干扰PPARγ基因对绵羊滋养层细胞增殖、凋亡、迁移和脂质积累的影响

doi:10.11843/j.issn.0366-6964.2024.06.014

Abstract

Abstract:

The aim of this study was to explore the effects of PPARγ on the cell viability, apoptosis, cell migration rate and lipid accumulation of sheep trophoblast cells by interfering with the expression of PPARγ gene, so as to provide a basis for further study of the molecular mechanism of PPARγ in trophoblast cells.To design and synthesize siRNA interference fragment of PPARγ gene, the siRNA with the highest interference efficiency was screened by Western blot and RT-qPCR. Then, the siRNA was transfected into the sheep trophoblast cells. After interfering with the PPARγ gene, the accumulation of lipid droplets in trophoblast cells was measured by BODIPY staining, the amount of triglycerides by colorimetric method, and the transcript levels of genes involved in lipid metabolism was detected using RT-qPCR (real-time quantitative PCR). CCK-8 assay, wound healing test and flow cytometry were used to detect the effects of PPARγ interference on cell viability, migration rate and apoptosis of trophoblast cells. siPPARγ-1 fragment with the highest interference efficiency was screened and used to transfect trophoblast cells to interfere with the expression of PPARγ gene. Interference with PPARγ gene expression in trophoblast cells reduced the accumulation capacity of lipid droplets and cellular triglyceride content (P < 0.01), and the transcription levels of related genes CD36, FABP4 and PLIN2 were also inhibited (P < 0.01). At the same time, interference with PPARγ gene expression significantly reduced cell viability (P < 0.01) and migration rate (P < 0.05), while the cell apoptosis rate was increased significantly (P < 0.01).This study showed that PPARγ gene plays an important role in regulating the function of trophoblast cells and its specific molecular mechanism can be studied in depth with a view to using it in reproductive breeding practices.

Key words: sheep, trophoblast cells, PPARγ, interference

CLC Number:

S826.3

Chang LIU, Kexing HAO, Yan CHEN, Weibin ZENG, Hengbin YU, Lei CHEN, Jing WANG, Guangdong HU. Effects of Interference with PPARγ Gene on Proliferation, Apoptosis, Migration and Lipid Accumulation of Trophoblast Cells in Sheep[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2421-2430.

