miR-646靶向调控FOXK1抑制胃癌增殖和侵袭转移分子机制的研究

发布时间：2018-05-10 16:20

本文选题：miR-646 + FOXK1　；参考：《南方医科大学》2017年硕士论文

【摘要】：研究背景和目的miRNA是一类内源性表达的单链非编码小RNA,其生物学功能是参与基因的转录后调控,且以负性调控为主。近年来的研究发现,miRNA的表达改变与包括胃癌在内的多种肿瘤的发生发展密切相关。miRNA主要通过靶向抑制下游关键分子的表达从而对肿瘤的增殖、侵袭及转移等发挥抑制作用。FOXK1作为FOX转录因子家族的一员,参与多种肿瘤的发生发展。本课题组此前的研究显示,FOXK1可作为癌基因促进胃癌的侵袭及转移。为了进一步了解miRNA对FOXK1介导的胃癌作用的影响,本课题组通过生物信息学手段预测对FOXK1具有调控作用的miRNA,选取了 miR-646作为研究对象。本课题旨在研究miR-646对胃癌细胞生物学特性的影响,并进一步明确miR-646是否可以通过靶向调控FOXK1抑制胃癌增殖和转移,阐明miR-646在胃癌增殖及转移中的分子机制,为寻找胃癌潜在的治疗靶点奠定理论基础。研究内容和方法首先,采用qRT-PCR和原位杂交实验检测胃癌细胞及胃癌组织中miR-646的表达并分析其与患者临床病理的相关性。通过转染miR-646 mimics或inhibitors人为过表达或敲减胃癌细胞中miR-646,再利用EdU增殖实验、Soft agar实验、Transwell侵袭实验和划痕实验评估其对胃癌细胞生物学特性的影响。接着,通过观察细胞表型、免疫荧光实验及Western blot检测EMT相关标志物来观察miR-646对胃癌细胞EMT的及TGF-β诱导的EMT的影响。采用双荧光素酶报告基因证实miR-646的靶基因及其作用位点,并通过体外细胞实验及动物实验模型进一步验证miR-646对FOXK1的靶向调控作用。最后,采取Western blot、IHC及ISH等手段进一步阐述miR-646对FOXK1靶向调控的分子机制。结果1.miR-646在胃癌细胞及胃癌组织中表达下调,其表达水平与肿瘤大小、浸润深度、淋巴结转移、TNM分期有关;过表达miR-646可抑制胃癌细胞的增殖和侵袭转移,敲减其表达则起促进作用。2.miR-646可抑制胃癌细胞的EMT,并阻断了 TGF-β诱导的EMT;反过来,胃癌细胞中miR-646的表达又受TGF-β的抑制,呈时间依赖性及剂量依赖性;3.过表达miR-646可抑制FOXK1 3'UTR双荧光素酶活性,突变其结合位点后,抑制作用消失;4.FOXK1与miR-646在胃癌组织的表达呈负相关,miR-646可抑制胃癌细胞FOXK1的表达,而过表达FOXK1可逆转miR-646对胃癌细胞侵袭转移活性的抑制作用;5.miR-646可抑制裸鼠皮下成瘤及转移瘤,FOXK1则可阻断其抑制作用。6.miR-646在胃癌组织中的表达与FOXK1呈负相关,与E-cadherin的表达正相关,FOXK1促进AKT/mTOR通路蛋白的磷酸化,miR-646则抑制AKT/mTOR通路蛋白的磷酸化。结论miR-646在胃癌细胞和组织中表达下调,并抑制胃癌细胞的增殖、侵袭及转移活性,在胃癌中发挥抑癌作用;miR-646通过靶向调控转录因子FOXK1及AKT/mTOR通路抑制胃癌细胞的EMT过程,进而抑制胃癌的侵袭转移活性及体内成瘤性。
[Abstract]:Background and objective miRNA is a class of endogenous single-stranded non-coding small RNAs whose biological function is to participate in the post-transcriptional regulation of genes and mainly to negative regulation. In recent years, it has been found that the change of miRNA expression is closely related to the occurrence and development of many kinds of tumors, including gastric cancer. As a member of FOX transcription factor family, FOXK1 is involved in the occurrence and development of many kinds of tumors. Previous studies by our team have shown that FOXK 1 can be used as a oncogene to promote invasion and metastasis of gastric cancer. In order to further understand the effect of miRNA on gastric cancer mediated by FOXK1, we used bioinformatics to predict the effect of miRNAs on FOXK1, and selected miR-646 as the research object. The purpose of this study was to investigate the effects of miR-646 on the biological characteristics of gastric cancer cells, and to further clarify whether miR-646 can inhibit the proliferation and metastasis of gastric cancer by targeting FOXK1, and to elucidate the molecular mechanism of miR-646 in the proliferation and metastasis of gastric cancer. In order to find potential therapeutic targets for gastric cancer lay a theoretical foundation. Methods first, qRT-PCR and in situ hybridization were used to detect the expression of miR-646 in gastric cancer cells and gastric cancer tissues, and the correlation between miR-646 expression and clinicopathology was analyzed. MiR-646 was overexpressed or knocked down by transfection of miR-646 mimics or inhibitors. The effect of miR-646 on the biological characteristics of gastric cancer cells was evaluated by EdU proliferation assay, soft agar assay, transwell invasion assay and scratch test. Then, the effects of miR-646 on EMT and EMT induced by TGF- 尾 in gastric cancer cells were observed by observing cell phenotype, immunofluorescence assay and Western blot detection. Double luciferase reporter gene was used to confirm the target gene and its action site of miR-646. The target regulation of miR-646 on FOXK1 was further verified by cell experiments in vitro and animal model. Finally, the molecular mechanism of miR-646 targeting regulation of FOXK1 was further elucidated by means of Western blotte IHC and ISH. Results the expression of 1.miR-646 was down-regulated in gastric cancer cells and gastric cancer tissues, and its expression level was related to tumor size, depth of invasion and lymph node metastasis stage, overexpression of miR-646 could inhibit the proliferation, invasion and metastasis of gastric cancer cells. In turn, the expression of miR-646 in gastric cancer cells was inhibited by TGF- 尾 and inhibited by TGF- 尾 in a time-and dose-dependent manner. Overexpression of miR-646 could inhibit the activity of FOXK1 3'UTR double luciferase. After mutation of the binding site, the inhibitory effect disappeared. 4. The expression of FOXK1 and miR-646 in gastric cancer tissues was negatively correlated with that of miR-646, which could inhibit the expression of FOXK1 in gastric cancer cells. Overexpression of FOXK1 could reverse the inhibitory effect of miR-646 on invasion and metastasis of gastric cancer cells. 5. MiR-646 could inhibit subcutaneous tumorigenesis and metastasis in nude mice, and FOXK1 could block its inhibitory effect. 6. The expression of miR-646 in gastric cancer was negatively correlated with FOXK1. FOXK1 promoted the phosphorylation of AKT/mTOR pathway proteins and inhibited the phosphorylation of AKT/mTOR pathway proteins. Conclusion the expression of miR-646 in gastric cancer cells and tissues is down-regulated, and the proliferation, invasion and metastasis of gastric cancer cells are inhibited. MiR-646 inhibits the EMT process of gastric cancer cells through the targeting regulation of transcription factor FOXK1 and AKT/mTOR pathway. And then inhibit the invasion and metastasis of gastric cancer activity and tumorigenesis in vivo.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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