MicroRNA-21-5p调控丝裂原活化蛋白激酶p38信号通路

MicroRNA-21-5p regulating p38 mitogen-activated protein kinases signaling pathway

目的验证microRNA（miR）-21-5p与丝裂原活化蛋白激酶激酶3（MKK3）的靶向调控关系。方法构建MKK33＇UTR报告载体（MKK3-3U）及突变载体（MKK3-3U-M）；化学合成miR．21-5p模拟物（mimics）、miR-21-5p抑制剂（inhibitor）、无意义miR（NC）。分别用NC（NCl组）、mimics（mimicl组）、inhibitor（inhibitorl组）与MKK3-3U共转染人胚肾（HEK293）细胞。分别用空载体（NC2组）、MKK3-3U（Wt组）、MKK3-3U-M（Mut组）与mimics共转染HEK293。双荧光素酶检测仪检测细胞荧光比值。予NC（对照组）、mimics（mimic2组）、inhibitor（inhibitor2组）分别干预人肾小管上皮（HK-2）细胞，反转录PCR、Westernblot法检测HK-2细胞中MKK3的mRNA及蛋白表达水平。结果1．NCl组、mimicl组、inhibitorl组荧光比值分别为100．00％、67．99％、133．17％，mimicl组与NCl组比较，差异有统计学意义（t=19．93，P〈0．01）。2．NC2组、Mt组、Mut组荧光比值分别为100．00％、65．30％、98．48％，Mt组与NC2组比较，差异有统计学意义（t=14．39，P〈0．01），而Mut组与NC2组间荧光比值差异无统计学意义（t=0．63，P〉0．05）。3．对照组、mimic2组、inhibi-tor2组MKK3mRNA相对表达量为1．36±0．02、1．01±0．04、1．43±0．06；蛋白质相对表达量为0．97±0．05、0．62±0．06、1．36±0．32，mimic2组较对照组MKK3的mRNA和蛋白表达均明显降低，差异均有统计学意义（F=85．98、118．26，P均〈0．01）。结论MKK3是miR-21-5p的靶基因，miR-21．5p在mRNA和蛋白质水平调控MKK3表达，miR-21-5p可能是调控p38丝裂原活化蛋白激酶信号通路的重要分子。

Objective To verify the targeting regulatory relationship between microRNA（miR）-21-5p and mi- togen-aetivated protein kinase kinase 3 （MKK3）. Methods miR-21-Sp and MKK3 targeted relationship was analyzed by bioinformaties database predict. Constructing MKK3 3 ＇UTR reporter vector （MKK3-3 U） and mutation reporter vec- tor （MKK3-3 U-M ） was constructed by synthesizing miR-21-Sp mimics （mimic） , miR-21-Sp inhibitor （inhibitor） , non- sense miR （NC）. NC （ NC1 group） , mimics （ mimicl group） , inhibitor （ inhibitorl group） together with MKK3-3 U co- transfeeted human embryonic kidney （HEK293） cells, respectively. Empty veetor-pYr-MirTarget （NC2 group） , MKK3- 3U （Mt group）, MKK3-3U-M （Mut group） together with mimic co-transfected HEK293 ,respectively. The fluorescence ratio was detected by dual lueiferase 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 NCI group,mimicsl group,and inhibitorl group were 100.00% ,67.99% , 133.17% , and there was difference between mimiel 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-Sp, miR-21-5p can regulate the expression of MKK3 both in mRNA and protein levels, miR-21-Sp may be an important regulatory molecule in p38 mitogen-aetivated protein kinase signaling pathway.