摘要目的 构建微小RNA-203(microRNA-203,miR-203)表达质粒和Ⅳ型胶原蛋白α4链(collagen typeⅣalpha 4,COL4A4) 3'UTR荧光素酶报告基因,探究miR-203对COL4A4的靶向调控作用及其在口腔黏膜下纤维化(oral submucous fibrosis,OSF)中的作用.方法 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.收集2016年1至4月在中南大学湘雅口腔医院黏膜科就诊、具有典型病理特征、未经治疗、不伴其他口腔疾病的9例OSF患者颊黏膜组织(OSF组)及其配对的正常组织(对照组),应用蛋白质印迹法检测两组标本中COL4A4的表达,实时定量聚合酶链反应法检测miR-203的相对表达量.通过生物信息学软件(http://www.targetscan.org/)寻找COL4A4基因3UTR与miR-203之间的作用位点.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共60 bp片段分别克隆入哺乳动物细胞miRNA表达报告载体(pMIR-REPORT Luciferase)荧光素酶报告基因载体,将其分别与β-半乳糖苷酶表达质粒及miR-203表达载体共转染293T细胞,24 h后检测报告基因活性.结果 COL4A4的相对表达水平OSF组(2.46±2.26)显著高于对照组(0.70±0.49)(P＜0.05);miR-203的相对表达水平(2-△△CT) OSF组(2.57±2.09)显著低于对照组(154.07±191.11)(P＜0.01).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(位点1),3个非保守作用位点(位点2,3,4);miR-203能明显抑制位点1介导的报告基因活性,能部分抑制位点2与位点4介导的报告基因活性,而对位点3介导的报告基因活性无显著影响.结论 在OSF组织中,miR-203与COL4A4的蛋白表达呈负相关,miR-203靶向结合COL4A4基因3'UTR,抑制COL4A4蛋白表达.

abstractsObjective To explore the effect of microRNA-203(miR-203) on the expression of collagen type Ⅳ alpha 4(COL4A4) and its role in oral submucous fibrosis(OSF).Methods It has been revealed that COL4A4 is the putative target of miR-203 by retrieving a variety of bioinformatics software tools.The expression of COL4A4 protein in the indicated tissues was examined by Western blotting analysis and the potential binding sites of miR-203 and COL4A4-3'UTR was analyzed by using bioinformatics.This study integrated a fragment of COL4A4 3'UTR containing the target sequence into the pMIR-REPORT luciferase vector.Then,the COL4A4 3'UTR-luciferase and miR-203 were contransfected into 293T cells.Beta-gal was used as a control to monitor transfection efficiency.Cells were harvested and luciferase activity was measured after 24 h of transfection.Results The relative expression mean valves of COL4A4 were 2.46±2.26 in OSF and 0.70±0.49 in normal control.The expression of COL4A4 in OSF was higher than normal control(P＜0.05) and the difference between groups was statistically significant.The relative expression mean valves of miR-203 were 2.57±2.09 in OSF and 154.07±191.11 in normal control.The expression of miR-203 in OSF was lower than in normal control(P＜0.01) and the difference between groups was statistically significant.Bioinformatics analysis revealed that the COL4A4 3'UTR had a conserved site (site 1) and 3 non-conserved sites(site 2,site 3,site4) complementary to the miR-203.MiR-203 could significantly inhibit site 1-luciferase reporter activity,partially inhibited site 3-and site 4-1uciferase reporter activities,while no significant effect on site 3-luciferase reporter activity.Conclusions MiR-203 was negatively correlated with the expression of COL4A4 protein in OSF.Moreover,miR-203 repressed the expression of COL4A4 by targeting 3'UTR site of COL4A4 mRNA.