过表达miRNA-424-5p靶向AKT3对奶牛子宫内膜上皮细胞凋亡的影响

doi:10.16431/j.cnki.1671-7236.2024.08.040

摘要/Abstract

摘要： 【目的】本试验通过过表达miRNA-424-5p,明确miRNA-424-5p与其靶基因丝氨酸/苏氨酸激酶3(AKT serine/threonine kinase 3,AKT3)对奶牛子宫内膜上皮细胞凋亡影响的机制,为阐明miRNA-424-5p在奶牛胎盘分离过程中的作用机制提供试验基础和理论依据。【方法】试验分为3组:空白对照组(Control)、阴性对照组(miRNA-424-5p mimics NC)和过表达组(miRNA-424-5p mimics)。采用茎环法实时荧光定量PCR检测转染miRNA-424-5p mimics后奶牛子宫内膜上皮细胞miRNA-424-5p的表达变化;采用Western blotting及实时荧光定量PCR检测过表达后AKT3和凋亡相关因子的表达变化,采用Annexin V-FITC/PI法检测奶牛子宫内膜上皮细胞凋亡情况。【结果】与空白对照及阴性对照组相比,转染miRNA-424-5p mimics后子宫内膜上皮细胞miRNA-424-5p mRNA的表达量极显著升高(P＜0.01),B淋巴细胞瘤-2 (B-cell lymphoma-2,Bcl-2)相关X蛋白(Bax)、半胱氨酸天冬氨酸蛋白酶3(cysteinyl aspartate specific proteinase 3,Caspase3)和Caspase9的mRNA表达量极显著升高(P＜0.01),AKT3和Bcl-2的mRNA表达量极显著降低(P＜0.01);Bax和Caspase3蛋白表达量极显著升高(P＜0.01),Caspase9蛋白表达量显著升高(P＜0.05),AKT3和Bcl-2 蛋白表达量极显著降低(P＜0.01);细胞凋亡率极显著升高(P＜0.01)。【结论】过表达miRNA-424-5p可通过靶向调控AKT3使奶牛子宫内膜上皮细胞凋亡因子Bax、Caspase3和Caspase9的表达量增加,使Bcl-2的表达量降低,进而促进细胞凋亡。

关键词: 奶牛; miRNA-424-5p; AKT3; 子宫内膜上皮细胞; 凋亡

Abstract: 【Objective】 In this study,overexpression of miRNA-424-5p was performed to clarify the mechanism of the effect of miRNA-424-5p and its target gene AKT3 on the apoptosis of cow endometrial epithelial cells,and provide experimental basis and theoretical basis for elucidating the mechanism of miRNA-424-5p in the process of cow placental separation.【Method】 The experiment was divided into three groups: Blank control group (Control), negative control group (miRNA-424-5p mimics NC) and overexpressed group (miRNA-424-5p mimics). The expression of miRNA-424-5p in cow endometrial epithelial cells after miRNA-424-5p mimics was detected by Real-time quantitative PCR using stem loop method.Western blotting and Real-time quantitative PCR were used to detect the expression changes of AKT3 and apoptosis-related factors after overexpression,and Annexin V-FITC/PI was used to detect the apoptosis of cow endometrial epithelial cells.【Result】 Compared with blank control and negative control groups,the expression of miRNA-424-5p mRNA in endometrial epithelial cells was extremely significantly increased (P＜0.01),the mRNA expression of Bax,Caspase3 and Caspase9 was extremely significantly increased (P＜0.01),and the mRNA expression of AKT3 and Bcl-2 was extremely significantly reduced (P＜0.01) after transfection with miRNA-424-5p mimics.The relative expression of Bax and Caspase3 proteins was extremely significantly increased (P＜0.01),Caspase9 protein was significantly increased (P＜0.05),and AKT3 and Bcl-2 proteins were extremely significantly reduced (P＜0.01).Apoptosis rate of cells was extremely significantly increased(P＜0.01).【Conclusion】 Overexpression of miRNA-424-5p could increase the expression of apoptotic factors Bax,Caspase3 and Caspase9,and decrease the expression of Bcl-2 in endometrial epithelial cells of dairy cows through targeted regulation of AKT3,thus leading to increased apoptosis.

