| 摘要: |
| 【目的】研究bta-miR-330对牛前体脂肪细胞增殖和分化的影响。【方法】利用实时荧光定量PCR(RT-qPCR)检测bta-miR-330在牛前体脂肪细胞分化进程中的表达量。根据bta-miR-330的成熟序列设计bta-miR-330的模拟物、抑制剂及其对照序列，通过lip3000转染至牛前体脂肪细胞，利用EdU染色、RT-qPCR和Western blot方法检测bta-miR-330过表达和抑制表达对牛前体脂肪细胞增殖阳性细胞数量、增殖相关基因P21、P27、CDK1、CCND1、CCNE1、MCM3和PCNA及增殖相关蛋白PCNA和CDK1表达量的影响。利用油红O染色、RT-qPCR和Western blot方法检测bta-miR-330过表达和抑制表达对牛前体脂肪细胞脂滴累积、细胞分化相关基因PPARγ、CEBPα、SREBP1和FABP4及分化相关蛋白PPARγ、CEBPα和FABP4表达量的影响。利用生物信息学方法预测bta-miR-330的靶基因，采用RT-qPCR及双萤光素报告酶方法检测bta-miR-330与靶基因的靶向关系。【结果】bta-miR-330在牛前体脂肪细胞分化过程中表达量存在显著差异。过表达bta-miR-330可降低牛前体脂肪细胞增殖阳性细胞数量，显著抑制细胞增殖相关基因MCM3、CCNE1和CCND1及相关蛋白PCNA和CDK1的表达。过表达bta-miR-330可促进牛前体脂肪细胞脂滴累积，显著促进细胞分化相关基因CEBPα和FABP4及相关蛋白PPARγ和FABP4的表达。抑制bta-miR-330的表达会在一定程度上出现与过表达相反的作用。在线软件预测长链酯酰辅酶A合成酶6（ACSL6）基因为bta-miR-330的靶基因，RT-qPCR和双萤光素报告酶载体试验结果表明，bta-miR-330和ACSL6存在靶向关系。【结论】bta-miR-330对牛前体脂肪细胞增殖和分化具有调控作用。 |
| 关键词: bta-miR-330 牛 前体脂肪细胞 ACSL6基因 |
| DOI：10.13207/j.cnki.jnwafu.2025.04.001 |
| 分类号: |
| 基金项目:国家肉牛牦牛产业技术体系项目（CARS-37）;陕西省畜禽育种“两链”融合重点专项 (2022GD-TSLD-46-0102);陕西省重点研发计划项目(2022NY-050,2022ZDLNY01-01) |
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| Effect of bta-miR-330 on the proliferation and differentiation of bovine preadipocytes |
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YANG Zhimei1, GAO Ni1, MA Xinhao1, ZAN Linsen1,2
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1.College of Animal Science and Technology,Northwest A&F University,Yangling，Shaanxi 712100,China;2.National Beef Cattle Improvement Center,Yangling，Shaanxi 712100,China
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| Abstract: |
| 【Objective】This research aimed to investigate the function of bta-miR-330 on the proliferation and differentiation of bovine preadipocytes.【Method】Real-time quantitative PCR (RT-qPCR) was used to detect the expression of bta-miR-330 in the differentiation process of preadipocytes.Based on the mature sequence of bta-miR-330,the mimics,inhibitors and control sequences of bta-miR-330 were designed and transfected into bovine preadipocytes by lip3000.The EdU staining,RT-qPCR,and Western blot were used to detect the effects of overexpression and inhibition of expression of bta-miR-330 on the number of positive cells,the expression of cell proliferation-related genes P21,P27,CDK1,CCND1,CCNE1,and proliferation-related proteins PCNA and CDK1.The Oil red O staining,RT-qPCR and Western blot were used to detect the effects of overexpression and inhibition of expression of bta-miR-330 on the accumulation of lipid droplets, the expression of cell differentiation-related genes PPARγ,CEBPα,FABP4,SREBP1 and differentiation-related proteins PPARγ,CEBPα and FABP4 in bovine preadipocytes.The bioinformatics was applied to predict the target genes of bta-miR-330,and the RT qPCR and dual luciferase reporter enzyme were used to detect the targeting relationship between bta-miR-330 and the target genes.【Result】The expression of bta-miR-330 in the differentiation process of bovine preadipocytes was significantly different.Overexpression of bta-miR-330 can reduce the number of positive cells in bovine preadipocytes,significantly inhibiting the expression of cell proliferation-related genes MCM3,CCNE1,CCND1,and related proteins PCNA and CDK1.Overexpression of bta-miR-330 can promote lipid droplet accumulation in bovine preadipocytes,significantly promoting the expression of cell differentiation related genes CEBPα and FABP4,and related proteins PPARγ and FABP4.Inhibiting the expression of bta-miR-330 can lead to effects that are somewhat opposite to those observed in overexpression.Online software predicted ACSL6 to be the target gene of bta-miR-330.Results of RT-qPCR and dual luciferin reporter enzyme vector tests showed that there was a targeting relationship between bta-miR-330 and ACSL6.【Conclusion】bta-miR-330 regulates the proliferation and differentiation processes of bovine preadipocytes. |
| Key words: bta-miR-330 bovine preadipocytes ACSL6 gene |
