本文選題：miR-192 + 胃腫瘤��； 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的為了探索miR-192-5p在體內(nèi)能否通過(guò)調(diào)控核苷酸切除修復(fù)(NER)途徑中ERCC3和ERCC4蛋白表達(dá)逆轉(zhuǎn)胃癌細(xì)胞的順鉑耐藥性。方法采用基因芯片微陣列方法檢測(cè)胃癌順鉑耐藥細(xì)胞SGC7901/DDP和親本細(xì)胞SGC7901中LncRNA、mRNA和miRNA的表達(dá)差異。通過(guò)生物信息學(xué)預(yù)測(cè)軟件分析核酸切除修復(fù)蛋白ERCC3和ERCC4的mRNA中存在miR-192-5p的靶向結(jié)合位點(diǎn)。利用慢病毒在SGC7901/DDP細(xì)胞中穩(wěn)定上調(diào)miR-192-5p的表達(dá)、在SGC7901細(xì)胞中穩(wěn)定下調(diào)miR-192-5p的表達(dá),應(yīng)用qRT-PCR驗(yàn)證轉(zhuǎn)染后miR-192-5p的表達(dá)水平。免疫蛋白印記技術(shù)(Western blot)方法檢測(cè)上調(diào)和下調(diào)miR-192-5p表達(dá)后細(xì)胞內(nèi)ERCC3和ERCC4的表達(dá)。用四甲基偶氮唑鹽微量酶反應(yīng)比色法(MTT)檢測(cè)調(diào)控miR-192-5p表達(dá)后細(xì)胞的順鉑耐藥性。將穩(wěn)定表達(dá)miR-192-5p的胃癌細(xì)胞、空載對(duì)照細(xì)胞和未轉(zhuǎn)細(xì)胞構(gòu)建人胃癌裸鼠皮下移植瘤模型,腹腔灌注順鉑,測(cè)量各組瘤體的生長(zhǎng)曲線及瘤體重量,免疫組化檢測(cè)ERCC3和ERCC4在瘤體內(nèi)的表達(dá)。結(jié)果基因芯片檢測(cè)結(jié)果提示,SGC7901/DDP和SGC7901細(xì)胞中存在多個(gè)LncRNA、mRNA和miRNA的表達(dá)差異。通過(guò)生物學(xué)軟件分析這些異常表達(dá)的基因可能與順鉑耐藥相關(guān),其中miR-192-5p在SGC7901/DDP中的表達(dá)較SGC7901中明顯降低,ERCC3和ERCC4的mRNA中存在miR-192-5p的靶向結(jié)合位點(diǎn)。上調(diào)miR-192-5p后,細(xì)胞內(nèi)ERCC3和ERCC4的表達(dá)下調(diào),順鉑的敏感性增強(qiáng);下調(diào)miR-192-5p后,細(xì)胞內(nèi)ERCC3和ERCC4的表達(dá)上調(diào),順鉑的耐藥性增強(qiáng)。動(dòng)物模型實(shí)驗(yàn)表明,上調(diào)miR-192-5p后,瘤體內(nèi)ERCC3和ERCC4的表達(dá)下調(diào),瘤體對(duì)順鉑化療的敏感性提高,而下調(diào)miR-192-5p表達(dá)后,瘤體內(nèi)ERCC3和ERCC4的表達(dá)上調(diào),瘤體對(duì)順鉑耐藥。結(jié)論MiR-192-5p通過(guò)調(diào)控NER途徑中ERCC3和ERCC4表達(dá)影響胃癌細(xì)胞的順鉑耐藥性
[Abstract]:Objective to investigate whether miR-192-5p can reverse cisplatin resistance in gastric cancer cells by regulating the expression of ERCC3 and ERCC4 protein in the nucleotide excision repair pathway.Methods cDNA microarray was used to detect the expression of LncRNA-mRNA and miRNA in cisplatin resistant gastric cancer cell line SGC7901/DDP and parental cell SGC7901.Bioinformatics prediction software was used to analyze the existence of targeted miR-192-5p binding sites in ERCC3 and ERCC4 mRNA.Lentivirus was used to steadily up-regulate the expression of miR-192-5p in SGC7901/DDP cells and down-regulate the expression of miR-192-5p in SGC7901 cells. QRT-PCR was used to verify the expression level of miR-192-5p after transfection.The expression of ERCC3 and ERCC4 in the cells after up-regulation and down-regulation of miR-192-5p expression was detected by Western blot.The cisplatin resistance of cells regulated by miR-192-5p was detected by microenzyme reaction of tetramethylazolium.Gastric cancer cells expressing miR-192-5p stably, unloaded control cells and untransformed cells were used to construct subcutaneous transplanted tumor model of human gastric cancer in nude mice. Cisplatin was infused intraperitoneally to measure the growth curve and weight of tumor in each group.The expression of ERCC3 and ERCC4 in the tumor was detected by immunohistochemistry.Results the results of gene chip analysis showed that there were differences in mRNA and miRNA expression of LncRNAs in SGC7901 / DDP and SGC7901 cells.The abnormal expression of these genes may be related to cisplatin resistance by biological software. The expression of miR-192-5p in SGC7901/DDP is significantly lower than that in mRNA of SGC7901 and ERCC3 and ERCC4 have targeted miR-192-5p binding sites.After upregulation of miR-192-5p, the expression of ERCC3 and ERCC4 was down-regulated, and the sensitivity of cisplatin was enhanced. After down-regulation of miR-192-5p, the expression of ERCC3 and ERCC4 was up-regulated, and the resistance of cisplatin was enhanced.After up-regulation of miR-192-5p, the expression of ERCC3 and ERCC4 was down-regulated, and the sensitivity of tumor to cisplatin chemotherapy was increased. After down-regulation of miR-192-5p, the expression of ERCC3 and ERCC4 was up-regulated, and the tumor was resistant to cisplatin.Conclusion MiR-192-5p affects cisplatin resistance of gastric cancer cells by regulating the expression of ERCC3 and ERCC4 in NER pathway.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2

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本文編號(hào)：1734375