhì)瘤　切入點：miRNA-451　出處：《天津醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:研究過表達(dá)mi RNA-451后調(diào)控人腦LN229和U251膠質(zhì)瘤細(xì)胞系上皮-間質(zhì)轉(zhuǎn)化的機制,以及過表達(dá)mi RNA-451后對前述細(xì)胞株的生物學(xué)行為的影響(侵襲、遷移、增殖等)。方法:第一部分:尋找并驗證mi RNA-451對人腦神經(jīng)膠質(zhì)瘤細(xì)胞系的上皮-間質(zhì)轉(zhuǎn)化作用及其影響路徑。具體方案是:選取LN229和U251人腦膠質(zhì)瘤細(xì)胞系進行培養(yǎng),Lipofectamine 2000轉(zhuǎn)染試劑進行轉(zhuǎn)染構(gòu)建高表達(dá)mi RNA-451細(xì)胞株。實驗分為空白對照組不進行任何處理;無義序列組:轉(zhuǎn)染寡核苷酸無意義序列為5-ACGUGACACGUUCGGAGAATT-3,5-UUCUCCGAACGUCACGUTT-3;和mi RNA-451處理組,轉(zhuǎn)染mi RNA-451擬似物(mi RNA-451mimics)序列為5-AAACCGUACCAUUACUGAGUU-3,3-CUCAGUAAUGGUAACGGUUUU-5,(簡稱mi RNA-451);實時定量聚合酶鏈反應(yīng)(RT-PCR)的研究能夠?qū)ι窠?jīng)膠質(zhì)瘤細(xì)胞系各組細(xì)胞中的轉(zhuǎn)染后mi RNA-451的表達(dá)水平進行清晰準(zhǔn)確的衡量,Western Blot實驗驗證E鈣黏素(E-cadherin),N鈣黏素(N-cadherin),波形蛋白(Vimentin),Twist,Snail的蛋白相對表達(dá)量;通過免疫熒光實驗進而比較分析mi RNA-451處理組與空白對照組的N-cadherin、Vimentin的蛋白表達(dá)量;計算機蛋白交聯(lián)網(wǎng)絡(luò)軟件以及前期研究成果構(gòu)建mi RNA-451調(diào)控EMT發(fā)生相關(guān)蛋白的信號通路;Western Blot實驗驗證mi RNA-451處理組中mi RNA-451調(diào)控細(xì)胞EMT發(fā)生信號通路中相關(guān)目的蛋白(CAB39、AMPK、PI3K、AKT、磷酸化AKT)的相對蛋白表達(dá)量。第二部分:分析mi RNA-451對人腦神經(jīng)神經(jīng)膠質(zhì)瘤細(xì)胞系生物行為的影響。按照第一部分的方法轉(zhuǎn)染并分組,定量聚合酶鏈反應(yīng)(RT-PCR)研究結(jié)果證明神經(jīng)膠質(zhì)瘤細(xì)胞系各組細(xì)胞中的mi RNA-451轉(zhuǎn)染后的表達(dá)水平;Transwell實驗對高表達(dá)mi RNA-451后抑制膠質(zhì)瘤細(xì)胞株的侵襲能力做出證實;細(xì)胞劃痕實驗對高表達(dá)mi RNA-451后膠質(zhì)瘤細(xì)胞株遷移能力做出有效檢測;使用細(xì)胞計數(shù)盒(CCK-8)和單克隆實驗驗證高表達(dá)mi RNA-451后膠質(zhì)瘤細(xì)胞株的增殖能力;Western blot實驗?zāi)軌驅(qū)ι窠?jīng)膠質(zhì)瘤細(xì)胞株生物學(xué)行為相關(guān)目的蛋白表達(dá)量做出蛋白層面的驗證。結(jié)果:第一部分結(jié)果顯示:在和空白對照組和無義序列組的分析對比中,mi RNA-451處理組的該基因表達(dá)量有顯著的增加;Western Blot表明mi RNA-451處理組中的E-cadherin表達(dá)量顯著上升,Twist、Snail、N-cadherin、Vimentin的蛋白表達(dá)量顯著減少;差異都具有統(tǒng)計學(xué)意義(P0.05);在免疫熒光實驗中,所取得實驗結(jié)果證實在mi RNA-451處理組中N-cadherin、Vimentin的蛋白表達(dá)量比空白對照組減少明顯,且結(jié)果具有顯著性(P0.05),與Western Blot實驗發(fā)現(xiàn)相同;在具體的信號通路圖中可以CAB39基因能夠通過AMPK,PI3K/AKT通路對EMT相關(guān)蛋白的表達(dá)產(chǎn)生影響,進而對癌細(xì)胞的行為的產(chǎn)生作用,我們前期研究已經(jīng)證實CAB39是mi RNA-451的靶標(biāo)基因;Western blot實驗結(jié)果證明mi RNA-451處理組mi RNA-451調(diào)控EMT相關(guān)蛋白信號通路中相關(guān)目的蛋白CAB39、AMPK、PI3K、磷酸化AKT等的表達(dá),且實驗結(jié)果發(fā)現(xiàn)表達(dá)量均減少了,并均有統(tǒng)計學(xué)意義(P0.05)。第二部分結(jié)果統(tǒng)計發(fā)現(xiàn):與空白對照組和無義序列相比,RT-PCR實驗有效說明了mi RNA-451處理組中其表達(dá)量的上升顯著;Transwell實驗有效說明了mi RNA-451處理組細(xì)胞侵襲能力的下降顯著;細(xì)胞劃痕實驗對mi RNA-451處理組細(xì)胞遷移能力的抑制減弱結(jié)果做出了充分有效說明;細(xì)胞計數(shù)盒(CCK-8)和單克隆實驗有效說明了mi RNA-451處理組的神經(jīng)膠質(zhì)瘤細(xì)胞殖能力被減弱;Western blot實驗對mi RNA-451組神經(jīng)膠質(zhì)瘤細(xì)胞生物學(xué)行為相關(guān)的蛋白Cyclin D1、MMP-2、MMP-9的表達(dá)量的顯著減少做出了有效充分說明,且實驗統(tǒng)計結(jié)果顯著性水平較高(P0.05)。結(jié)論:通過上述實驗及實驗結(jié)果統(tǒng)計分析,可以發(fā)現(xiàn)mi RNA-451靶向的CAB39能夠通過AMPK、PI3K/AKT通路對膠質(zhì)瘤細(xì)胞系中上皮-間質(zhì)轉(zhuǎn)化的發(fā)生產(chǎn)生較為顯著的抑制作用;并在其基礎(chǔ)上對神經(jīng)膠質(zhì)瘤細(xì)胞株的侵襲和遷移能力產(chǎn)生明顯的限制;并且可以使其克隆增殖能力受限。
