MicroRNA-21-5p regulating p38 mitogen-activated protein kinases signaling pathway
10.3760/cma.j.issn.2095-428X.2014.05.016
- VernacularTitle:MicroRNA-21-5p调控丝裂原活化蛋白激酶p38信号通路
- Author:
Zhihui LI
;
Xu DENG
- Publication Type:Journal Article
- Keywords:
MicroRNA-21-5p;
Mitogen-activated protein kinase kinase 3;
p38 mitogen-activated protein kinase kinase;
Target gene;
Regulation
- From:
Chinese Journal of Applied Clinical Pediatrics
2014;29(5):375-379
- CountryChina
- Language:Chinese
-
Abstract:
Objective To verify the targeting regulatory relationship between microRNA (miR)-21-5p and mitogen-activated protein kinase kinase 3 (MKK3).Methods miR-21-5p and MKK3 targeted relationship was analyzed by bioinformatics database predict.Constructing MKK3 3'UTR reporter vector (MKK3-3U) and mutation reporter vector (MKK3-3 U-M) was constructed by synthesizing miR-21-5p mimics (mimic),miR-2 1-5p inhibitor (inhibitor),nonsense miR (NC).NC (NC1 group),mimics (mimic1 group),inhibitor (inhibitor1 group) together with MKK3-3U cotransfected human embryonic kidney (HEK293) cells,respectively.Empty vector-pYr-MirTarget (NC2 group),MKK33U (Mt group),M KK3-3U-M (Mut group) together with mimic co-transfected HEK293,respectively.The fluorescence ratio was detected by dual luciferase assay.NC,mimics,inhibitor intervened human renal tubular epithelial (HK-2) cells,respectively.RT-PCR and Western blot were used to test cell MKK3 expression level in HK-2.Results 1.The fluorescence ratios of NC1 group,mimicsl group,and inhibitor1 group were 100.00％,67.99％,133.17％,and there was difference between mimic1 group and NC1 group(t =19.93,P ＜ 0.05).2.The fluorescence ratios of NC2 group,Mt group and Mut group were 100.00％,65.30％,98.48％,and there was difference between Mt group and NC2 group (t =14.39,P ＜ 0.05).There was no significant difference between NC2 and Mut group (t =0.63,P ＞ 0.05).3.MKK3 mRNA relative expression levels of the control group mimic2 group and inhibitor2 group were 1.36 ± 0.02,1.01 ± 0.04,1.43 ± 0.06 ; relative protein expression levels were 0.97 ± 0.05,0.62 ± 0.06,1.36 ± 0.32.MKK3 mRNA and protein levels of mimic2 group were significantly lower compared with the control group (F =85.98,118.26,all P ＜ 0.01).Conclusions MKK3 is the target gene of miR-21-5p.miR-21-5p can regulate the expression of MKK3 both in mRNA and protein levels.miR-21-5p may be an important regulatory molecule in p38 mitogen-activated protein kinase signaling pathway.
