miR-626下调HepG2细胞载脂蛋白(a)的表达

Hsa-miR-626 down-regulates expression of apolipoprotein(a) in HepG2 cells

目的筛选高效调控LPA基因表达的miRNA。方法用在线预测工具预测作用于LPA基因3'UTR的miRNA,RT-PCR和Western blot检测转染预测所得miRNA mimic后HepG2细胞载脂蛋白(a)[Apo(a)]mRNA和蛋白表达水平。实验分为对照组、miR-626 mimic组、miR-626 mimic+miR-626 inhibitor组和miR-626 inhibitor组共4组。使用荧光素酶报告系统进行miR-626靶标验证实验。结果可作用于LPA基因3'UTR的miRNA有9个。mimic转染组与对照组相比,Apo(a)的mRNA表达水平差异不显著,但miR-626能显著下调Apo(a)蛋白的表达。使用miR-626 inhibitor后,miR-626对Apo(a)蛋白的下调作用减弱。转染miR-626后细胞裂解液的荧光强度显著下降。结论miR-626能显著下调Apo(a)蛋白表达水平,其通过与LPA mRNA 3'UTR序列结合,抑制LPA mRNA翻译实现的。

Aim To screen miRNA for efficient regulation of LPA gene expression. Methods The miRNA of LPA gene 3＇UTR were predicted by on-line prediction tools. The mRNA and protein expression of apolipoprotein （a） （ Apo （a） ） were detected by RT-PCR and Western blot after transfeetion. The experiment was divided into 4 groups ： control group, miR-626 mimic group, miR-626 mimie＋miR-626 inhibitor group and miR-626 inhibitor group. The miR-626 target validation assay was performed using a lueiferase reporter system. Results There were 9 miRNA binding 3＇UTR of LPA gene. Compared with the control group, the expression of Apo（a） mRNA was not significantly different between the mimic transfeeted group and the control group, but miR-626 significantly down-regulated the expression of Apo（a） pro-tein. MiR-626 attenuated Apo（a） protein down-regulation by miR-626 inhibitor. The fluorescence intensity of the miR- 626 cells was significantly lower than that of the control group after cell lysis. Conclusion MiR-626 down-regulates the expression of Apo（a） in HepG2 cells. MiR-626 down-regulates the expression of Apo（a） protein in LPA mRNA by direct binding to LPA mRNA 3＇UTR.