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口腔粘膜下纤维性变相关miRNA表达谱的建立及其功能的初步探讨
Establishment of MiRNA Expression Proming of Oral Submucous Fibrosis and Preliminary Investigation of Its Function
【作者】 刘斌杰；
【导师】 翦新春；
【作者基本信息】 中南大学 ， 外科学， 2012， 博士
【摘要】 研究背景及目的：口腔粘膜下纤维性变(oral submucous fibrosis, OSF)是一种慢性、隐匿性、具有癌变倾向的口腔粘膜病,癌变率高达7.6%。最近的流行病学调查表明,OSF的发病地区、发病率和癌变率均有上升趋势。目前认为OSF与咀嚼槟榔、胶原代谢紊乱、免疫、遗传、微循环及血液流变学等因素有关,本课题组前期研究发现,丹参联合小剂量泼尼松龙治疗具有明显的临床效果,但口腔粘膜下纤维性变的确切发病机制及治疗显效获得的机制仍不清楚。miRNA是近年来发现的转录后调节基因表达的内源性非编码单链小RNA分子,参与了机体生长发育,器官形成,造血,细胞增殖、分化及凋亡等众多生理活动和病理过程。近年来miRNA在心肌纤维化、肾脏纤维化和肝纤维化等方面已有许多报道,至于miRNA在OSF发病中的功能及机制研究报道较少。本研究旨在探讨miRNA在OSF发病及临床治疗转归中的功能及作用机制,为阐明OSF的发病机理及临床治疗提供新的线索。研究方法：(1)选取1例临床上具有典型病理特征的中期OSF组织及其配对正常组织,以正常组织为对照,部分病变组织行Affymetrix miRNA芯片实验,建立OSF相关miRNA表达谱。原配对组织及选取的初诊OSF配对中、晚期各10例组织经niRNA专用抽提试剂盒抽提miRNA后,经专用miRNA逆转录试剂盒逆转录后,用实时定量PCR验证miRNA芯片结果。(2)针对感兴趣的miRNA,根据下列三个国际公认的、拥有不同计算方法的数据库：www.targetscan.org、pictar.mdc-berlin.de/cgi-bin/PicTar_vertebrate.cgi、www.mirbase.org进行miRNA靶标的初步预测,寻找三个数据库都预测为靶的基因。(3)从正常口腔粘膜组织获得原代成纤维细胞,以槟榔碱(50μg/m1)诱导,于72h后检测差异miRNA的表达。原代培养OSF-Fb细胞,经丹参(90mg/ml)联合小剂量泼尼松龙作用,于72h后检测差异miRNA的表达。(4)通过生物信息学软件对miR-203可能调控的靶基因进行预测,结合其生物学功能,筛选出miR-203可能的靶基因COL4A4。Western blot检测上述收集的配对标本组织、槟榔碱诱导前后的正常口腔粘膜成纤维细胞及丹参联合小剂量泼尼松龙处理前后的OSF-Fb细胞中COL4A4的表达。(5)将COL4A4基因3’UTR包括miR-203结合位点、上下游部分侧翼序列以及酶切位点在内的共60bp片段分别克隆入pMIR-REPORT荧光素酶报告基因载体,将其分别与β-gal表达质粒及miR-203表达载体共转染293T细胞24h后检测报告基因活性。结果：(1)所选病变组织属于OSF病变中期,miRNA芯片显示差异1.5倍以上的miRNA共有18个,其中OSF病变组织中上调12个,下调6个,miRNA特异性实时定量PCR验证结果与芯片结果一致;(2)临床中、晚期OSF组织中差异miRNA的变化趋势与芯片结果基本一致；(3)生物信息学分析结果显示其中的miR-203、 miR-23b及miR-200c等可能参与了OSF病变过程及其后的癌变；(4)槟榔碱能诱导正常口腔粘膜成纤维细胞中miRNA的表达改变,丹参联合小剂量泼尼松龙能逆转OSF成纤维细胞中相关miRNA的表达；(5)生物信息预测显示COL4A4基因3’UTR与miR-203之间有一物种间保守作用位点(site1),3个非保守作用位点(site2, site3, site4);(6)OSF组织中,COL4A4的蛋白表达量明显高于配对正常组织,槟榔碱能诱导上调正常口腔粘膜成纤维细胞中COL4A4的表达,而丹参联合小剂量泼尼松龙能下调OSF成纤维细胞中COL4A4的表达;(7)miR-203能明显抑制site1介导的报告基因活性,能部分抑制site2与site4介导的报告基因活性,而对site3介导的报告基因活性无明显影响。结论：(1)首次构建了OSF相关的miRNA表达谱；(2)临床组织验证提示差异miRNA在OSF的发生发展中发挥作用；(3)槟榔碱体外能诱导正常口腔粘膜成纤维细胞中miRNA的表达改变,而丹参联合泼尼松龙能逆转OSF成纤维细胞中相关miRNA的表达；(4)miR-203与COL4A4的蛋白表达呈负相关,miR-203能直接靶向抑制COL4A4的蛋白表达。
【Abstract】 Background and Objective:Oral submucous fibrosis (OSF) is a chronic pre-cancerous disease of insidious onset characterized by the deposition of fibrous tissues in the oral submucosa, with about7.6%malignant transformation rate. Recent epidemiological studies show an increase in the high incidence area, incidence rate and malignant transformation of OSF. many factors such as chewing betel nut, disorders of collagen metabolism, immune, genetic, microcirculation and hemorheology play important roles in the pathogenesis of OSF. We have previous found that Salvia in combination with low-dose prednisolone was effective in the treatment of OSF. However, the exact pathogenesis of OSF is not yet known, as well as the treatment.MicroRNAs (miRNAs) are a recently discovered class of small non-coding RNAs that post-transcriptionally regulate gene expression, involved in regulation of various physiological and pathological processes such as growth and development, organogenesis, hematopoiesis, cell proliferation, differentiation and apoptosis. Many recent reports have demonstrated the role of miRNAs in myocardial fibrosis, kidney fibrosis and liver fibrosis. However, the relationship between miRNA and OSF is not clearly. To provide new clues to the pathogenesis and treatment of OSF, our study investigates the function and mechanism of miRNA in the OSF.Methods:(1) select three case with typical clinical pathological features of OSF and its paired normal tissue (internal control). The miRNA expression profiles between the OSF and its paired control were compared by using the Affymetrix analysis. After extraction miRNA from the mid-late OSF tissue specimens and the control normal tissue, some of the differentially expressed miRNAs were validated by real-time quantitative PCR.