Key words: dairy cows; miRNA-424-5p; AKT3; endometrial epithelial cells; apoptosis

中图分类号:

S858.23

姚伟佳, 罗春海, 刘佳金, 王薇, 李丹阳, 刘炳琦, 付世新. 过表达miRNA-424-5p靶向AKT3对奶牛子宫内膜上皮细胞凋亡的影响[J]. 中国畜牧兽医, 2024, 51(8): 3635-3642.

YAO Weijia, LUO Chunhai, LIU Jiajin, WANG Wei, LI Danyang, LIU Bingqi, FU Shixin. Effect of Overexpression of miRNA-424-5p Targeting AKT3 on Apoptosis of Endometrial Epithelial Cells in Dairy Cows[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(8): 3635-3642.

参考文献

[1] 黄光红.奶牛胎衣不下的不同治疗方法对其生产和繁殖性能的影响[J].上海畜牧兽医通讯,2013,4:52-53. HUANG G H.Effects of different treatment methods on the production and reproductive performance of dairy cows with fetal overgrowth[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2013,4:52-53.(in Chinese)
[2] KELTON D F,LISSEMORE K D,MARTIN R E.Recommendations for recording and calculating the incidence of selected clinical diseases of dairy cattle[J].Journal of Dairy Science,1998,81(9):2502-2509.
[3] 刘新,李金容,鲁必均.奶牛胎衣不下的病因及治疗[J].中国畜牧兽医文摘,2018,34(1):194. LIU X,LI J R,LU B J.Etiology and treatment of stillbirth in dairy cows[J].Chinese Abstracts of Animal Husbandry and Veterinary Medicine,2018,34(1):194.(in Chinese)
[4] 张国华,吕思文,金振华,等.奶牛胎衣不下的病因、临床症状和防治措施[J].现代畜牧科技,2021,10:84-85. ZHANG G H,LYU S W,JIN Z H,et al.Etiology,clinical symptoms and prevention measures of stillbirth in dairy cows[J]. Modern Animal Husbandry Science and Technology,2021,10:84-85.(in Chinese)
[5] HOOSHMANDABBASI R,ZERBE H,BAUERSACHS S,et al.Pregnancy-associated glycoproteins in cows with retained fetal membranes[J].Theriogenology,2018,105:158-163.
[6] MAHNANI A,SADEGHI-SEFIDMAZGI A,ANSARI-MAHYARI S,et al.Assessing the consequences and economic impact of retained placenta in Holstein dairy cattle[J]. Theriogenology,2021,175(12):61-68.
[7] LIN X,LIN Q,LIN X,et al.miRNA-495-3p attenuates TNF-α induced apoptosis and inflammation in human nucleus pulposus cells by targeting IL5RA[J].Inflammation,2020,43(5):1797-1805.
[8] 郑程远.胎衣不下奶牛母体胎盘组织差异表达microRNA的筛选及验证[D].大庆:黑龙江八一农垦大学,2015. ZHENG C Y.Screening and verification of differential expression of microRNA in maternal placental tissue of dairy cows without fetal coat[D].Daqing:Heilongjiang Bayi Agricultural University,2015.(in Chinese)
[9] UMAR T,MA X,YIN B,et al.miR-424-5p overexpression inhibits LPS-stimulated inflammatory response in bovine endometrial epithelial cells by targeting IRAK2[J].Journal of Reproductive Immunology,2022,150:103471.
[10] ZHAO C,ZHAO F,CHEN H,et al.microRNA-424-5p inhibits the proliferation,migration,and invasion of nasopharyngeal carcinoma cells by decreasing AKT3 expression[J]. Brazilian Journal of Medical and Biological Research,2020,53(7):e9029.
[11] KAMEMORI Y,WAKAMIYA K,NISHIMURA R,et al.Expressions of apoptosis-regulating factors in bovine retained placenta[J].Placenta,2011,32(1):20-26.
