本文選題：miR-106b + 喉癌��； 參考：《山西醫(yī)科大學》2013年博士論文

【摘要】：喉癌是頭頸部常見的惡性腫瘤之一,其全世界發(fā)病率呈逐年增長趨勢。目前喉癌的治療方法主要是手術治療,雖然人們一直致力于對喉癌早診斷、早治療,并在切除癌腫的前提下,盡可能保留或重建喉的功能,但是,患者術后仍有不同程度發(fā)音障礙和吞咽困難,影響到患者生存質量。為了改善療效及提高預后的生存率,盡管人們不斷探索喉癌發(fā)生、發(fā)展機制,但喉癌發(fā)生的分子機制尚不明確。 microRNA(miRNA)是一類存在于生物體內非編碼單鏈小分子RNA,長度約為21-25個核苷酸,它們通過堿基互補配對方式與靶基因mRNA的3’非翻譯區(qū)(3'-untranslated region,3'UTR)完全或不完全結合,導致靶基因mRNA降解或翻譯抑制,從而調控靶基因的表達。目前研究認為,miRNA通過類似癌基因、抑癌基因或其他方式的作用對細胞生長、凋亡和細胞周期調控,導致腫瘤發(fā)生發(fā)展。我們前期通過miRNA芯片技術篩選到了一系列在喉癌中表達差異的miRNA,如：miR-106b、miR-151-3p、miR-19b、miR-185、miR-139-5p等。后期我們通過熒光定量PCR進一步研究發(fā)現(xiàn)miR-106b在喉癌中顯著性表達。因此,本研究以miR-106b為研究對象,揭示其在喉癌發(fā)生發(fā)展中的作用。 第一部分miR-106b在喉癌組織中的表達研究 目的 研究miR-106b在喉癌、癌旁組織中表達水平。 方法 提取14例配對喉癌手術新鮮標本(喉癌組織和癌旁組織)RNA,用莖環(huán)實時熒光定量PCR法檢測niR-106b在喉癌和癌旁組織中表達水平。 結果 喉癌組織中miR-106b表達水平較癌旁組織顯著增高。 結論 miR-106b在喉癌組織中高表達,推測niR-106b可能與喉癌發(fā)生發(fā)展有關。 第二部分niR-106b對喉癌細胞增殖、侵襲和體內生長能力的影響 目的 研究miR-106b對喉癌細胞體外增殖、侵襲和體內生長能力的影響。 方法 采用實時熒光定量PCR檢測轉染miR-106b ASO后,Hep-2和TU-212喉癌細胞miR-106b表達水平,采用體外克隆形成實驗、細胞侵襲實驗和體內生長實驗觀察miR-106b對Hep-2和TU-212喉癌細胞增殖和侵襲能力影響。 結果 轉染了miR-106b ASO的Hep-2和TU-212喉癌細胞其miR-106b表達水平降低了約80%,抑制niR-106b表達可以降低喉癌細胞體內外生長和體外侵襲能力。 結論 miR-106b在喉癌細胞株中高表達,而抑制其表達可以部分逆轉喉癌細胞體內外生長和體外侵襲能力。 第三部分miR-106b靶向調控RUNX3的研究 目的 研究miR-106b是否可以直接靶向調控RUNX3。 方法 生物信息學方法篩選RUNX3的候選miRNA,熒光素酶報告基因實驗檢測候選miRNA對RUNX3的調控作用,Western blot檢測轉染了miR-106b模擬劑和抑制劑的喉癌細胞中RUNX3蛋白表達變化,采用熒光素酶報告基因實驗驗證miR-106b對RUNX3直接調控作用。 結果 預測的RUNX3上游的11個可能調控性miRNA,熒光素酶報告基因實驗顯示共轉染miR-130b、miR-106b mimics和RUNX3-3'UTR能夠顯著降低報告質粒熒光素酶的活性,其中miR-130b能夠直接靶定RUNX3在胃癌細胞中已有報導。因此,我們進一步研究rmiR-106b的作用。轉染miR-106b ASO的喉癌細胞中RUNX3蛋白表達水平升高2-3倍,轉染miR-106b mimics的喉癌細胞中RUNX3蛋白表達水平降低約40%～70%。熒光素酶報告基因實驗顯示niR-106b功能被ASO減弱后,含有野生型RUNX3-3'UTR報告質粒熒光素酶活性顯著增強,轉染miR-106b mimics后,報告質粒熒光素酶活性顯著降低,而miR-106b mimics和miR-106b ASO對含有突變型RUNX3-3'UTR的報告質粒的熒光素酶活性則無顯著影響。 結論 RUNX3為miR-106b的靶基因。 