miR-455-3p对奶山羊化学诱导乳腺上皮细胞乳脂代谢的影响

doi:10.16431/j.cnki.1671-7236.2024.05.018

摘要/Abstract

摘要： 【目的】探讨miR-455-3p对奶山羊化学诱导乳腺上皮细胞(CiMECs)乳脂代谢的影响,为研究微小RNA(miRNA)对山羊乳腺功能的调控机制提供理论依据。【方法】利用单一小分子化合物RepSox(TGFβR-1/ALK5抑制剂)诱导山羊成纤维细胞(GEFs),诱导8 d后观察细胞形态变化,采用特异性标记物免疫荧光染色和油红O染色鉴定CiMECs是否诱导成功。使用脂质体将miR-455-3p的过表达载体(miR-455-3p mimics、miR-455-3p mimics-NC)和干扰载体(miR-455-3p inhibitors、miR-455-3p inhibitors-NC)转染至CiMECs,通过细胞增殖及毒性检测(CCK-8)试剂盒、甘油三酯检测试剂盒、实时荧光定量PCR检测miR-455-3p对于CiMECs的增殖率、甘油三酯分泌情况及乳脂代谢基因表达量。【结果】GEFs诱导8 d后形成类似于山羊乳腺上皮细胞的岛状、多核和鹅卵石状细胞形状。免疫荧光结果显示,CiMECs可表达上皮特异性标志物抗原,包括细胞角蛋白14(CK14)和CK18;油红O染色结果显示,诱导后的细胞具有分泌脂滴的功能。CiMECs转染结果显示,miR-455-3p mimics极显著促进了miR-455-3p的表达(P＜0.01),miR-455-3p inhibitors显著抑制了miR-455-3p的表达(P＜0.05)。CCK-8及甘油三酯检测结果表明,与对照组相比,miR-455-3p mimics组细胞增殖率、甘油三酯分泌量均极显著降低(P＜0.01),miR-455-3p inhibitors组细胞增殖率、甘油三酯分泌量均显著增加(P＜0.05)。实时荧光定量PCR结果显示,与对照组相比,诱导后CiMECs中HADHB基因表达量极显著降低(P＜0.01);miR-455-3p mimics组HADHB基因表达量极显著升高(P＜0.01),miR-455-3p inhibitors组HADHB基因表达量显著降低(P＜0.05)。【结论】通过抑制miR-455-3p可促进奶山羊CiMECs增殖,降低脂肪代谢基因HADHB的表达,从而提高山羊奶中脂肪酸含量。研究结果可为后续利用miRNA改善山羊乳品质提供参考。

关键词: 奶山羊; 化学诱导乳腺上皮细胞(CiMECs); miR-455-3p; 乳脂

Abstract: 【Objective】 This study was aimed to explore the effect of miR-455-3p on milk fat metabolism in chemically induced mammary epithelial cells (CiMECs) of dairy goats,so as to provide a theoretical basis for the study of microRNA (miRNA) regulation of mammary gland function in goats.【Method】 Goat fibroblasts (GEFs) were induced by a single small molecule compound RepSox (TGFβR-1/ALK5 inhibitor),and the morphological changes of the cells were observed after 8 days of induction,and immunofluorescence staining and oil red O staining,which were specific markers,were used to identify whether the mammary epithelial cells were successfully induced.The overexpression vectors (miR-455-3p mimics and miR-455-3p mimics-NC) and interference vectors (miR-455-3p inhibitors and miR-455-3p inhibitors-NC) for miR-455-3p were transfected into CiMECs using liposomes,which were analyzed by the cell proliferation and toxicity assay (CCK-8) kit,triglyceride assay kit and Real-time quantitative PCR to detect the proliferation rate,triglyceride secretion and the expression of milk fat metabolism genes of miR-455-3p for CiMECs.【Result】 GEFs induced the formation of insular,multinucleated and cobblestone cell shapes similar to goat mammary epithelial cells after 8 days of induction.The immunofluorescence results showed that CiMECs could express epithelial-specific marker antigens,including cytokeratin 14 (CK14) and CK18.The results of oil-red O staining showed that CiMECs possessed the function of secretion of lipid droplets.The CiMECs transfection results showed that compared with control group,miR-455-3p mimics extremely significantly promoted the expression of miR-455-3p (P＜0.01),and miR-455-3p inhibitors significantly inhibited the expression of miR-455-3p expression (P＜0.05).The results of CCK-8 and triglyceride assay showed that compared with control group,the cell proliferation rate and triglyceride secretion were extremely significantly decreased in miR-455-3p mimics group (P＜0.01),and the cell proliferation rate and triglyceride secretion were significantly increased in miR-455-3p inhibitors group (P＜0.05).Real-time quantitative PCR results showed that compared with control group,the expression of HADHB gene was extremely significantly decreased in CiMECs after induction (P＜0.01),was extremely significantly increased in miR-455-3p mimics group (P＜0.01),and was significantly decreased in miR-455-3p inhibitors group (P＜0.05).【Conclusion】 The inhibition of miR-455-3p promoted CiMECs proliferation and reduced the expression of the fat metabolism gene HADHB in dairy goats,thereby increasing the fatty acid content of goat milk.The results could provide a basis for the subsequent use of miRNA to improve the milk quality in goats.

Key words: dairy goats; chemically induced mammary epithelial cells (CiMECs); miR-455-3p; milk fat

中图分类号:

S814.8

李卫清, 王国栋, 吕丹薇, 张丹丹, 刘权辉, 潘思尧, 黄奔. miR-455-3p对奶山羊化学诱导乳腺上皮细胞乳脂代谢的影响[J]. 中国畜牧兽医, 2024, 51(5): 1969-1978.

LI Weiqing, WANG Guodong, LYU Danwei, ZHANG Dandan, LIU Quanhui, PAN Siyao, HUANG Ben. Effect of miR-455-3p on Milk Fat Metabolism in Chemically Induced Mammary Epithelial Cells in Dairy Goats[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(5): 1969-1978.

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