China Animal Husbandry and Veterinary Medicine ›› 2022, Vol. 49 ›› Issue (8): 2869-2879.doi: 10.16431/j.cnki.1671-7236.2022.08.003

• Biotechnology • Previous Articles     Next Articles

Effects of WIP1 Gene on Proliferation and Differentiation of 3T3-L1 Preadipocytes and Its Expression in Different Growth Stages of Mice

WANG Nan1, FENG Baoliang2, ZHENG Yunxi3, HUANG Lei4, WANG Yue1, XU Songsong1,4, ZHANG Xiuling1, LIU Zhiguo1, LI Kui1,4, MU Yulian1   

  1. 1. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
    2. Tianjin Ningheyuan Swine Breeding Farm Co., Ltd., Tianjin 301504, China;
    3. Queen Mary School, Nanchang University, Nanchang 330031, China;
    4. Agricultural Genomics Institute, Chinese Academy of Agricultural Sciences, Shenzhen 518120, China
  • Received:2022-03-01 Online:2022-08-05 Published:2022-07-21

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

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