第四部分喉癌中niR-106b靶向調控RUNX3作用機制研究 實驗一喉癌中RUNX3基因啟動子甲基化及其蛋白表達的研究 目的 研究RUNX3基因啟動子甲基化狀態(tài)及其蛋白表達與喉癌發(fā)生發(fā)展的關系。 方法 采用Western blot檢測RUNX3在喉癌組織中的表達水平。用甲基化特異性PCR(MSP)方法檢測RUNX3基因啟動子甲基化發(fā)生狀況。用Western Blot檢測RUNX3蛋白在RUNX3啟動子甲基化喉癌組織、RUNX3啟動子非甲基化喉癌組織和正常喉上皮中的表達情況。免疫組化檢測正常喉上皮組織、癌旁組織、RUNX3啟動子甲基化喉癌組織和RUNX3啟動子非甲基化喉癌組織中RUNX3的表達情況。 結果 RUNX3啟動子甲基化發(fā)生率約為41.67%,無論RUNX3啟動子是否發(fā)生甲基化,RUNX3在喉癌組織中均低表達。 結論 除RUNX3基因啟動子甲基化在喉癌發(fā)生發(fā)展中起重要作用外,可能還有其它機制如miR-106b的調控引起喉癌的發(fā)生發(fā)展。 實驗二研究RUNX3在喉癌細胞中的作用 目的 過表達RUNX3,研究RUNX3在喉癌細胞中的作用 方法 構建過表達RUNX3的pCMV6/RUNX3,Western blot檢測pCMV6/RUNX3質粒轉染Hep-2和TU-212喉癌細胞后RUNX3蛋白表達水平,MTT實驗、克隆形成實驗和Transwell侵襲實驗觀察pCMV6/RUNX3質粒轉染細胞后生長曲線、增殖和侵襲能力變化。 結果 過表達RUNX3后,喉癌細胞生長侵襲能力明顯下降。 結論 上調RUNX3表達可以抑制喉癌細胞的生長、增殖和侵襲能力。 實驗三miR-106b靶向調控RUNX3在喉癌細胞中作用機制 目的 研究miR-106b通過靶向調控RUNX3對喉癌細胞功能的影響。 方法 向喉癌細胞株中同時轉染RUNX3-siRNA和ASO-miR-106b,使得miR-106b和RUNX3的表達同時下調,Western Blot檢測RUNX3表達水平,實時熒光定量PCR法檢測mR-106b表達水平,克隆形成實驗和Transwell侵襲實驗檢測細胞增殖和侵襲能力變化。 結果 喉癌細胞轉染ASO-miR-106b+siRNA Control的增殖和侵襲能力顯著低于轉染ASO-miR-106b+RUNX3siRNA組,證明RUNX3siRNA能夠部分挽救ASO-miR-106b對喉癌細胞的生物學功能影響。 結論 miR-106b通過靶向作用RUNX3參與喉癌的發(fā)生。
[Abstract]:Laryngeal cancer is one of the most common malignant tumors in the head and neck, and its worldwide incidence is increasing year by year. At present, the main treatment method of larynx cancer is surgical treatment. Although people have been working on early diagnosis and treatment of larynx cancer, the function of larynx is retained or reconstructed as much as possible under the premise of cancerous excision, but there are still different degrees after surgery. Dysphonia and dysphagia affect the quality of life of the patient. In order to improve the curative effect and improve the survival rate of the prognosis, although people continue to explore the occurrence and mechanism of larynx, the molecular mechanism of the occurrence of larynx is not clear.
