miR⁃7靶向调控CTSK对贵州黑山羊卵巢颗粒细胞增殖和迁移的影响  

Effect of miR⁃7 targeted regulation of CTSK on proliferation and migration of ovarian granulosa cells in Guizhou Capra hircus

作　　者：刘彬[1] 陆情梅 杨永鲜 周明帅 温晓艳 赵佳福[1] LIU Bin;LU Qing-mei;YANG Yong-xian;ZHOU Ming-shuai;WEN Xiao-yan;ZHAO Jia-fu(College of Animal Science,Guizhou University/Key Laboratory of Genetic Breeding and Reproduction of Plateau Mountain Animals of Ministry of Education,Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院/贵州大学高原山地动物遗传育种与繁殖教育部重点实验室,贵阳550025

出　　处：《西南农业学报》2024年第2期429-435,共7页Southwest China Journal of Agricultural Sciences

基　　金：贵州省科技计划项目(黔科合支撑〔2022〕一般089、黔科合支撑〔2022〕重点033);国家级大学生创新创业训练计划项目(202110657012)。

摘　　要：【目的】探明miR⁃7与CTSK基因相互作用关系及其对贵州黑山羊卵巢颗粒细胞增殖和凋亡的影响,为贵州黑山羊优良品种保藏、选育与开发利用提供依据。【方法】采用在线数据库筛选miR⁃7与CTSK基因3′UTR的结合靶点,通过将CTSK基因3′UTR插入双荧光素酶报告系统中进行互作位点检测;采用CCK⁃8和划痕试验检测miR⁃7是否靶向调控CTSK基因影响贵州黑山羊卵巢颗粒细胞的增殖和迁移;过表达miR⁃7后采用RT⁃qPCR检测其对促增殖蛋白PCNA、抗凋亡蛋白BCL2和促凋亡相关蛋白BAX、caspase⁃3、caspase⁃9表达水平的影响。【结果】成功构建CTSK基因3′⁃UTR 2个预测靶点的野生型和突变型双荧光素酶报告载体;双荧光素酶试验结果表明CTSK基因3′UTR区的2个预测靶位点均受miR⁃7调控;CCK⁃8和细胞划痕结果表明,转染miR⁃7试验组的卵巢颗粒细胞增殖和迁移能力均显著高于对照(NC)组;RT⁃qPCR结果表明,上调miR⁃7能极显著上调PCNA和BCL2的表达,抑制BAX、caspase⁃3和caspase⁃9的表达。【结论】miR⁃7可以靶向结合CTSK的3′UTR区2个靶位点并极显著抑制CTSK基因的表达;过表达miR⁃7可以显著促进贵州黑山羊卵巢颗粒细胞增殖和迁移能力,并促进增殖标志基因、抗凋亡基因的表达和抑制促凋亡相关基因的表达。研究结果可为进一步研究miR⁃7靶向调控CTSK表达影响贵州黑山羊繁殖性能的分子机制提供理论依据。【Objective】The interaction between miR⁃7 and CTSK genes and their effects on the proliferation and apoptosis of ovarian granulosa cells in Capra hircus were explored,so as to provide a basis for the preservation,breeding,development and utilization of good breeds of C.hircus.【Method】The online database was used to screen the binding targets of miR⁃7 and CTSK gene 3′UTR,and the interaction sites were detected by inserting the CTSK gene 3′UTR into the dual luciferase reporter system.CCK⁃8 and scratch test were used to detect whether miR⁃7 targeted CTSK gene to affect the proliferation and migration of ovarian granulosa cells in C.hircus.After overexpression of miR⁃7,RT⁃qPCR was used to detect its effect on the expression levels of PCNA,BCL2,BAX,caspase⁃3 and caspase⁃9.【Result】The study successfully constructed wild⁃type and mutant dual⁃luciferase reporter vectors for the 2 predicted targets in the 3′UTR region of the CTSK gene.The results of dual⁃luciferase assay showed that the 2 predicted targets in the 3′UTR region of the CTSK gene were regulated by miR⁃7.The results of CCK⁃8 and cell scratch showed that the proliferation and migration of the ovarian granulosa cells of the test group transfected with miR⁃7 were significantly higher than the control group.RT⁃qPCR results showed that up⁃regulation of miR⁃7 could significantly up⁃regulate the ex⁃pression of PCNA and BCL2,and inhibit the expression of BAX,caspase⁃3 and caspase⁃9.【Conclusion】miR⁃7 can target two target sites in the 3′UTR region of CTSK and inhibit the expression of CTSK gene.Overexpression of miR⁃7 can significantly promote the proliferation and migration of ovarian granulosa cells in C.hircus,and promote the expression of proliferation marker genes,anti⁃apoptotic genes and inhibit the expression of pro⁃apoptotic genes.The related research results can provide a basis for further study on the molecular mechanism of miR⁃7 targeted regulation of CTSK expression affecting the r

关 键 词：贵州黑山羊 miRNA CTSK 卵巢颗粒细胞 细胞增殖和迁移 

分 类 号：S827[农业科学—畜牧学]