乌梅总黄酮对MPP+诱导的SH-SY5Y细胞中miR-145-3p/CREB5轴的影响

Effects of Fructus mume total flavone on miR-145-3p/CREB5 axis in SH-SY5Y cells induced by MPP+

目的研究乌梅总黄酮(Fructus mume total flavone,FMF)对MPP^(+)诱导人神经母细胞瘤SH-SY5Y细胞基因表达的影响。方法采用CCK-8筛选FMF及MPP^(+)作用于SH-SY5Y细胞的最佳浓度与时间。收集对照组、模型组(MPP^(+)诱导)和FMF组(MPP^(+)诱导+FMF干预)SH-SY5Y细胞,进行全基因转录组高通量测序,并筛选差异表达的miRNAs和mRNAs。通过生信软件分析miR-145-3p潜在靶基因,并与差异表达mRNAs相交取交集。将SH-SY5Y细胞分为6组:对照组、模型组、miR-145-3p mimics组、mimics NC组、miR-145-3p inhibitor组、inhibitor NC组。qRT-PCR检测细胞miR-145-3p表达水平,Western blot检测细胞CREB5蛋白表达水平,双荧光素酶报告基因实验检测细胞荧光活性。结果对照组与模型组间比较,存在33个差异表达miRNAs,模型组与FMF组间比较,存在16个差异表达miRNAs,取交集后,得到7个差异表达的miRNAs,其中miR-145-3p在模型组下调,在FMF组上调。经分析获到4个miR-145-3p调控的差异靶基因:CREB5、EGLN1、NTNG1和SLC22A15。与对照组比较,模型组miR-145-3p表达水平显著降低,CREB5蛋白表达水平显著升高。与NC组比较,miR-145-3p mimics组miR-145-3p表达水平显著升高,CREB5蛋白表达水平显著降低,miR-145-3p inhibitor组miR-145-3p表达水平显著降低,CREB5蛋白表达水平显著升高。双荧光素酶报告基因结果显示miR-145-3p与CREB5存在靶向调控关系。结论miR-145-3p和CREB5均在MPP^(+)诱导的SH-SY5Y细胞中差异表达,FMF可调控miR-145-3p/CREB5轴在细胞中的表达水平。

Objective To study the effects of Fructus mume total flavone(FMF)on gene expression of MPP^(+)induced human neuroblastoma SH-SY5Y cells.Methods CCK-8 was used to screen the optimal concentrations and time points of FMF and MPP^(+)acting on SH-SY5Y cells.SH-SY5Y cells from control group,model group(MPP^(+)induction)and FMF group(MPP^(+)induction+FMF intervention)were collected for high-throughput sequencing of whole gene transcriptome,and the differentially expressed miRNAs and mRNAs were screened.The potential target genes of miR-145-3p were analyzed by bioinformatics software,and intersected with differentially expressed mRNAs.SH-SY5Y cells were divided into 6 groups:control,model,miR-145-3p mimics,mimics NC,miR-145-3p inhibitor,and inhibitor NC.qRT-PCR was used to detect the expression level of miR-145-3p in cells.Western blot was used to detect the expression level of CREB5 protein in cells.And double luciferase reporter gene assay was used to detect the fluorescence activity of cells.Results There were 33 differentially expressed miRNAs between the control group and the model group,and 16 differentially expressed miRNAs between the model group and the FMF group.After taking the intersection,seven differentially expressed miRNAs were obtained,of which miR-145-3p was down-regulated in the model group and up-regulated in the FMF group.After analysis,four differential target genes regulated by miR-145-3p were obtained:CREB5,EGLN1,NTNG1,and SLC22A15.Compared with the control group,the expression level of miR-145-3p in the model group decreased significantly,and the expression level of CREB5 protein increased significantly.Compared with NC group,miR-145-3p expression level in miR-145-3p mimics group was significantly higher,CREB5 protein expression level was significantly lower,miR-145-3p expression level in miR-145-3p inhibitor group was significantly lower,CREB5 protein expression level was significantly higher.The results of double luciferase reporter gene showed that there was a targeted regulatory relationship between miR-145-3p and CREB5.Conclusion Both miR-145-3p and CREB5 are differentially expressed in SH-SY5Y cells induced by MPP^(+).FMF can regulate the expression level of miR-145-3p/CREB5 axis in cells.