论文部分内容阅读
目的:探讨micRNA-224(miR-224)在卵巢癌(ovarian cancer，OV)中的表达以及其对于OV患者预后的影响。方法:应用GEO以及TCGA数据库筛选OV中异常表达的miRNA。qRT-PCR以及Western blot检测正常细胞以及卵巢癌细胞中miR-224和TNRC6A的表达水平。通过miR-224 inhibitor以及其阴性对照(negative control，NC)转染至SKOV3细胞中。通过CCK-8检测miR-224 inhibitor转染后SKOV3细胞活性的影响。流式细胞术以及AO/EB染色检测转染miR-224 inhibitor后对SKOV3细胞凋亡的作用。将n TNRC6A(trinucleotide repeat-containing gene 6a)野生型(WT)和突变型(Mut)荧光素酶报告质粒与miR-224 inhibitor或miR-NC inhibitor共转染，采用荧光素酶报告系统分析n TNRC6A在SKOV3细胞中的荧光素酶活性。TCGA数据库验证miR-224与n TNRC6A在卵巢癌中的相关性以及n TNRC6A在OV患者的表达以及对于不良预后的影响。n 结果:miR-224在OV患者中高表达(n P<0.001)，且低表达miR-224卵巢癌患者生存时间明显高于miR-224高表达卵巢癌患者(n P＝0.004)。低表达miR-224能够抑制SKOV3细胞活性(n P＝0.024)，并诱导SKOV3细胞凋亡。生物信息学筛选n TNRC6A是miR-224的潜在靶基因。OV患者中n TNRC6A与miR-224表达呈负相关(n P＝0.001)。同时，Western blot以及qRT-PCR结果显示低表达miR-224能够使SKOV3细胞中n TNRC6A的表达升高。荧光素酶活性检测结果显示miR-224能与n TNRC6A基因的3′-UTR结合并负向调控(n P＝0.024)。TCGA数据库结果表明，n TNRC6A在OV患者中低表达(n P＝0.001)，过表达n TNRC6A的OV患者生存时间较低表达的时间长(n P＝0.018)。n 结论:miR-224靶向调控n TNRC6A调节卵巢癌细胞活性，参与OV患者不良预后。n “,”Objective:To investigate the expression of microRNA-224(miR-224)in ovarian cancer(OV)and its effect on the prognosis of OV patients.Methods:GEO and TCGA databases were used to screen differentially expressed miRNAs in OV.The expression levels of miR-224 and TNRC6A in normal cells and ovarian cancer cells were detected by quantitative reverse transcription polymerase chain reaction(qRT-PCR)and Western blot.SKOV3 cells were transfected with miR-224 inhibitor and its negative control (negative control, NC). The effect of miR-224 inhibitor transfection on SKOV3 activity was detected by CCK-8.The effect of miR-224 inhibitor transfection on SKOV3 cell apoptosis was detected by flow cytometry and AO/EB staining.n TNRC6A wild-type(WT)and mutant(Mut)luciferase reporter plasmids were co-transfected with miR-224 inhibitor or miR-NC inhibitor, and luciferase activity was analyzed by luciferase reporter system.TCGA database was used verity the correlation between miR-224 and n TNRC6A in OV, the expression of TNRC6A in OV patients and the impact of prognosis.n Results:miR-224 was highly expressed in OV patients (n P<0.001), and the survival time of patients with low expression of miR-224 of OV patients was significantly higher than that of patients with high expression of miR-224 (n P=0.004). Low expression of miR-224 could inhibit cell viability of SKOV3 cells (n P=0.024) and induce SKOV3 cell apoptosis.Bioinformatics screening n TNRC6A is a potential target gene of miR-224.Moreover, there was a negative correlation between TNRC6A and miR-224 expression in OV patients (n P=0.001). Western blot and qRT-PCR showed that silence of miR-224 could increase the expression of n TNRC6A in SKOV3 cells.The results of luciferase activity showed that miR-224 could bind to the 3′-UTR of n TNRC6A gene and negatively regulate it (n P=0.024). The results of TCGA database showed that n TNRC6A was expressed lower levels in OV patients (n P=0.001), and the survival time of OV patients with high expression of n TNRC6A A was longer than that of OV patients with low expression (n P=0.018).n Conclusion:miR-224 regulates OV activity by targeting n TNRC6A and affects the survival time of OV patients.n