miR-138-5p靶向调控DEK抑制舌癌迁移

发布时间：2018-01-07 15:30

本文关键词：miR-138-5p靶向调控DEK抑制舌癌迁移　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

更多相关文章： miR-138-5p DEK 舌癌 细胞迁移

【摘要】：目的阐明miR-138-5p/DEK的生物学功能,探讨miR-138-5p靶向DEK抑制舌癌细胞迁移的机制。方法进行免疫组化,研究DEK表达与区域性淋巴结转移是否存在相关性;构建舌癌SCC6、SCC15细胞过表达和敲低DEK的稳定克隆,并进行划痕实验和Transwell实验,研究DEK过表达、敲低对细胞迁移的影响;用此稳定克隆细胞进行Cyclin-A蛋白检测,研究DEK表达与Cyclin-A表达相关性;对靶向DEK的miRNA进行筛选,得到能够调控DEK基因的miRNA,即miR-138-5p,合成miR-138-5p mimic/inhibitor,进行qRT-PCR实验,研究miR-138-5p调控DEK蛋白表达主要是发生在转录水平还是转录后水平;构建野生型DEK的3’UTR(DEK WT)和突变体DEK的3’UTR(DEK MUT),进行转录活性实验,验证miR-138-5p通过下调DEK的3’UTR的活性来调控DEK蛋白及其下游相关基因的表达;收集舌癌标本,进行原位杂交实验,研究miR-138-5p在舌癌中的生物学功能;在舌癌细胞中,转染miR-138-5p及其inhibitor,利用划痕和Transwell等方法检测miR-138-5p对细胞迁移的作用和影响;对机制作深入研究,在不同细胞系中进行DEK敲低后回复实验、Western Blot等实验研究mi R-138-5p/DEK对舌癌迁移作用的具体机制。结果免疫组化结果发现在有区域性淋巴结转移时,DEK表达高,DEK会促进癌细胞转移向其他部位;DEK过表达促进细胞迁移,DEK敲低可抑制细胞迁移;在SCC6和SCC15细胞中,Cyclin-A高表达于PCDH-DEK组细胞,而在PSIH-DEK中则低表达;miR-138-5p能够调控DEK基因,过表达miR-138-5p后,DEK的mRNA水平明显下降,说明miR-138-5p调控DEK蛋白表达主要发生在转录水平;miR-138-5p通过下调DEK的3’UTR的活性来调控DEK蛋白及其下游相关基因的表达;原位杂交试验发现在有区域性淋巴结转移的舌癌标本中,miR-138-5p的表达水平较低,与DEK表达成负相关;miR-138-5p抑制舌癌细胞迁移;DEK通过EMT途径促进舌癌细胞迁移,miR-138-5p对舌癌迁移的影响依赖于DEK,其对DEK下游基因也进行调控。结论miRNA-138-5p在舌癌中发挥着抑癌基因的作用,通过作用于DEK而影响舌癌细胞的迁移。本实验首次证实了miR-138-5p对舌癌细胞迁移的抑制作用,并创新性阐明了其作用靶点DEK及部分作用机制,miR-138-5p/DEK的研究丰富了舌癌发生发展的理论,有望给舌癌的生物靶向治疗带来新希望。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.86

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