人软骨肉瘤中miR-140-5p靶基因预测验证及对靶基因表达的调控作用观察

Prediction and verification of target gene of miR-140-5p in human chondrosarcoma and regulation of miR-140-5p on expression of target gene

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。

Objective To predict and identify the target gene of miR-140-5p and to observe the regulation of miR-140-5p on its target gene in human chondrosarcoma.Methods①Target gene prediction and verification of miR-140-5p in human chondrosarcoma:The miR-140-5p 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-140-5p in human chondrosarcoma.Glucose transporter 1(GLUT-1)was identified as the likely target gene of miR-140-5p in human chondrosarcoma.GLUT-1 3′UTR sequences were retrieved by NCBI GenBank.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 293T were divided into groups A and B,which were transfected with miR-140-5p mimics(promoting miR-140-5p expression)+WT GLUT-1 3′UTR fluorescein reporter vector,miR-140-5p 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-140-5p 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-140-5p 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.Results①The direct target gene of miR-140-5p 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<0.05.②The 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.ConclusionsThe miR-140-5p could directly target GLUT-1 in human chondrosarcoma.The miR-140-5p 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-140-5p inhibits the mRNA transcription and protein translation process of GLUT-1 primarily by direct targeting to the GLUT-1 3′UTR.