干扰PTEN基因对奶山羊乳腺上皮细胞脂质合成相关基因的转录及脂肪酸组成的影响

doi:10.11843/j.issn.0366-6964.2020.04.022

Abstract

Abstract: The aim of this study was to investigate the effect of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene on milk fat synthesis and fatty acid composition in goat mammary gland epithelial cells by RNA interference. The complete CDS region of PTEN gene (GenBank accession number:MK158074.1) was amplified from the mammary gland tissues of Xinong Saanen dairy goat by RT-PCR. Sequence analysis and expression analysis of different lactation stages were performed. Then the siRNA targeting the PTEN gene was synthesized and screened by RT-qPCR to further detect PTEN interference on the transcription level of lipid synthesis-related genes and fatty acid composition. Results showed that:the CDS region of Capra hircus PTEN gene was cloned for the first time, with a total length of 1 212 bp. Compared with cattle (Bos Taurus), porcine (Sus scrofa) and human (Homo sapiens), the nucleic acid sequence homology of dairy goat PTEN was 99%, 98% and 97%,and the amino acid sequence homology was all reach up to 99%. The protein structure analysis showed that there was no transmembrane structure in PTEN protein. Subcellular localization analysis showed PTEN located in the cytoplasm. N terminal of PTEN protein has a phosphatase catalytic domain. The transcription of PTEN gene decreased dramatically by 51.5% in peak lactation compared with dry period. By transfecting the synthesized siRNA into mammary epithelial cells,the ideal siRNA was successfully screened of which the interference efficiency reached 94% (P<0.01). Compared with the control group,the mRNA transcriptions of SREBP1, FASN, ACACA and SCD1 were significantly up-regulated with PTEN interference (P<0.01 or P<0.05),and the mRNA transcriptions of LPL, FABP3, ACOX1, CPT1B, GPAM, DGAT2 and HSL were significantly down-regulated (P<0.01 or P<0.05). Fatty acid analysis showed that PTEN interference significantly up-regulated the desaturation index of C16:1 (P<0.05), but didn't affect C18:1. In summary, PTEN can regulate the transcription of lipid synthesis-related genes and fatty acid composition in mammary epithelial cells,which play an important role in milk fat metabolism in dairy goat.

Key words: dairy goat, GMEC, PTEN, RNA interference, lipid synthesis, fatty acid composition

CLC Number:

S287
S813.3

YAO Dawei, MA Jing, CHEN Lili, WANG Tianzhen, SUN Huan, SONG Wenqin, MA Yi. Effects of Interfering PTEN Gene on the Transcription of Lipid Synthesis-related Genes and Fatty Acid Composition in Goat Mammary Epithelial Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(4): 851-860.