[Abstract]:Objective: To study the expression of MI RNA-451 and U251 LN229 after the regulation of human brain glioma cell lines of epithelial mesenchymal transition mechanism, and the effect of overexpression of MI RNA-451 on the cell biological behavior (invasion, migration, proliferation, etc.). Methods: the first part: to find and verify mi RNA-451 on human glioma tumor cells of epithelial mesenchymal transition and its influence path. Specific programs are: LN229 and U251 were cultured from human glioma cell line, Lipofectamine 2000 transfection reagent were transfected with MI expressing RNA-451 cells were divided into blank control group without any treatment; nonsense sequence group: nonsense oligonucleotide transfection sequence 5-ACGUGACACGUUCGGAGAATT-3,5-UUCUCCGAACGUCACGUTT-3; and MI RNA-451 treatment group, Mi transfection of RNA-451 analogues (MI RNA-451mimics) sequence for 5-AAACCGUACCAUUACUGAGUU-3,3-CUCAGUAAUGGUAACGG UUUU-5 (MI RNA-451); real time quantitative polymerase chain reaction (RT-PCR) to study the expression level of MI RNA-451 of transfected glioma cell line cells were measured in the clear, Western Blot experimental verification of E cadherin (E-cadherin), N cadherin (N-cadherin), vimentin (Vimentin, Twist), the relative expression of Snail protein by immunofluorescence; experimental and comparative analysis of MI RNA-451 treatment group and blank control group N-cadherin, the expression of Vimentin protein; computer network software and the previous research results to construct protein cross-linking signaling pathway mi RNA-451 regulation EMT related protein; MI RNA-451 regulate cell associated EMT the purpose of signal pathway of Western protein in Blot experiment mi RNA-451 treatment group (CAB39, AMPK, PI3K, AKT, phosphorylated AKT) expression of relative protein content. The second part: the analysis of MI RNA-451 Effect on the human brain glioma cell line biological behavior. Grouped according to the first part of the method of transfection and quantitative polymerase chain reaction (RT-PCR) results show that the expression level of MI RNA-451 was transfected into glioma cell line cells were in the post; the invasion ability of Transwell experiments on the high expression of MI inhibits glioma cell line RNA-451 make validation; cell scratch experiments on expression of MI after RNA-451 glioma cell migration ability to make effective use of detection; cell (CCK-8) and experimental verification of high expression of monoclonal proliferation of glioma cells line mi RNA-451 Western blot; experiments on biological behavior of glioma cell lines related to protein expression the amount of protein to test level. Results: the results showed that: in the first part and the blank control group and antisense sequence group