【摘要】 目的预测并验证人软骨肉瘤中miR-140-5p的靶基因,观察人软骨肉瘤中miR-140-5p对靶基因的调控作用。方法①miR-140-5p在人软骨肉瘤中的靶基因预测及验证:采用microRNA. org软件检索miR-140-5p序列,miRBase、miRWalk靶基因预测分析数据库预测miR-140-5p在人软骨肉瘤中的靶基因,认为miR-140-5p在人软骨肉瘤中的靶基因可能为葡萄糖转运蛋白1(GLUT-1)。通过NCBI Gen Bank检索GLUT-1 3’UTR序列。构建野生型(WT,正常表达)、突变型(MUT,抑制表达) GLUT-1 3’UTR双荧光素酶报告载体。取人胚肾细胞HEK 293T分为A、B组,分别转染miR-140-5p mimics(促进miR-140-5p表达)+WT型GLUT-1 3’UTR荧光素报告载体、miR-140-5p mimics+MUT型GLUT-1 3’UTR荧光素报告载体,利用荧光素酶活性检测试剂盒测算萤火虫荧光素酶相对活性。②转染miR-140-5p对人软骨肉瘤细胞株JJ012的GLUT-1 mRNA及蛋白表达影响:取对数生长期JJ012细胞分为观察组、对照组,分别转染miR-140-5p mimics、NC mimics(空白无意义),转染24 h时采用qRT-PCR法检测两组细胞GLUT-1 mRNA,采用Western blotting法检测两组细胞GLUT-1蛋白。结果①miR-140-5p在人软骨肉瘤中的直接靶基因可能为GLUT-1。A、B组荧光素酶活性分别为0. 37±0. 03、0. 93±0. 01,二者比较,P <0. 05。②观察组、对照组细胞GLUT-1 mRNA相对表达量分别为0. 31±0. 10、0. 95±0. 06,二者比较,P <0. 05。观察组、对照组细胞GLUT-1蛋白相对表达量分别为0. 42±0. 13、0. 93±0. 07,二者比较,P <0. 05。结论 miR-140-5p在人软骨肉瘤中的靶基因可能为GLUT-1。miR-140-5p主要通过靶向结合GLUT-1 3’UTR区域进行mRNA转录后调控。miR-140-5p主要通过直接靶向作用于GLUT-1 3’UTR进而抑制蛋白翻译过程。更多还原

【Abstract】 Objective To predict and identify the target gene of miR-140-5 p and to observe the regulation of miR-140-5 p on its target gene in human chondrosarcoma. Methods ① Target gene prediction and verification of miR-140-5 p in human chondrosarcoma: The miR-140-5 p sequence was retrieved by using the microRNA. org software,and the miRBase and miRWalk target gene predictive analysis databases were used to predict the target gene of miR-140-5 p in human chondrosarcoma. Glucose transporter 1( GLUT-1) was identified as the likely target gene of miR-140-5 p in human chondrosarcoma. GLUT-1 3’UTR sequences were retrieved by NCBI Gen Bank. The wild type( WT,normal expression) and mutant type( MUT,inhibited expression) GLUT-1 3’-UTR dual-luciferase reporter vector was constructed. Human embryonic kidney cells HEK 293 T were divided into groups A and B,which were transfected with miR-140-5 p mimics( promoting miR-140-5 p expression) + WT GLUT-1 3’ UTR fluorescein reporter vector,miR-140-5 p mimics + MUT type GLUT-1 3’ UTR fluorescein reporter vector,and the relative activity of firefly luciferase was measured by luciferase activity assay kit. ②Effect of transfection of miR-140-5 p on GLUT-1 mRNA and protein expression in human chondrosarcoma cell line JJ012:JJ012 cells in the logarithmic growth phase were divided into the observation group and control group,which were transfected with miR-140-5 p mimics and NC mimics,respectively. After transfection for 24 h,the GLUT-1 mRNA expression was detected by qRT-PCR,and GLUT-1 protein expression was detected by Western blotting. Results ①The direct target gene of miR-140-5 p in human chondrosarcoma may be GLUT-1. The luciferase activities of group A and group B were 0. 37± 0. 03 and 0. 93 ± 0. 01,respectively,with statistically significant difference,P < 0. 05. ② The relative expression levels of GLUT-1 mRNA in the observation group and the control group were 0. 31 ± 0. 10 and 0. 95 ± 0. 06,respectively,with statistically significant difference,P < 0. 05. The relative expression levels of GLUT-1 protein in the observation group and the control group were 0. 42 ± 0. 13 and 0. 93 ± 0. 07,respectively,with statistically significant difference,P < 0. 05. Conclusions The miR-140-5 p could directly target GLUT-1 in human chondrosarcoma. The miR-140-5 p primarily regulates post-transcriptional regulation of mRNA by targeting to the GLUT-1 3’ UTR region,and inhibits the protein translation process primarily by directly targeting on the GLUT-1 3’UTR. The miR-140-5 p inhibits the mRNA transcription and protein translation process of GLUT-1 primarily by direct targeting to the GLUT-1 3’ UTR.更多还原