miR-535靶向GAB2基因通过激活PI3K/AKT信号通路促进山羊颗粒细胞增殖  

miR-535 Targets the GAB2 Gene to Promote Goat Granulosa Cell Proliferation Through Activation of the PI3K/AKT Signaling Pathway

作　　者：王鹏[1] 刘子嶷 刘玉芳 储明星[1] WANG Peng;LIU ZiYi;LIU YuFang;CHU MingXing(Institute of Animal Science,Chinese Academy of Agricultural Sciences/State Key Laboratory of Animal Biotech Breeding,Beijing 100193)

机构地区：[1]中国农业科学院北京畜牧兽医研究所/畜禽生物育种全国重点实验室,北京100193

出　　处：《中国农业科学》2023年第23期4757-4771,共15页Scientia Agricultura Sinica

基　　金：国家自然科学基金(32102509);财政部和农业农村部国家现代农业产业技术体系(CARS-38);中国农业科学院科技创新工程(CAAS-ZDRW202106和ASTIP-IAS13)。

摘　　要：【背景】MicroRNA(miRNA)是长度为18—25 nt的短RNA分子,在哺乳动物卵巢颗粒细胞调控卵泡发育过程中起着重要作用。团队前期对云上黑山羊高、低产羔数个体卵巢转录组测序结果显示,miR-535能够影响山羊产羔数,但具体调控机制尚不清楚。【目的】通过研究miR-535靶向GAB2(GRB2 associated binding protein 2)及其相关信号通路PI3K/AKT对山羊颗粒细胞增殖的影响,进一步探讨其分子生物学调控机制。【方法】在本研究中,选择具有两胎以上产羔记录的高、低产云上黑山羊各3只,同期发情处理后采集卵泡期卵巢组织,收集原代颗粒细胞。使用荧光定量PCR(reverse transcription-quantitativePCR,RT-qPCR)检测miR-535和GAB2在云上黑山羊高、低产卵巢组织中的表达量。构建GAB2的过表达/干扰载体,使用RT-qPCR、Western blot、免疫荧光、CCK8、EdU和细胞凋亡检测候选基因GAB2对山羊卵巢颗粒细胞增殖的影响。使用miRDB和miRanda软件预测miR-535和GAB2的靶向关系,构建GAB2的野生型和突变型载体,利用双荧光素酶活性检验检测miR-535和GAB2的靶向关系。构建过表达/干扰miR-535载体,探究其颗粒细胞增殖及下游基因功能的影响。【结果】RT-qPCR结果显示,GAB2在高产云上黑山羊卵巢组织中的表达量显著低于低产个体,miR-535的表达则相反(P<0.05);随后,RT-qPCR和Westernblot结果表明,在颗粒细胞中过表达GAB2后,CCND2、CDK4和BCL2的表达量显著升高(P<0.05),BAX的表达量显著降低(P<0.05),抑制其表达则与之相反;EdU和CCK8检测显示,GAB2过表达后显著促进颗粒细胞增殖(P<0.05),抑制其表达后则相反;双荧光素酶报告试验表明,miR-535抑制了GAB2基因3’UTR区域的双荧光素酶活性。RT-qPCR和Western blot结果显示,在颗粒细胞中过表达miR-535后,GAB2、CCND2、CDK4和BCL2的表达量显著降低(P<0.05),BAX的表达量显著升高(P<0.05),抑制其表达后则与之相反;EdU和CCK8检测显示,miR-535【Background】MicroRNAs(miRNAs)are short RNA molecules of 18-25 nt in length that play an important role in the regulation of follicle development in mammalian ovary granulosa cells(GCs).The previous sequencing of the transcriptome of the ovaries of high and low kidding individuals in Yunshang black goats showed that miR-535 was able to influence the kidding number of goats,but the specific regulatory mechanism was not yet clear.【Objective】The aim of this study was to investigate the molecular mechanisms of miR-535 targeting the GRB2 associated binding protein 2(GAB2)and its associated signaling pathway PI3K/AKT affected the proliferation of goat GCs,so as to further investigate the molecular biological regulation mechanism.【Method】In this study,three high-and low-fertility Yunshang black goats with the kidding number record of more than two litters were selected,and their follicular ovarian tissues were collected after synchronous estrus treatment for collecting primary GCs.The expression of miR-535 and GAB2 in high-and low-yield ovary tissues of Yunshang black goats was detected by reverse transcription-quantitative PCR(RT-qPCR).The overexpression/inhibitor of GAB2 vector was constructed and the effect of candidate GAB2 on the proliferation of goat GCs was detected using RT-qPCR,Western blot,immunofluorescence,CCK8,EdU and Apoptosis,respectively.The prediction of the targeted relationship between miR-535 and GAB2 was performed with miRDB and miRanda software.The Wild-type and Mutant vectors of GAB2 were constructed and the targeting relationship between miR-535 and GAB2 was detected by the dual luciferase activity assay.The overexpression/inhibitor miR-535 vector was constructed to explore the effect of its GCs proliferation and downstream gene function.【Result】The RT-qPCR results showed that the expression of GAB2 was significantly lower in ovarian tissues of Yunshang black goats with high-fertility than that in low-fertility groups,and the expression of miR-535 was the opposite(P<0.05).The resul

关 键 词：山羊产羔数 卵巢颗粒细胞增殖 GAB2基因 miR-535 P13K/AKT信号通路 

分 类 号：S827[农业科学—畜牧学]