(2) Choose the computational prediction of miRNA targets from the TargetScan sites (www.targetscan.org、pictar.mdc-berlin.de/cgi-bin/PicTar vertebrate.cg、www.mirbase.org).(3) Treatment the primary normal oral mucoses cells by Arecoline, we detected the interesting miRNA expression at72h. Following, the primary cells OSF-Fb were treated with a combination of Salvia and low-dose prednisolone, then detection the different miRNAs at72h.(4) Using a variety of bioinformatics software tools, it realved that COL4A4was the putative target of miR-203. Examine the expreesion of COL4A4in the indcated cells by Western blot analysis.(5) we integrated a fragment of the COL4A43’UTR containing the target sequence into the pMIR-REPORT luciferase vector. Then, the COL4A43’UTR-luciferase construct and miR-203were contransfected into293T cells. Beta-gal was used as a control to monitor transfection efficiency.24h after transfection, cells were harvested and luciferase activity was measured.Results:(1) The miRNA microarray chip analysis identified19miRNAs differentially expressed with1.5-fold or greater change. Of these,13microRNAs were upregulated and6were downregulated. The results of expression data obtained by microarray analysis were in good agreement with those obtained by real-time quantitative PCR analyses.(2) The expression of miRNAs from the samples of middle-and late-stage OSF were consistent with the miRNA expression profiles observed from the microarray data. (3) Microarray-Bioinformatics analysis revealed that the miR-203, miR-23b, miR-200c of Oral Submucous Fibrosis play important roles in the development of OSF and subsequently malignant transformation.(4) Arecoline exposure induced changes in the expression of miRNAs in normal mucosal cells. Salvia in combination with low doses of prednisolone could reverse the miRNA expression.(5)Bioinformatics analysis revealed that the COL4A43’UTR has a conserved site (site1) and3non-conserved sites (site2, site3, site4) complementary to the miR-203.(6) The expression of COL4A4in OSF was significantly higher than normal control. Arecoline induced the expression of COL4A4in normal mucosal cells. Salvia in combination with low-dose prednisolone downregulated the expression of COL4A4in OSF cells.(7) miR-203could significantly inhibit site1-luciferase reporter activity, partially inhibited site3-and site4-luciferase reporter activity, while no significant effect on site3-luciferase reporter activity.Conclusion:(1) construction of miRNA expression profiling of the OSF.(2) The clinical evidences proved that miRNAs play essential role in the OSF.(3) In vitro, Arecoline changed the expression of miRNAs in normal oral mucosal cells, while the Salvia in combination with prednisolone could reversed it.(4) miR-203was negatively correlated with the expression of COL4A4protein in OSF. Moreover, miR-203can directly repress COL4A4mRNA.