WIP1基因对3T3-L1前脂肪细胞增殖分化的影响及其在小鼠不同生长阶段的表达

doi:10.16431/j.cnki.1671-7236.2022.08.003

摘要/Abstract

摘要： 【目的】探究野生型p53诱导磷酸酶1(WIP1)基因与脂肪细胞增殖和分化的关系,以期为猪优质性状育种提供新基因素材。【方法】利用脂质体转染方法将合成的WIP1基因的3对siRNAs (WIP1-790、WIP1-893和WIP1-1845)和阴性对照NC-siRNA分别转染3T3-L1前脂肪细胞,通过实时荧光定量PCR检测WIP1基因的表达水平,比较不同siRNA的干扰效率。然后利用筛选到的干扰效率最高的siRNA敲减WIP1基因在3T3-L1前脂肪细胞中的表达,通过CCK-8检测siRNA敲减细胞和NC-siRNA细胞不同生长时期(0、12、24、48、72、96和120 h)的增殖情况,并通过实时荧光定量PCR检测上述2种细胞24和48 h的细胞周期蛋白B1(Cyclin B1)和Cyclin D1的表达水平。对干扰效率最高的siRNA和NC-siRNA组3T3-L1前脂肪细胞进行成脂诱导分化,通过油红O染色和甘油三酯定量法评估成脂分化效率,利用实时荧光定量PCR检测过氧化物酶体增殖物活化受体γ(PPARγ)、CCAAT/增强子结合蛋白α(C/EBPα)、脂肪酸结合蛋白4(FABP4)、硬脂酰辅酶A去饱和酶(SCD1)的表达水平,Western blotting法检测PPARγ蛋白的表达水平;通过实时荧光定量PCR检测WIP1基因在21日龄、8周龄和6月龄小鼠腹股沟皮下脂肪组织和性腺脂肪组织的时空表达情况。【结果】基因干扰试验结果显示,3对siRNAs对WIP1基因的表达均具有极显著的干扰作用(P＜0.01),其中WIP1-790的干扰效率最高,达到了70%以上。CCK-8检测结果显示,与NC-siRNA组相比,WIP1-790干扰组细胞的增殖速率在各生长时期均极显著降低(P＜0.01),且细胞周期基因Cyclin B1和Cyclin D1的表达量在24和48 h均极显著下调(P＜0.01)。成脂诱导分化结果显示,与NC-siRNA组相比,WIP1-790干扰组细胞在成脂分化第8天油红O染色阳性细胞明显减少,脂滴含量极显著降低(P＜0.01),甘油三酯含量显著降低(P＜0.05);PPARγ、C/EBPα、FABP4、SCD1等标记基因mRNA表达水平均极显著降低(P＜0.01),PPARγ蛋白表达水平极显著降低(P＜0.01)。WIP1基因在8周龄和6月龄小鼠的腹股沟皮下脂肪组织中的表达量分别显著或极显著高于21日龄小鼠(P＜0.05;P＜0.01),且在6月龄小鼠的性腺脂肪组织中,WIP1基因的表达量极显著高于21日龄和8周龄的小鼠(P＜0.01)。【结论】WIP1基因通过调控Cyclin B1、Cyclin D1和PPARγ等细胞周期和成脂分化相关基因的表达影响3T3-L1细胞的增殖和分化,且参与小鼠脂肪沉积过程,结果可为深入解析WIP1基因调控脂肪发生的分子机制提供参考。