MicroRNA (miRNA) is a class of non coded single strand small molecules, RNA, with 21-25 nucleotides in length. They are completely or incompletely combined with the 3 'non translation region (3'-untranslated region, 3'UTR) of the target gene mRNA by the complementary pairing of bases, causing the target gene mRNA degradation or translation inhibition, thus regulating the target gene table. Da. Current research suggests that miRNA, through the role of oncogenes, tumor suppressor genes, or other ways of regulating cell growth, apoptosis and cell cycle regulation, leads to the development of tumor. We screened a series of miRNA, such as miR-106b, miR-151-3p, miR-19b, miR-185, miR-139-5p, and so on, through miRNA chip technology. In the later period, we found the significant expression of miR-106b in larynx cancer by fluorescence quantitative PCR. Therefore, this study was based on miR-106b and revealed its role in the development of larynx cancer.
Part one expression of miR-106b in laryngeal carcinoma
objective
To study the expression level of miR-106b in laryngeal carcinoma and paracancerous tissues.
Method
RNA was extracted from 14 cases of paired laryngectomy (laryngeal carcinoma tissue and para cancer tissue), and the expression level of niR-106b in laryngeal and paracancerous tissues was detected by real time PCR method.
Result
The expression level of miR-106b in laryngeal carcinoma tissues was significantly higher than that in adjacent tissues.
conclusion
MiR-106b is highly expressed in laryngeal carcinoma. It is speculated that niR-106b may be related to the development of laryngeal carcinoma.
The second part is the effect of niR-106b on the proliferation, invasion and in vivo growth of laryngeal carcinoma cells.
objective
To study the effects of miR-106b on proliferation, invasion and growth of laryngeal carcinoma cells in vitro.
Method
After transfection of miR-106b ASO with real-time fluorescent quantitative PCR, the miR-106b expression level of Hep-2 and TU-212 larynx cancer cells was detected. The effects of miR-106b on the proliferation and invasion ability of Hep-2 and TU-212 larynx cancer cells were observed by in vitro cloning and formation experiments.
Result
The expression level of miR-106b in Hep-2 and TU-212 cells transfected with miR-106b ASO was reduced by about 80%. The inhibition of niR-106b expression could reduce the growth and invasion ability of larynx cells in vitro and in vitro.
conclusion
MiR-106b is highly expressed in laryngeal cancer cell lines, but inhibiting its expression can partially reverse the growth and invasion ability of laryngeal cancer cells in vivo and in vitro.
Study on the third part of miR-106b targeting RUNX3
objective
Whether miR-106b can directly target RUNX3.
Method
The Bioinformatics Method screened the candidate miRNA for RUNX3, and the luciferase reporter gene test detected the role of the candidate miRNA for the regulation of RUNX3. Western blot detected the changes in the expression of RUNX3 protein in the larynx cells transfected with miR-106b analogue and inhibitor, and the luciferase reporter gene was used to verify the direct regulation of miR-106b on RUNX3.
Result
11 possible regulatory miRNA in the upstream of RUNX3 is predicted. The luciferase reporter gene experiment shows that CO transfection of miR-130b, miR-106b mimics and RUNX3-3'UTR can significantly reduce the activity of the reporter plasmid luciferase, and miR-130b can directly target RUNX3 in gastric cancer cells. Therefore, we further study the effect of rmiR-106b. The expression level of RUNX3 protein in the larynx cells transfected with miR-106b ASO increased by 2-3 times. The expression level of RUNX3 protein in the larynx cells transfected with miR-106b mimics decreased by about 40% ~ 70%. luciferase reporter gene experiment showed that the function of niR-106b was weakened by ASO, and the luciferase activity of the wild type RUNX3-3'UTR plasmid was significantly enhanced and the transfection miR-1 was carried out. After 06B mimics, the luciferase activity of the reported plasmid was significantly reduced, while the miR-106b mimics and miR-106b ASO had no significant effect on the luciferase activity of the reported plasmid containing the mutant RUNX3-3'UTR.
conclusion
RUNX3 is the target gene for miR-106b.
