miR-106b在喉癌中对RUNX3表达的调控及其机制研究

发布时间：2018-04-22 07:02

本文选题：miR-106b + 喉癌　；参考：《山西医科大学》2013年博士论文

【摘要】：喉癌是头颈部常见的恶性肿瘤之一,其全世界发病率呈逐年增长趋势。目前喉癌的治疗方法主要是手术治疗,虽然人们一直致力于对喉癌早诊断、早治疗,并在切除癌肿的前提下,尽可能保留或重建喉的功能,但是,患者术后仍有不同程度发音障碍和吞咽困难,影响到患者生存质量。为了改善疗效及提高预后的生存率,尽管人们不断探索喉癌发生、发展机制,但喉癌发生的分子机制尚不明确。 microRNA(miRNA)是一类存在于生物体内非编码单链小分子RNA,长度约为21-25个核苷酸,它们通过碱基互补配对方式与靶基因mRNA的3’非翻译区(3'-untranslated region,3'UTR)完全或不完全结合,导致靶基因mRNA降解或翻译抑制,从而调控靶基因的表达。目前研究认为,miRNA通过类似癌基因、抑癌基因或其他方式的作用对细胞生长、凋亡和细胞周期调控,导致肿瘤发生发展。我们前期通过miRNA芯片技术筛选到了一系列在喉癌中表达差异的miRNA,如：miR-106b、miR-151-3p、miR-19b、miR-185、miR-139-5p等。后期我们通过荧光定量PCR进一步研究发现miR-106b在喉癌中显著性表达。因此,本研究以miR-106b为研究对象,揭示其在喉癌发生发展中的作用。第一部分miR-106b在喉癌组织中的表达研究目的研究miR-106b在喉癌、癌旁组织中表达水平。方法提取14例配对喉癌手术新鲜标本(喉癌组织和癌旁组织)RNA,用茎环实时荧光定量PCR法检测niR-106b在喉癌和癌旁组织中表达水平。结果喉癌组织中miR-106b表达水平较癌旁组织显著增高。结论 miR-106b在喉癌组织中高表达,推测niR-106b可能与喉癌发生发展有关。第二部分niR-106b对喉癌细胞增殖、侵袭和体内生长能力的影响目的研究miR-106b对喉癌细胞体外增殖、侵袭和体内生长能力的影响。方法采用实时荧光定量PCR检测转染miR-106b ASO后,Hep-2和TU-212喉癌细胞miR-106b表达水平,采用体外克隆形成实验、细胞侵袭实验和体内生长实验观察miR-106b对Hep-2和TU-212喉癌细胞增殖和侵袭能力影响。结果转染了miR-106b ASO的Hep-2和TU-212喉癌细胞其miR-106b表达水平降低了约80%,抑制niR-106b表达可以降低喉癌细胞体内外生长和体外侵袭能力。结论 miR-106b在喉癌细胞株中高表达,而抑制其表达可以部分逆转喉癌细胞体内外生长和体外侵袭能力。第三部分miR-106b靶向调控RUNX3的研究目的研究miR-106b是否可以直接靶向调控RUNX3。方法生物信息学方法筛选RUNX3的候选miRNA,荧光素酶报告基因实验检测候选miRNA对RUNX3的调控作用,Western blot检测转染了miR-106b模拟剂和抑制剂的喉癌细胞中RUNX3蛋白表达变化,采用荧光素酶报告基因实验验证miR-106b对RUNX3直接调控作用。结果预测的RUNX3上游的11个可能调控性miRNA,荧光素酶报告基因实验显示共转染miR-130b、miR-106b mimics和RUNX3-3'UTR能够显著降低报告质粒荧光素酶的活性,其中miR-130b能够直接靶定RUNX3在胃癌细胞中已有报导。因此,我们进一步研究rmiR-106b的作用。转染miR-106b ASO的喉癌细胞中RUNX3蛋白表达水平升高2-3倍,转染miR-106b mimics的喉癌细胞中RUNX3蛋白表达水平降低约40%～70%。荧光素酶报告基因实验显示niR-106b功能被ASO减弱后,含有野生型RUNX3-3'UTR报告质粒荧光素酶活性显著增强,转染miR-106b mimics后,报告质粒荧光素酶活性显著降低,而miR-106b mimics和miR-106b ASO对含有突变型RUNX3-3'UTR的报告质粒的荧光素酶活性则无显著影响。结论 RUNX3为miR-106b的靶基因。第四部分喉癌中niR-106b靶向调控RUNX3作用机制研究实验一喉癌中RUNX3基因启动子甲基化及其蛋白表达的研究目的研究RUNX3基因启动子甲基化状态及其蛋白表达与喉癌发生发展的关系。方法采用Western blot检测RUNX3在喉癌组织中的表达水平。用甲基化特异性PCR(MSP)方法检测RUNX3基因启动子甲基化发生状况。用Western Blot检测RUNX3蛋白在RUNX3启动子甲基化喉癌组织、RUNX3启动子非甲基化喉癌组织和正常喉上皮中的表达情况。免疫组化检测正常喉上皮组织、癌旁组织、RUNX3启动子甲基化喉癌组织和RUNX3启动子非甲基化喉癌组织中RUNX3的表达情况。结果 RUNX3启动子甲基化发生率约为41.67%,无论RUNX3启动子是否发生甲基化,RUNX3在喉癌组织中均低表达。结论除RUNX3基因启动子甲基化在喉癌发生发展中起重要作用外,可能还有其它机制如miR-106b的调控引起喉癌的发生发展。实验二研究RUNX3在喉癌细胞中的作用目的过表达RUNX3,研究RUNX3在喉癌细胞中的作用方法构建过表达RUNX3的pCMV6/RUNX3,Western blot检测pCMV6/RUNX3质粒转染Hep-2和TU-212喉癌细胞后RUNX3蛋白表达水平,MTT实验、克隆形成实验和Transwell侵袭实验观察pCMV6/RUNX3质粒转染细胞后生长曲线、增殖和侵袭能力变化。结果过表达RUNX3后,喉癌细胞生长侵袭能力明显下降。结论上调RUNX3表达可以抑制喉癌细胞的生长、增殖和侵袭能力。实验三miR-106b靶向调控RUNX3在喉癌细胞中作用机制目的研究miR-106b通过靶向调控RUNX3对喉癌细胞功能的影响。方法向喉癌细胞株中同时转染RUNX3-siRNA和ASO-miR-106b,使得miR-106b和RUNX3的表达同时下调,Western Blot检测RUNX3表达水平,实时荧光定量PCR法检测mR-106b表达水平,克隆形成实验和Transwell侵袭实验检测细胞增殖和侵袭能力变化。结果喉癌细胞转染ASO-miR-106b+siRNA Control的增殖和侵袭能力显著低于转染ASO-miR-106b+RUNX3siRNA组,证明RUNX3siRNA能够部分挽救ASO-miR-106b对喉癌细胞的生物学功能影响。结论 miR-106b通过靶向作用RUNX3参与喉癌的发生。
[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.

【学位授予单位】：山西医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R739.65

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