关键词: WIP1基因; 细胞增殖; 成脂分化; 脂肪沉积

Abstract: 【Objective】 The aim of this study was to explore the relationship between WIP1 gene and adipocyte proliferation and differentiation,in order to provide new genetic material for pig quality trait breeding.【Method】 Three pairs siRNAs of WIP1 gene (WIP1-790,WIP1-893 and WIP1-1845) and negative control NC-siRNA were transfected into 3T3-L1 preadipocytes by lipofection respectively.The expression level of WIP1 gene was detected by Real-time quantitative PCR to compare the interference efficiency of different siRNAs.Then the siRNA with the highest interference efficiency was used to interfere with the expression of WIP1 gene in 3T3-L1 preadipocytes.The cell proliferation of siRNA knockdown cells and NC-siRNA cells at different growth stages (0,12,24,48,72,96 and 120 h) was detected by CCK-8,and the expression of Cyclin B1 and Cyclin D1 genes at 24 and 48 h were also determinated by Real-time quantitative PCR.In addition,the 3T3-L1 preadipocytes of NC-siRNA and siRNA with the highest interference efficiency groups were induced to adipogenic differentiation.The efficiency of adipogenic differentiation was evaluated by oil red O staining and triglyceride quantification,respectively.The expression of peroxisome proliferator-activated receptor γ(PPARγ),CCAAT/Enhancer binding protein (C/EBPα),fatty acid binding protein 4 (FABP4),and stearoyl-coenzme A desaturase 1 (SCD1) were detected by Real-time quantitative PCR.The expression of PPARγ protein was detected by Western blotting.The temporal and spatial expression of WIP1 gene in inguinal white adipose tissues and perigonadal white adipose tissues of mice at different ages (21 days,8 weeks and 6 months) was detected by Real-time quantitative PCR.【Result】 The results of gene interference test showed that compared with NC-siRNA group the three pairs of siRNAs had an extremely significant interference effect on the expression of WIP1 (P<0.01),and the interference efficiency of WIP1-790 was the highest,reaching more than 70%.The CCK-8 test results showed that compared with NC-siRNA group,the proliferation rate of WIP1-790 interference group was extremely significantly decreased at all growth stages(P<0.01),and the expression of Cyclin B1 and Cyclin D1 genes were also significantly down regulated at 24 and 48 h (P<0.01).The results of adipogenic differentiation experiment showed that compared with NC-siRNA group,the oil red O positive cells were obvious reduced,the contents of lipid droplets in was extremely significantly decreased (P<0.01),and the content of triglyceride was significantly decreased (P<0.05) in WIP1-790 interference group on the 8 d of adipogenic differentiation.The mRNA expression levels of PPARγ,C/EBPα,FABP4 and SCD1 genes were extremely significantly lower in the WIP1 gene interfering cells than those in the negative control cells(P<0.01).The level of PPARγ protein in the WIP1 gene interfering cells was also extremely significantly lower than that of negative control cells(P<0.01).At the individual level of mice,the expression of WIP1 gene in inguinal white adipose tissues of 8-week-old and 6-month-old mice was extremely significantly higher than that of 21-day-old mice(P<0.05;P<0.01),and the expression of WIP1 gene in perigonadal white adipose tissues of 6-month-old mice was extremely significantly higher than that of 21-day-old and 8-week-old mice(P<0.01).【Conclusion】 The results revealed that WIP1 gene could affect the proliferation and differentiation of 3T3-L1 cells by regulating the expression of cell cycle and adipogenic differentiation-related genes such as Cyclin B1,Cyclin D1 and PPARγ,and it was also participated the process of fat deposition.This study would provide basic data for further analysis of the molecular mechanism of adipogenesis.

Key words: WIP1 gene; cell proliferation; adipogenic differentiation; fat accumulation

中图分类号:

S829.9

王楠, 冯保亮, 郑云曦, 黄雷, 王悦, 徐松松, 张秀玲, 刘志国, 李奎, 牟玉莲. WIP1基因对3T3-L1前脂肪细胞增殖分化的影响及其在小鼠不同生长阶段的表达[J]. 中国畜牧兽医, 2022, 49(8): 2869-2879.

WANG Nan, FENG Baoliang, ZHENG Yunxi, HUANG Lei, WANG Yue, XU Songsong, ZHANG Xiuling, LIU Zhiguo, LI Kui, MU Yulian. Effects of WIP1 Gene on Proliferation and Differentiation of 3T3-L1 Preadipocytes and Its Expression in Different Growth Stages of Mice[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 2869-2879.

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