Figures/Tables 7

Table 1

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References 37

1	DHAR R , SINGH S , MUKHERJEE I , et al. MTiness in pseudo-malignant behavior of trophoblasts during embryo implantation[J]. Front Biosci (Landmark Ed), 2021, 26 (4): 717- 743. doi: 10.2741/4915
2	骆金红, 陈祥, 尚以顺, 等. 转录组测序筛选山羊妊娠早期胚胎附植相关基因[J]. 畜牧兽医学报, 2022, 53 (5): 1465- 1474. doi: 10.11843/j.issn.0366-6964.2022.05.014
	LUO J H , CHEN X , SHANG Y S , et al. Transcriptome sequencing screening the genes related to goat embryo attachment in early pregnancy[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53 (5): 1465- 1474. doi: 10.11843/j.issn.0366-6964.2022.05.014
3	宋美君, 郝科兴, 海思妤, 等. SRIF-14对绵羊子宫内膜上皮细胞增殖和凋亡的影响[J]. 畜牧兽医学报, 2023, 54 (8): 3325- 3334. doi: 10.11843/j.issn.0366-6964.2023.08.018
	SONG M J , HAO K X , HAI S Y , et al. Effects of SRIF-14 on proliferation and apoptosis of endometrial epithelial cells in sheep[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54 (8): 3325- 3334. doi: 10.11843/j.issn.0366-6964.2023.08.018
4	CAMPANILE G , BARUSELLI P S , LIMONE A , et al. Local action of cytokines and immune cells in communication between the conceptus and uterus during the critical period of early embryo development, attachment and implantation-implications for embryo survival in cattle: a review[J]. Theriogenology, 2021, 167, 1- 12. doi: 10.1016/j.theriogenology.2021.02.020
5	PADMANABHAN R A , LALORAYA M . Estrogen-initiated protein interactomes during embryo implantation[J]. Am J Reprod Immunol, 2016, 75 (3): 256- 262. doi: 10.1111/aji.12455
6	KADAM L , KILBURN B , BACZYK D , et al. Rosiglitazone blocks first trimester in-vitro placental injury caused by NF-κB-mediated inflammation[J]. Sci Rep, 2019, 9 (1): 2018. doi: 10.1038/s41598-018-38336-2
7	YU J , HU Y P , SHENG M Z , et al. Selective PPARγ modulator diosmin improves insulin sensitivity and promotes browning of white fat[J]. J Biol Chem, 2023, 299 (4): 103059. doi: 10.1016/j.jbc.2023.103059
8	盘道兴, 王振, 杨茂林, 等. 不同品种猪PPARγ和C/EBPα基因表达规律与肌内脂肪含量的相关[J]. 中国农业科学, 2017, 50 (1): 171- 182.
	PAN D X , WANG Z , YANG M L , et al. Association of the PPARγ and C/EBPα gene expression with intramuscular fat content in different varieties of pig[J]. Scientia Agricultura Sinica, 2017, 50 (1): 171- 182.
9	TAKADA I , MAKISHIMA M . Peroxisome proliferator-activated receptor agonists and antagonists: a patent review (2014-present)[J]. Expert Opin Ther Pat, 2020, 30 (1): 1- 13. doi: 10.1080/13543776.2020.1703952
10	LEVYTSKA K , DREWLO S , BACZYK D , et al. PPAR- γ regulates Trophoblast differentiation in the BeWo cell model[J]. PPAR Res, 2014, 2014, 637251.
11	BLITEK A , SZYMANSKA M . Expression and role of peroxisome proliferator-activated receptors in the porcine early placenta trophoblast[J]. Domest Anim Endocrinol, 2019, 67, 42- 53. doi: 10.1016/j.domaniend.2018.12.001
12	BLITEK A , SZYMANSKA M . Peroxisome proliferator-activated receptor (PPAR) isoforms are differentially expressed in peri-implantation porcine conceptuses[J]. Theriogenology, 2017, 101, 53- 61. doi: 10.1016/j.theriogenology.2017.06.013
13	SZYMANSKA M , BLITEK A . Prostacyclin synthesis and prostacyclin receptor expression in the porcine corpus luteum: evidence for a luteotropic role in vitro[J]. Biol Reprod, 2019, 100 (1): 162- 174. doi: 10.1093/biolre/ioy178
14	DUAN S Z , IVASHCHENKO C Y , WHITESALL S E , et al. Hypotension, lipodystrophy, and insulin resistance in generalized PPARγ-deficient mice rescued from embryonic lethality[J]. J Clin Invest, 2007, 117 (3): 812- 822. doi: 10.1172/JCI28859
15	BO Q L , CHEN Y H , YU Z , et al. Rosiglitazone pretreatment protects against lipopolysaccharide-induced fetal demise through inhibiting placental inflammation[J]. Mol Cell Endocrinol, 2016, 423, 51- 59. doi: 10.1016/j.mce.2016.01.004
16	RIBEIRO E S , GRECO L F , BISINOTTO R S , et al. Biology of Preimplantation Conceptus at the onset of elongation in dairy cows[J]. Biol Reprod, 2016, 94 (4): 97.
17	BROOKS K E , BURNS G W , SPENCER T E . Peroxisome proliferator activator receptor gamma (PPARG) regulates conceptus elongation in sheep[J]. Biol Reprod, 2015, 92 (2): 42.
18	HAO K X , LIU X X , CHEN Y , et al. PPARγ regulates lipid metabolism and viability of sheep trophoblast cells[J]. Reprod Domest Anim, 2023, 58 (11): 1559- 1568. doi: 10.1111/rda.14471
19	EL DAIRI R , HUUSKONEN P , PASANEN M , et al. Corrigendum to "Peroxisome proliferator activated receptor gamma (PPAR-γ) ligand pioglitazone regulated gene networks in term human primary trophoblast cells" [Reprod.Toxicol.81 (2018) 99-107][J]. Reprod Toxicol, 2021, 104, 169. doi: 10.1016/j.reprotox.2021.02.001
20	ZHANG Z , YANG Y H , LV X M , et al. Interleukin-17 promotes proliferation, migration, and invasion of trophoblasts via regulating PPAR-γ/RXR-α/Wnt signaling[J]. Bioengineered, 2022, 13 (1): 1224- 1234. doi: 10.1080/21655979.2021.2020468