[12] 陈焘.microRNA-29c过表达介导Akt3对P19细胞增殖、凋亡和分化的影响[D].南京:南京医科大学,2021. CHEN T.Effects of microRNA-29c overexpression mediating Akt3 on proliferation,apoptosis and differentiation of P19 cells[D].Nanjing:Nanjing Medical University,2021.(in Chinese)
[13] YEGANEH P N,RICHARDSON C,BAHRANI-MOSTAFAVI Z,et al.Dysregulation of AKT3 along with a small panel of mRNAs stratifies high-grade serous ovarian cancer from both normal epithelia and benign tumor tissues[J].Genes Cancer,2017,8(11-12):784-798.
[14] 李丕宝.miR-150通过靶向抑制AKT3减轻LPS诱导急性肺损伤的机制研究[D].苏州:苏州大学,2021. LI P B.Mechanism of miR-150 alleviating LPS-induced acute lung injury by targeting AKT3 inhibition[D].Suzhou:Soochow University,2021.(in Chinese)
[15] XUE D,WANG H,CHEN Y,et al.Circ-AKT3 inhibits clear cell renal cell carcinoma metastasis via altering miR-296-3p/E-cadherin signals[J].Molecular Cancer,2019,8(1):151.
[16] 周丽亚.FEZF1-AS1和AKT3在中晚期肺腺癌中的表达及与EGFR-TKIs疗效关系的研究[D].蚌埠:蚌埠医学院,2023. ZHOU L Y.The expression of FEZF1-AS1 and AKT3 in advanced lung adenocarcinoma and their relationship with the efficacy of EGFR-TKIs[D].Bengbu:Bengbu Medical College,2023.(in Chinese)
[17] ATTUPURAM N M,KUMARESAN A,NARAYANAN K,et al.Cellular and molecular mechanisms involved in placental separation in the bovine:A review[J]. Molecular Reproduction & Development,2016,83(4):287-297.
[18] 邢菲菲.高NEFA对奶牛子宫内膜上皮细胞ECM降解关键因子的影响[D].大庆:黑龙江八一农垦大学,2023. XING F F.Effects of high NEFA on key factors of ECM degradation in dairy cow endometrial epithelial cells[D].Daqing:Heilongjiang Bayi Agricultural University,2023.(in Chinese)
[19] BOOS A,JANSSEN V,MULLING C.Proliferation and apoptosis in bovine placentomes during pregnancy and around induced and spontaneous parturition as well as in cows retaining the fetal membranes[J].Reproduction,2003,126(4):469-480.
[20] 温小庆.高NEFA经FoxO1途径诱导奶牛子宫内膜上皮细胞凋亡的调节机制[D].大庆:黑龙江八一农垦大学,2023. WEN X Q.Regulation mechanism of apoptosis induced by high NEFA via FoxO1 pathway in dairy cow endometrial epithelial cells[D].Daqing:Heilongjiang Bayi Agricultural University,2023.(in Chinese)
[21] IORIO M V,FERRACIN M,LIU C G,et al.microRNA gene expression deregulation in human breast cancer [J].Cancer Research,2005,65(16):7065-7070.
[22] HE L,THOMSON J M,HEMANN M T,et al.A microRNA polycistron as a potential human oncogene [J]. Nature,2005,435(7043):828-833.
[23] DENG S,CALIN G A,CROCE C M,et al.Mechanisms of microRNA deregulation in human cancer[J].Cell Cycle,2008,7(17):2643-2646.
[24] 刘嘉欣,黎同明,黄慧贤,等.基于PI3K/Akt信号通路探讨姜黄素诱导A549细胞凋亡的机制[J].中国临床药理学杂志,2023,39(17):2487-2491. LIU J X,LI T M,HUANG H X,et al.Study on the mechanism of curcumin-induced apoptosis of A549 cells based on PI3K/Akt signaling pathway[J].Chinese Journal of Clinical Pharmacology,2023,39(17):2487-2491.(in Chinese)