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.
Objective 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.
出处
《中华口腔医学杂志》
CAS
CSCD
北大核心
2016年第9期526-531,共6页
Chinese Journal of Stomatology
