摘要
目的探讨miR-30a-5p调节BDNF/Trk B信号对口腔鳞状细胞癌(OSCC)的作用及分子机制。方法人OSCC CAL-27细胞转染后,设置为miR-30a-5p mimic组(miR-30a-5p的模拟物)、NC mimic组(阴性对照模拟物),同时设未转染CAL-27细胞为Control组,采用实时荧光定量PCR检测各组细胞miR-30a-5p和脑源性神经营养因子(BDNF)基因表达,采用Western blot实验检测各组细胞BDNF、酪氨酸激酶受体(Trk B)及磷酸化酪氨酸激酶受体(p-TrkB)蛋白表达;取CAL-27细胞,建立小鼠皮下移植瘤模型,分为miR-30a-5p agomir(miR-30a-5p激动剂)组及NC agomir(阴性对照激动剂)组,测量干预后第7~28天肿瘤体积和质量的变化,采用实时荧光定量聚合酶链式反应(Real-time,PCR)检测各组小鼠瘤组织miR-30a-5p和BDNF基因表达,采用Western blot实验检测各组瘤组织BDNF、Trk B及p-TrkB蛋白表达,采用苏木素伊红染色(HE)观察各组瘤组织学变化,采用双荧光素酶报告基因实验检测miR-30a-5p与BDNF的靶向关系。结果与NC mimic组比较,miR-30a-5p mimic组CAL-27细胞miR-30a-5p表达明显上调,BDNF mRNA及BDNF、Trk B、p-TrkB蛋白均明显下调(P<0.01);与NC agomir组比较,miR-30a-5p agomir组小鼠第14~28天肿瘤体积和质量降低(P<0.05或P<0.01);HE染色结果显示,NC agomir组小鼠肿瘤组织的肿瘤细胞排列紧密且不规则、可见核大深染、核固缩及病理性核分裂,miR-30a-5p agomir组肿瘤细胞减少、可见部分核分裂、核深染、核固缩、并出现核坏死;与NC agomir组比较,miR-30a-5p agomir组小鼠瘤组织miR-30a-5p表达明显上调,BDNF mRNA及BDNF、Trk B、p-TrkB蛋白均明显下调(P<0.01);双荧光素酶报告基因实验结果显示,miR-30a-5p与BDNF能够靶向结合。结论 miR-30a-5p能够靶向结合BDNF,抑制BDNF/Trk B信号活化,进而抑制OSCC的进展。
Objective To explore the effect of the regulation of miR-30 a-5 p of BDNF/TrkB signaling on oral squamous cell carcinoma( OSCC) and its molecular mechanism. Methods After transfection of human OSCC CAL-27 cells,a miR-30 a-5 p mimic group( miR-30 a-5 p mimic) and an NC mimic group( negative control mimic) were set up. At the same time,untransfected CAL-27 cells were set up as the control group. Real-time fluorescence quantitative PCR was used to detect the gene expression of miR-30 a-5 p and brain-derived neurotrophic factor( BDNF) in each group. Western blot experiments were used to detect the protein expression of BDNF,Tyrosine kinase receptor( Trk B) and Phosphorylated tyrosine kinase receptor( p-TrkB) in each group. CAL-27 cells were taken to establish a mouse subcutaneous xenograft model,which was divided into a miR-30 a-5 p Agomir( miR-30 a-5 p agonist) group and an NC Agomir( negative control agonist) group. The changes of tumor volume and mass from 7 to 28 days after intervention were measured,real-time fluorescence quantitative PCR was used to detect the gene expression of miR-30 a-5 p and BDNF of tumor tissues in mice in each group.Western blot experiments were used to detect the expression of BDNF,Trk B,and p-TrkB proteins of tumor tissues in each group,and the pathological changes of tumor tissues were detected by HE staining. The targeting relationship between miR-30 a-5 p and BDNF was verified by dual-luciferase reporter gene assay. Results Compared with the NC mimic group,miR-30 a-5 p expression in CAL-27 cells of the miR-30 a-5 p mimic group was significantly up-regulated,BDNF mRNA,BDNF,TrkB,and p-TrkB proteins were significantly down-regulated( P < 0. 01);Compared with the NC agomir group,the tumor volume and quality of the miR-30 a-5 p agomir mice group were significantly reduced from 14 days to 28 days( P < 0. 05 or P < 0. 01);HE staining results showed that the tumor cells in the NC Agomir mice group were arranged closely and irregularly,with large hyperchromatic nuclei,nuclear pyknosis,and pat
作者
马明
刘滨
MA Ming;LIU Bin(The Second Affiliated Hospital of Shenyang Medical College,Shenyang 110002,Liaoning,China)
出处
《贵州医科大学学报》
CAS
2021年第2期190-197,共8页
Journal of Guizhou Medical University
基金
沈阳医学院科学基金项目(20181009)。
