苜蓿源miR168b通过靶向肉碱棕榈酰转移酶1A对奶牛乳腺上皮细胞增殖、凋亡和脂滴积累的调控研究

Alfalfa miR168b Regulates Proliferation, Apoptosis and Lipid DropletProduction in Bovine Mammary Epithelial Cells through Target GeneCarnitine Palmityl Transferase 1A

本试验旨在探究苜蓿源miRNAs(mtr-miRNAs)对奶牛乳腺上皮细胞(BMECs)增殖和凋亡的影响。试验首先通过CCK8、5-乙炔基-2′脱氧尿嘧啶核苷(EdU)、流式细胞术、基因和蛋白定量等方法检测苜蓿源-miR168b(mtr-miR168b)对BMECs增殖、凋亡和周期的影响。然后,敲低mtr-miR168b靶基因肉碱棕榈酰转移酶1A(CPT1A)表达后,检测了BMECs的活力和凋亡。通过诱导转染后细胞,用氟硼二吡咯(Bodipy)染色和油红O染色检测mtr-miR168b和CPT1A基因干扰对BMECs脂滴合成的影响。结果表明:1)mtr-miR168b高表达极显著降低了BMECs活力(P<0.01)。2)mtr-miR168b高表达极显著抑制了BMECs增殖(P<0.01),延长了细胞G1~S期进程,极显著促进了细胞凋亡(P<0.01)。3)mtr-miR168b在BMECs中高表达极显著抑制了细胞增殖基因周期蛋白依赖性激酶4(CDK4)、增殖细胞核抗原(PCNA)、细胞周期蛋白D1(Cyclin D1)和细胞周期蛋白D2(Cyclin D2)的基因的表达水平(P<0.01),极显著增加了凋亡标志基因Bcl2关联X蛋白(BAX)基因的表达水平(P<0.01),极显著抑制了PCNA蛋白表达水平(P<0.01),极显著增加了BAX蛋白表达水平(P<0.01),并且mtr-miR168b显著抑制了蛋白激酶B(AKT)和哺乳动物雷帕霉素靶蛋白(mTOR)基因的表达水平(P<0.05)。4)mtr-miR168b高表达抑制了BMECs中脂滴的积累。5)干扰mtr-miR168b的靶基因CPT1A,对BMECs脂滴积累也有抑制作用。综上所述,mtr-miR168b可通过抑制BMECs增殖,促进细胞凋亡,并通过靶向CPT1A基因抑制BMECs中脂滴的生成。试验结果可为mtr-miRNAs调控牛奶乳脂合成提供分子层面的技术支撑。

This experiment was aimed to investigate the effects of alfalfa-derived exogenous miRNAs(mtr-miRNAs)on the proliferation and apoptosis of bovine mammary epithelial cells(BMECs)in dairy cows.The assay examined the effect of mtr-miR168b on proliferation,apoptosis and cycle of BMECs by a combination of CCK8,EdU,flow cytometry,gene and protein quantification assays.Meanwhile,the viability and apoptosis of BMECs were examined after knocking down the expression level of mtr-miR168b target genecarnitine palmityl transferase 1A(CPT1A).Transfected cells were induced and the effects of mtr-miR168b and si-CPT1A on lipid droplet synthesis in BMECs were examined by Bodipy staining and Oil Red O staining.The results were as follows:1)mtr-miR168b overexpression highly significantly reduced the viability of BMECs(P<0.01).2)Overexpression of mtr-miR168b highly significantly inhibited the proliferation of BMECs(P<0.01),prolonged G1 to S phase progression,and highly significantly promoted apoptosis(P<0.01).3)mtr-miR168b overexpression in BMECs inhibited the expression levels of cell proliferation gene cyclin dependent kinase 4(CDK4),proliferating cell nuclear antigen(PCNA),Cyclin D 1 and Cyclin D 2,increased the expression level of apoptosis marker gene BCL2-associated X protein(BAX),suppressed PCNA protein expression level and increased BAX protein expression level,and mtr-miR168b inhibited protein kinase B(AKT)and mammalian target protein of rapamycin(mTOR)gene expression level(P<0.01).4)mtr-miR168b inhibited the accumulation of lipid droplets in BMECs.5)Interference with CPT1A,the target gene of mtr-miR168b,also had an inhibitory effect on the accumulation of lipid droplets in BMECs.In conclusion,mtr-miR168b can inhibit the production of lipid droplets in BMECs by suppressing the proliferation of BMECs,promoting apoptosis,and inhibiting the production of lipid droplets in BMECs by targeting the CPT1A gene.The results of the experiment can provide technical information at the molecular level for alfalfa-derived miRNAs to regulate milk fat synthesis.