microRNA-96-5p靶向调控羊驼黑色素细胞中MITF基因的表达

doi:10.11843/j.issn.0366-6964.2020.007

摘要/Abstract

摘要： 旨在预测并验证调控小眼畸形相关转录因子（microphthalmia-associated transcription factor， MITF）基因表达的关键miRNA。使用在线软件对与MITF具有靶向关系的miRNAs进行预测。在羊驼黑色素细胞中，利用荧光定量PCR检测过表达和抑制表达miR-96-5p后MITF基因的表达量；通过免疫细胞化学技术对过表达和抑制表达miR-96-5p的细胞进行MITF特异抗体的染色。结果表明，miR-96-5p是调控MITF表达的一个关键miRNA，经荧光定量PCR、免疫细胞化学技术检测，当过表达miR-96-5p后，MITF mRNA水平不变，但其下游基因表达量极显著降低（P<0.01），MITF蛋白水平表达量与其下游基因的蛋白水平表达量全部极显著低于空白组（P<0.01），当抑制表达miRNA-96-5p后，MITF mRNA水平不变，但其下游基因表达量显著升高（P<0.01），MITF蛋白水平表达量与其下游基因的蛋白水平表达量全部极显著高于空白组（P<0.01）。miR-96-5p可以靶向结合MITF基因，在MITF转录后水平发挥调控作用，抑制MITF的转录，抑制MITF及其下游基因TYR、TYRP1、TYRP2的表达。

关键词: miR-96-5p, MITF, miRNAs预测, 靶基因验证

Abstract: The study aimed to predict and verify the key miRNA regulating the expression of microphthalmia-associated transcription factor (MITF). The online softwares were used to predict miRNAs with a targeted relationship to MITF in this study. In the melanocytes of alpaca, the expression of MITF gene after overexpression and inhibition of miR-96-5p was detected by fluorescence quantitative PCR. MITF-specific antibody staining was performed on cells in which the expression of miR-96-5p was overexpressed and inhibited by immunocytochemical techniques. The results showed that miR-96-5p was a key miRNA regulating the expression of MITF. The results of fluorescence quantitative PCR and immunocytochemical detection showed that when miR-96-5p was overexpressed, MITF mRNA level had no significant change, but the expressions of its downstream genes were extremely significantly reduced (P<0.01). MITF protein and its downstream proteins levels were extremely significantly lower than those in the blank groups (P<0.01). When miR-96-5p was inhibited, MITF mRNA level had no significant change, but the expressions of its downstream genes were extremely significantly increased (P<0.01). MITF protein and its downstream proteins levels were extremely significantly higher than those in the blank groups (P<0.01). In conclusion, miR-96-5p can target MITF gene and play a regulatory role at the post-transcriptional level of MITF, inhibiting the transcription of MITF and the expressions of its downstream genes of TYR, TYRP1 and TYRP2.

Key words: miR-96-5p, MITF, prediction of miRNAs, validation of target gene

中图分类号:

S829.9

张利环, 马悦悦, 刘文艳, 蓝吴涛, 朱芷葳. microRNA-96-5p靶向调控羊驼黑色素细胞中MITF基因的表达[J]. 畜牧兽医学报, 2020, 51(6): 1229-1237.

ZHANG Lihuan, MA Yueyue, LIU Wenyan, LAN Wutao, ZHU Zhiwei. microRNA-96-5p Targets MITF Gene in Alpaca Melanocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(6): 1229-1237.

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