The fourth part is the mechanism of niR-106b targeting RUNX3 in laryngeal carcinoma.
Methylation of RUNX3 gene promoter and its protein expression in laryngeal carcinoma
objective
To study the relationship between the methylation status of RUNX3 gene promoter and protein expression and the occurrence and development of laryngeal carcinoma.
Method
The expression level of RUNX3 in the carcinoma of larynx was detected by Western blot. Methylation specific PCR (MSP) method was used to detect the methylation of RUNX3 gene promoter. The expression of RUNX3 protein in RUNX3 promoter methylation laryngeal carcinoma tissue, RUNX3 promoter in non methylated larynx tissues and normal laryngeal epithelium was detected by Western Blot. The expression of RUNX3 in normal laryngeal epithelium, paracancerous tissue, RUNX3 promoter methylation laryngeal carcinoma tissue and RUNX3 promoter non methylation laryngeal carcinoma tissue was detected by histochemical method.
Result
The methylation rate of RUNX3 promoter was about 41.67%. Whether RUNX3 promoter methylation occurred, RUNX3 expression was low in laryngeal carcinoma.
conclusion
Besides the methylation of RUNX3 gene plays an important role in the development of laryngeal carcinoma, there may be other mechanisms, such as the regulation of miR-106b, which may lead to the development of laryngeal carcinoma.
Experiment two to study the role of RUNX3 in laryngeal cancer cells
objective
Overexpression of RUNX3 to study the role of RUNX3 in laryngeal carcinoma cells
Method
The expression of RUNX3 pCMV6/RUNX3 and Western blot were constructed to detect the RUNX3 protein expression level after pCMV6/RUNX3 plasmid transfected to Hep-2 and TU-212 carcinoma cells, MTT experiment, clone formation experiment and Transwell invasion test to observe the growth curve, proliferation and invasiveness of pCMV6/RUNX3 plasmid transfected cells.
Result
After over expression of RUNX3, the growth and invasion ability of laryngeal cancer cells decreased significantly.
conclusion
Up regulation of RUNX3 expression can inhibit the growth, proliferation and invasion of laryngeal cancer cells.
Experiment three the mechanism of miR-106b targeting RUNX3 in laryngeal cancer cells
objective
Objective to study the effect of miR-106b on the function of laryngeal cancer cells by targeting RUNX3.
Method
RUNX3-siRNA and ASO-miR-106b were transfected into the larynx cell line. The expression of miR-106b and RUNX3 was downregulated simultaneously. The expression level of RUNX3 was detected by Western Blot. The level of mR-106b expression was detected by real time fluorescence quantitative PCR method. The colonization and invasion ability of the cells were detected by clone formation experiment and Transwell invasion test.
Result
The proliferation and invasion ability of ASO-miR-106b+siRNA Control transfected by larynx cancer cells were significantly lower than that of transfected ASO-miR-106b+RUNX3siRNA group. It was proved that RUNX3siRNA could partly save the biological function of ASO-miR-106b on larynx cancer cells.
conclusion
MiR-106b participates in laryngeal carcinomas by targeting RUNX3.

【學位授予單位】：山西醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R739.65

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1 江黎珠;FoxM1在喉癌中的表達及其在姜黃素抑制喉癌細胞株Hep-2增殖、侵襲、轉移中的作用[D];重慶醫(yī)科大學;2011年

2 溫曉霞;喉癌大體分型的病理特點與臨床意義[D];中國醫(yī)科大學;2004年

3 周q，

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