本文關(guān)鍵詞：miR-138-5p靶向調(diào)控DEK抑制舌癌遷移　出處：《安徽醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

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【摘要】：目的闡明miR-138-5p/DEK的生物學(xué)功能,探討miR-138-5p靶向DEK抑制舌癌細(xì)胞遷移的機(jī)制。方法進(jìn)行免疫組化,研究DEK表達(dá)與區(qū)域性淋巴結(jié)轉(zhuǎn)移是否存在相關(guān)性;構(gòu)建舌癌SCC6、SCC15細(xì)胞過(guò)表達(dá)和敲低DEK的穩(wěn)定克隆,并進(jìn)行劃痕實(shí)驗(yàn)和Transwell實(shí)驗(yàn),研究DEK過(guò)表達(dá)、敲低對(duì)細(xì)胞遷移的影響;用此穩(wěn)定克隆細(xì)胞進(jìn)行Cyclin-A蛋白檢測(cè),研究DEK表達(dá)與Cyclin-A表達(dá)相關(guān)性;對(duì)靶向DEK的miRNA進(jìn)行篩選,得到能夠調(diào)控DEK基因的miRNA,即miR-138-5p,合成miR-138-5p mimic/inhibitor,進(jìn)行qRT-PCR實(shí)驗(yàn),研究miR-138-5p調(diào)控DEK蛋白表達(dá)主要是發(fā)生在轉(zhuǎn)錄水平還是轉(zhuǎn)錄后水平;構(gòu)建野生型DEK的3’UTR(DEK WT)和突變體DEK的3’UTR(DEK MUT),進(jìn)行轉(zhuǎn)錄活性實(shí)驗(yàn),驗(yàn)證miR-138-5p通過(guò)下調(diào)DEK的3’UTR的活性來(lái)調(diào)控DEK蛋白及其下游相關(guān)基因的表達(dá);收集舌癌標(biāo)本,進(jìn)行原位雜交實(shí)驗(yàn),研究miR-138-5p在舌癌中的生物學(xué)功能;在舌癌細(xì)胞中,轉(zhuǎn)染miR-138-5p及其inhibitor,利用劃痕和Transwell等方法檢測(cè)miR-138-5p對(duì)細(xì)胞遷移的作用和影響;對(duì)機(jī)制作深入研究,在不同細(xì)胞系中進(jìn)行DEK敲低后回復(fù)實(shí)驗(yàn)、Western Blot等實(shí)驗(yàn)研究mi R-138-5p/DEK對(duì)舌癌遷移作用的具體機(jī)制。結(jié)果免疫組化結(jié)果發(fā)現(xiàn)在有區(qū)域性淋巴結(jié)轉(zhuǎn)移時(shí),DEK表達(dá)高,DEK會(huì)促進(jìn)癌細(xì)胞轉(zhuǎn)移向其他部位;DEK過(guò)表達(dá)促進(jìn)細(xì)胞遷移,DEK敲低可抑制細(xì)胞遷移;在SCC6和SCC15細(xì)胞中,Cyclin-A高表達(dá)于PCDH-DEK組細(xì)胞,而在PSIH-DEK中則低表達(dá);miR-138-5p能夠調(diào)控DEK基因,過(guò)表達(dá)miR-138-5p后,DEK的mRNA水平明顯下降,說(shuō)明miR-138-5p調(diào)控DEK蛋白表達(dá)主要發(fā)生在轉(zhuǎn)錄水平;miR-138-5p通過(guò)下調(diào)DEK的3’UTR的活性來(lái)調(diào)控DEK蛋白及其下游相關(guān)基因的表達(dá);原位雜交試驗(yàn)發(fā)現(xiàn)在有區(qū)域性淋巴結(jié)轉(zhuǎn)移的舌癌標(biāo)本中,miR-138-5p的表達(dá)水平較低,與DEK表達(dá)成負(fù)相關(guān);miR-138-5p抑制舌癌細(xì)胞遷移;DEK通過(guò)EMT途徑促進(jìn)舌癌細(xì)胞遷移,miR-138-5p對(duì)舌癌遷移的影響依賴于DEK,其對(duì)DEK下游基因也進(jìn)行調(diào)控。結(jié)論miRNA-138-5p在舌癌中發(fā)揮著抑癌基因的作用,通過(guò)作用于DEK而影響舌癌細(xì)胞的遷移。本實(shí)驗(yàn)首次證實(shí)了miR-138-5p對(duì)舌癌細(xì)胞遷移的抑制作用,并創(chuàng)新性闡明了其作用靶點(diǎn)DEK及部分作用機(jī)制,miR-138-5p/DEK的研究豐富了舌癌發(fā)生發(fā)展的理論,有望給舌癌的生物靶向治療帶來(lái)新希望。
[Abstract]:Objective to elucidate the biological function of miR-138-5p/DEK and to explore the mechanism of miR-138-5p targeting DEK in inhibiting the migration of tongue cancer cells. To investigate the correlation between DEK expression and regional lymph node metastasis. To construct the stable clone of overexpression and knockout DEK of SCC6 and SCC15 cells, and to study the overexpression of DEK by scratch test and Transwell assay. The effect of knockout on cell migration; The stable clone cells were used to detect the Cyclin-A protein and to study the correlation between DEK expression and Cyclin-A expression. The miRNA targeting DEK was screened to obtain the miR-138-5p that could regulate the DEK gene. MiR-138-5p mimicr inhibitor was synthesized and qRT-PCR experiments were carried out. To study whether miR-138-5p regulates the expression of DEK mainly at the transcriptional level or posttranscriptional level. The transcriptional activity of wild-type DEK and mutant DEK was studied. It was verified that miR-138-5p regulated the expression of DEK protein and its downstream related genes by down-regulating the activity of DEK 3-UTR. The biological function of miR-138-5p in tongue cancer was studied by in situ hybridization. MiR-138-5p and its inhibitor were transfected into tongue cancer cells. The effects and effects of miR-138-5p on cell migration were detected by scratch and Transwell. The mechanism of DEK knockout in different cell lines was studied. Western Blot and other experiments were used to study the specific mechanism of mi R-138-5p / DEK on the migration of tongue cancer. Results Immunohistochemical results showed that there were regional lymph node metastasis. High expression of DEK can promote metastasis of cancer cells to other sites. Overexpression of DEK could promote cell migration and inhibit cell migration. The expression of Cyclin-A in SCC6 and SCC15 cells was high in PCDH-DEK group, but low in PSIH-DEK. MiR-138-5p could regulate the DEK gene, and the mRNA level of dek decreased significantly after overexpression of miR-138-5p. The results showed that miR-138-5p regulated the expression of DEK protein mainly at the transcriptional level. MiR-138-5p down-regulates the expression of DEK protein and its downstream genes by down-regulating the activity of DEK 3-UTR. In situ hybridization test showed that the expression level of miR-138-5p was low in tongue carcinoma with regional lymph node metastasis, and negatively correlated with DEK expression. MiR-138-5p inhibited the migration of tongue cancer cells. DEK promotes the migration of tongue cancer cells via EMT pathway. The effect of miR-138-5p on the migration of tongue cancer depends on DEK. Conclusion miRNA-138-5p plays an important role as a tumor suppressor gene in tongue cancer. The effect of miR-138-5p on the migration of tongue cancer cells was confirmed for the first time by the action of miR-138-5p on the migration of tongue cancer cells. The research on the action target DEK and partial action mechanism of miR-138-5p / DEK has enriched the theory of the development of tongue cancer. It is expected to bring new hope to the biological target therapy of tongue cancer.
【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)】：R739.86

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