References 28

[1]	AWAD A C, SHIN H S, ROMSOS D R, et al. Direct desaturation of free myristic acid by hen liver microsomal Δ9-desaturase without prior activation to myristoyl-CoA derivative[J]. J Agric Food Chem, 2004, 52(10):3194-3201.
[2]	BHATTACHARYA A, BANU J, RAHMAN M, et al. Biological effects of conjugated linoleic acids in health and disease[J]. J Nutr Biochem, 2006, 17(12):789-810.
[3]	BICHI E, TORAL P G, HERVAS G, et al. Inhibition of Δ9-desaturase activity with sterculic acid:effect on the endogenous synthesis of cis-918:1 and cis-9, trans-1118:2 in dairy sheep[J]. J Dairy Sci, 2012, 95(9):5242-5252.
[4]	HAN L Q, GAO T Y, YANG G Y, et al. Overexpression of SREBF chaperone (SCAP) enhances nuclear SREBP1 translocation to upregulate fatty acid synthase (FASN) gene expression in bovine mammary epithelial cells[J]. J Dairy Sci, 2018, 101(7):6523-6531.
[5]	XIE F C, LI B X, BROUSSARD C, et al. Identification, synthesis and evaluation of substituted benzofurazans as inhibitors of CREB-mediated gene transcription[J]. Bioorg Med Chem Lett, 2013, 23(19):5371-5375.
[6]	FANG M G, PAK M L, CHAMBERLAIN L, et al. The CREB coactivator CRTC2 is a lymphoma tumor suppressor that preserves genome integrity through transcription of DNA mismatch repair genes[J]. Cell Rep, 2015, 11(9):1350-1357.
[7]	HORIE Y, SUZUKI A, KATAOKA E, et al. Hepatocyte-specific Pten deficiency results in steatohepatitis and hepatocellular carcinomas[J]. J Clin Invest, 2004, 113(12):1774-1783.
[8]	BOOSANI C S, GUNASEKAR P, AGRAWAL D K. An update on PTEN modulators-a patent review[J]. Expert Opin Ther Pat, 2019, 29(11):881-889.
[9]	WANG Z R, HOU X M, QU B, et al. Pten regulates development and lactation in the mammary glands of dairy cows[J]. PLoS One, 2014, 9(7):e0102118.
[10]	YAO D W, LUO J, HE Q Y, et al. Characterization of the liver X receptor-dependent regulatory mechanism of goat stearoyl-coenzyme A desaturase 1 gene by linoleic acid[J]. J Dairy Sci, 2016, 99(5):3945-3957.
[11]	SHI H P, SHI H B, LUO J, et al. Establishment and characterization of a dairy goat mammary epithelial cell line with human telomerase (hT-MECs)[J]. Anim Sci J, 2014, 85(7):735-743.
[12]	YAO D W, LUO J, HE Q Y, et al. Thyroid hormone responsive (THRSP) promotes the synthesis of medium-chain fatty acids in goat mammary epithelial cells[J]. J Dairy Sci, 2016, 99(4):3124-3133.
[13]	BIONAZ M, LOOR J J. Identification of reference genes for quantitative real-time PCR in the bovine mammary gland during the lactation cycle[J]. Physiol Genomics, 2007, 29(3):312-319.
[14]	SHI H B, LUO J, YAO D W, et al. Peroxisome proliferator-activated receptor-γ stimulates the synthesis of monounsaturated fatty acids in dairy goat mammary epithelial cells via the control of stearoyl-coenzyme A desaturase[J]. J Dairy Sci, 2013, 96(12):7844-7853.
[15]	MALEK M, KIELKOWSKA A, CHESSA T, et al. PTEN regulates PI(3, 4)P2 signaling downstream of class I PI3K[J]. Mol Cell, 2017, 68(3):556-580.
[16]	YUE S H, LI J J, LEE S Y, et al. Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness[J]. Cell Metab, 2014, 19(3):393-406.
[17]	QIU T, TIAN Y Q, GAO Y J, et al. PTEN loss regulates alveolar epithelial cell senescence in pulmonary fibrosis depending on Akt activation[J]. Aging (Albany NY), 2019, 11(18):7492-7509.
[18]	CHEN C C, STAIRS D B, BOXER R B, et al. Autocrine prolactin induced by the Pten-Akt pathway is required for lactation initiation and provides a direct link between the Akt and Stat5 pathways[J]. Genes Dev, 2012, 26(19):2154-2168.
[19]	RODGERS S J, FERGUSON D T, MITCHELL C A, et al. Regulation of PI3K effector signalling in cancer by the phosphoinositide phosphatases[J]. Biosci Rep, 2017, 37(1):BSR20160432.
[20]	WANG C M, YUAN R S, ZHUANG W Y, et al. Schisandra polysaccharide inhibits hepatic lipid accumulation by downregulating expression of SREBPs in NAFLD mice[J]. Lipids Health Dis, 2016, 15(1):195.
[21]	CARCANGIU V, MURA M C, DAGA C, et al. Association between SREBP-1 gene expression in mammary gland and milk fat yield in Sarda breed sheep[J]. Meta Gene, 2013, 1:43-49.
[22]	CAPUTO M, DE ROSA M C, RESCIGNO T, et al. Binding of polyunsaturated fatty acids to LXRα and modulation of SREBP-1 interaction with a specific SCD1 promoter element[J]. Cell Biochem Funct, 2014, 32(8):637-646.
[23]	HAO J, ZHU L, ZHAO S, et al. PTEN ameliorates high glucose-induced lipid deposits through regulating SREBP-1/FASN/ACC pathway in renal proximal tubular cells[J]. Exp Cell Res, 2011, 317(11):1629-1639.
[24]	OLINER J D, ANDRESEN J M, HANSEN S K, et al. SREBP transcriptional activity is mediated through an interaction with the CREB-binding protein[J]. Genes Dev, 1996, 10(22):2903-2911.
[25]	SHEN Y H, SONG G X, LIU Y Q, et al. Silencing of FABP3 promotes apoptosis and induces mitochondrion impairment in embryonic carcinoma cells[J]. J Bioenerg Biomembr, 2012, 44(3):317-323.
[26]	XU H L, HERTZEL A V, STEEN K A, et al. Uncoupling lipid metabolism from inflammation through fatty acid binding protein-dependent expression of UCP2[J]. Mol Cell Biol, 2015, 35(6):1055-1065.
[27]	RAN H, ZHU Y M, DENG R Y, et al. Stearoyl-CoA desaturase-1 promotes colorectal cancer metastasis in response to glucose by suppressing PTEN[J]. J Exp Clin Cancer Res, 2018, 37(1):54.
[28]	BERNARD L, LEROUX C, BONNET M, et al. Expression and nutritional regulation of lipogenic genes in mammary gland and adipose tissues of lactating goats[J]. J Dairy Res, 2005, 72(2):250-255.

