發(fā)布時間：2018-05-10 16:20

  本文選題：miR-646 + FOXK1　； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景和目的miRNA是一類內(nèi)源性表達的單鏈非編碼小RNA,其生物學(xué)功能是參與基因的轉(zhuǎn)錄后調(diào)控,且以負性調(diào)控為主。近年來的研究發(fā)現(xiàn),miRNA的表達改變與包括胃癌在內(nèi)的多種腫瘤的發(fā)生發(fā)展密切相關(guān)。miRNA主要通過靶向抑制下游關(guān)鍵分子的表達從而對腫瘤的增殖、侵襲及轉(zhuǎn)移等發(fā)揮抑制作用。FOXK1作為FOX轉(zhuǎn)錄因子家族的一員,參與多種腫瘤的發(fā)生發(fā)展。本課題組此前的研究顯示,FOXK1可作為癌基因促進胃癌的侵襲及轉(zhuǎn)移。為了進一步了解miRNA對FOXK1介導(dǎo)的胃癌作用的影響,本課題組通過生物信息學(xué)手段預(yù)測對FOXK1具有調(diào)控作用的miRNA,選取了 miR-646作為研究對象。本課題旨在研究miR-646對胃癌細胞生物學(xué)特性的影響,并進一步明確miR-646是否可以通過靶向調(diào)控FOXK1抑制胃癌增殖和轉(zhuǎn)移,闡明miR-646在胃癌增殖及轉(zhuǎn)移中的分子機制,為尋找胃癌潛在的治療靶點奠定理論基礎(chǔ)。研究內(nèi)容和方法首先,采用qRT-PCR和原位雜交實驗檢測胃癌細胞及胃癌組織中miR-646的表達并分析其與患者臨床病理的相關(guān)性。通過轉(zhuǎn)染miR-646 mimics或inhibitors人為過表達或敲減胃癌細胞中miR-646,再利用EdU增殖實驗、Soft agar實驗、Transwell侵襲實驗和劃痕實驗評估其對胃癌細胞生物學(xué)特性的影響。接著,通過觀察細胞表型、免疫熒光實驗及Western blot檢測EMT相關(guān)標志物來觀察miR-646對胃癌細胞EMT的及TGF-β誘導(dǎo)的EMT的影響。采用雙熒光素酶報告基因證實miR-646的靶基因及其作用位點,并通過體外細胞實驗及動物實驗?zāi)Ｐ瓦M一步驗證miR-646對FOXK1的靶向調(diào)控作用。最后,采取Western blot、IHC及ISH等手段進一步闡述miR-646對FOXK1靶向調(diào)控的分子機制。結(jié)果1.miR-646在胃癌細胞及胃癌組織中表達下調(diào),其表達水平與腫瘤大小、浸潤深度、淋巴結(jié)轉(zhuǎn)移、TNM分期有關(guān);過表達miR-646可抑制胃癌細胞的增殖和侵襲轉(zhuǎn)移,敲減其表達則起促進作用。2.miR-646可抑制胃癌細胞的EMT,并阻斷了 TGF-β誘導(dǎo)的EMT;反過來,胃癌細胞中miR-646的表達又受TGF-β的抑制,呈時間依賴性及劑量依賴性;3.過表達miR-646可抑制FOXK1 3'UTR雙熒光素酶活性,突變其結(jié)合位點后,抑制作用消失;4.FOXK1與miR-646在胃癌組織的表達呈負相關(guān),miR-646可抑制胃癌細胞FOXK1的表達,而過表達FOXK1可逆轉(zhuǎn)miR-646對胃癌細胞侵襲轉(zhuǎn)移活性的抑制作用;5.miR-646可抑制裸鼠皮下成瘤及轉(zhuǎn)移瘤,FOXK1則可阻斷其抑制作用。6.miR-646在胃癌組織中的表達與FOXK1呈負相關(guān),與E-cadherin的表達正相關(guān),FOXK1促進AKT/mTOR通路蛋白的磷酸化,miR-646則抑制AKT/mTOR通路蛋白的磷酸化。結(jié)論miR-646在胃癌細胞和組織中表達下調(diào),并抑制胃癌細胞的增殖、侵襲及轉(zhuǎn)移活性,在胃癌中發(fā)揮抑癌作用;miR-646通過靶向調(diào)控轉(zhuǎn)錄因子FOXK1及AKT/mTOR通路抑制胃癌細胞的EMT過程,進而抑制胃癌的侵襲轉(zhuǎn)移活性及體內(nèi)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.2

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本文編號：1870006