comparative analysis, MI RNA-451 The expression of the gene group is significantly increased; Western Blot showed significantly increased expression of MI, RNA-451 group in E-cadherin Twist, Snail, N-cadherin, Vimentin protein expression was significantly reduced; all the differences were statistically significant (P0.05); in immunofluorescence experiments, the experimental results show that N-cadherin in MI RNA-451 treatment group, the expression of Vimentin protein decreased significantly than the control group, and the result was significant (P0.05), Western and Blot found the same experiment; in signaling pathway specific in CAB39 gene by AMPK, affect the expression of PI3K/AKT pathway related proteins of EMT, and then influences the behavior of the cancer cells, we demonstrated that CAB39 is the target gene mi RNA-451 Western blot; the experimental results show that the MI RNA-451 mi RNA-451 EMT control group related protein signal pathway In the related protein CAB39, AMPK, PI3K, the expression of phosphorylated AKT, and the experimental results show that the expression amount was reduced, and had statistical significance (P0.05). Some results of second statistics: compared with the blank control group and antisense sequence, RT-PCR experiment shows mi RNA-451 treatment group in the expression the Transwell increased significantly; experimental effective description of the MI RNA-451 group cell invasion ability decreased significantly; the inhibition of cell scratch experiment on MI RNA-451 treated cell migration results adequately weakened effective instruction; cell count Kit (CCK-8) and the effective description of the MI monoclonal RNA-451 treated glioma cell proliferation the ability of MMP-2 Western blot has been weakened; experimental group RNA-451 on MI glioma cell biological behavior of protein Cyclin, D1 significantly reduced the expression of MMP-9 has made the full said In Ming Dynasty, and the experimental results were significantly higher (P0.05). Conclusion: through the analysis of the experiment and the experimental results of statistics, can be found in CAB39 Mi to target RNA-451 through AMPK, PI3K/AKT pathway on epithelial glioma cell lines between matter transformation inhibited more significantly; and on the basis of on the migration and invasion of glioma cell lines have been limited; and the clonal proliferation ability is limited.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.41

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Young Jin Jun;Se Min Jang;Kang Hong Lee;Ki-Seok Jang;Seung Sam Paik;;Clinicopathologic significance of GLUT1 expression and its correlation with Apaf-1 in colorectal adenocarcinomas[J];World Journal of Gastroenterology;2011年14期

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本文編號：1596840