21	BLITEK A , SZYMANSKA M . Peroxisome proliferator-activated receptor β/δ and γ agonists differentially affect prostaglandin E2 and cytokine synthesis and nutrient transporter expression in porcine trophoblast cells during implantation[J]. Theriogenology, 2020, 152, 36- 46. doi: 10.1016/j.theriogenology.2020.04.024
22	陈慧慧, 郝科兴, 龙德智, 等. 哈萨克羊胎盘原代滋养层细胞的分离、纯化与基本特征鉴定[J]. 畜牧与兽医, 2022, 54 (11): 5- 11.
	CHEN H H , HAO K X , LONG D Z , et al. Isolation and purification primary trophoblast cells from Kazakh sheep plancnta and identification of their basic characteristics[J]. Animal Husbandry & Veterinary Medicine, 2022, 54 (11): 5- 11.
23	BLASCHITZ A , WEISS U , DOHR G , et al. Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening[J]. Placenta, 2000, 21 (7): 733- 741. doi: 10.1053/plac.2000.0559
24	KOWALEWSKI M P , MEYER A , HOFFMANN B , et al. Expression and functional implications of peroxisome proliferator-activated receptor gamma (PPARγ) in canine reproductive tissues during normal pregnancy and parturition and at antiprogestin induced abortion[J]. Theriogenology, 2011, 75 (5): 877- 886. doi: 10.1016/j.theriogenology.2010.10.030
25	HAO K X , WANG J , LI Z Y , et al. PPARγ/mTOR regulates the synthesis and release of prostaglandins in ovine trophoblast cells in early pregnancy[J]. Vet Sci, 2022, 9 (11): 649. doi: 10.3390/vetsci9110649
26	HERNANDEZ-QUILES M , BROEKEMA M F , KALKHOVEN E . PPARgamma in metabolism, immunity, and cancer: unified and diverse mechanisms of action[J]. Front Endocrinol (Lausanne), 2021, 12, 624112. doi: 10.3389/fendo.2021.624112
27	TIAN S C , LEI P , TENG C Y , et al. Targeting PLIN2/PLIN5-PPARγ: sulforaphane disturbs the maturation of lipid droplets[J]. Mol Nutr Food Res, 2019, 63 (20): 1900183. doi: 10.1002/mnfr.201900183
28	NARUSE K , NOGUCHI T , SADO T , et al. Chemokine and free fatty acid levels in insulin-resistant state of successful pregnancy: a preliminary observation[J]. Mediators Inflamm, 2012, 2012, 432575.
29	MATHEW D J , PETERSON K D , SENN L K , et al. Ruminant conceptus-maternal interactions: interferon-tau and beyond[J]. J Anim Sci, 2022, 100 (7): skac123. doi: 10.1093/jas/skac123
30	ARMISTEAD B , KADAM L , SIEGWALD E , et al. Induction of the PPARγ (Peroxisome Proliferator-Activated Receptor γ)-GCM1 (Glial Cell Missing 1) syncytialization axis reduces sFLT1 (Soluble fms-Like Tyrosine Kinase 1) in the preeclamptic placenta[J]. Hypertension, 2021, 78 (1): 230- 240. doi: 10.1161/HYPERTENSIONAHA.121.17267
31	BHATTACHARYA B , HOME P , GANGULY A , et al. Atypical protein kinase C iota (PKCλ/ι) ensures mammalian development by establishing the maternal-fetal exchange interface[J]. Proc Natl Acad Sci U S A, 2020, 117 (25): 14280- 14291. doi: 10.1073/pnas.1920201117
32	GRIMALDI B , KOHAN-GHADR H R , DREWLO S . The potential for placental activation of PPARγ to improve the angiogenic profile in preeclampsia[J]. Cells, 2022, 11 (21): 3514. doi: 10.3390/cells11213514
33	GAO F M , HU W X , LI Y , et al. Mono-2-ethylhexyl phthalate inhibits human extravillous trophoblast invasion via the PPARγ pathway[J]. Toxicol Appl Pharmacol, 2017, 327, 23- 29. doi: 10.1016/j.taap.2017.04.014
34	KOLBEN T M , ROGATSCH E , VATTAI A , et al. PPARγ expression is diminished in macrophages of recurrent miscarriage placentas[J]. Int J Mol Sci, 2018, 19 (7): 1872. doi: 10.3390/ijms19071872
35	YAN S S , DING J L , WANG Z H , et al. CTRP6 regulates M1 macrophage polarization via the PPAR-γ/NF-κB pathway and reprogramming glycolysis in recurrent spontaneous abortion[J]. Int Immunopharmacol, 2023, 124, 110840. doi: 10.1016/j.intimp.2023.110840
36	SHOAITO H , PETIT J , CHISSEY A , et al. The role of peroxisome proliferator-activated receptor gamma (PPARγ) in Mono(2-ethylhexyl) Phthalate (MEHP)-mediated cytotrophoblast differentiation[J]. Environ Health Perspect, 2019, 127 (2): 027003. doi: 10.1289/EHP3730
37	OJOSNEGROS S , SERIOLA A , GODEAU A L , et al. Embryo implantation in the laboratory: an update on current techniques[J]. Hum Reprod Update, 2021, 27 (3): 501- 530. doi: 10.1093/humupd/dmaa054

基因 Gene	GenBank号 GenBank No.	序列(5′→3′) Sequence	产物长度/bp Product length
CD36	XM_027968558.1	F：TGCCAGTTGGAGACATGCTCAT	154
		R：ACTGCCACTTCGTCTGGATTCT
FABP4	NM_001114667.1	F：ATGAAGGTGCTCTGGTACAAGT	135
		R：ATGCTCTCTCGTAAACTCTGGT
PLIN2	NM_001104932.1	F：CGCAATGCTGCCTCCTTT	131
		R：AGCCAGTTGAGGGGTGTGTT
PPARγ	KF727439.1	F：CTCATAATGCCATCAGGTTCG	102
		R：CAGCAGACTCTGGGTTCAGTTG

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Effects of Interference with PPARγ Gene on Proliferation, Apoptosis, Migration and Lipid Accumulation of Trophoblast Cells in Sheep

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