Effects of Interfering PTEN Gene on the Transcription of Lipid Synthesis-related Genes and Fatty Acid Composition in Goat Mammary Epithelial Cells

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 28

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	TANG Yinmei, LI Qi, LI Haiyang, LIN Yaqiu, WANG Yong, XIANG Hua, HUANG Lian, ZHU Jiangjiang. Cloning of the Goat FATP2 Gene and Its Regulatory Effect on Lipid Deposition in Precursor Adipocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3642-3652.
[2]	SHAO Peng, TANG Yinmei, LIN Yaqiu, WANG Yong, XIANG Hua, HUANG Lian, ZHU Jiangjiang. Regulation Effect of PSMD9 on Lipid Deposition in Goat Precursor Adipocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3653-3663.
[3]	SHAO Yuexin, ZHANG Xinyu, GE Liyan, SHI Huaiping. Cloning and RNA Interference Analysis of ATF4 Gene in Xinong Saanen Dairy Goat [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2353-2364.
[4]	BO Luqi, WANG Xueying, HOU Jiaming, ZHANG Li, ZHANG Jianhui, RUAN Rulin, ZHANG Bohan, WANG Shuang, SONG Mingxin, ZHANG Ziqun. Interference of Ts-ODC Gene of Trichinella spiralis by siRNA-757 Effect on Its Acid Resistance [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2114-2125.
[5]	WANG Han, MENG Lijie, LIU Wenjiao, XU Yongjian, GONG Ting. Effect of TAS1R3 Gene Interference on Autophagy Related Factors in Leydig Cells of Xiang Pig [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1525-1534.
[6]	HE Zhicheng, QIN Xiaochen, Lü Yongqiang, LI Xiujin, BIAN Huilong, LUO Jun, LI Cong. Multiple Stepwise Regression Analysis and Growth Curve Fitting of Body Measurement Traits in Saanen Dairy Goats [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5301-5311.
[7]	HE Xue, WANG Ning, HUA Ruiqi, DU Xiaodi, YANG Guangyou. Cloning, Expression of Echinococcus granulosus BAG3 and EB1 and Their Roles in the Apoptosis of Protoscoleces [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1562-1575.
[8]	HUANG Min, XIE Xiaogang, HE Qifu, CAO Xuyang, DONG Xiangchen, KANG Jian, LIU Jun, QUAN Fusheng. Cloning Analysis and Construction of Knockout Vector of Zfy Gene in Goat [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 711-721.
[9]	WANG Yidan, CHEN Dishi, XIANG Hua, ZHANG Huanrong, REN Yupeng. Inhibitory Effect of Duck Interferon-induced Transmembrane Proteins against Proliferation of DHAV-3 [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 822-833.
[10]	HAN Yawen, LAI Zhenlin, ZHANG Mengmei, ZHAO Huiying, LIU Tengfei. Effect of 4℃ Liquid Storage on Ultrastructure of Dairy Goat Spermatozoa [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 964-971.
[11]	WANG Wei, HUA Min, ZHANG Yun, LI Tao, ZHANG Qiandong, YUAN Yang, CHENG Zhentao, WEN Ming. Effect of RNAi Targeting cofilin2 Gene on Duck Enteritis Virus Proliferation [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 983-989.
[12]	WU Ke, FENG Hang, WANG Juan, YANG Zengqi. Whole Genome Sequencing and Molecular Characterization Analysis of Clostridium perfringens Type D Strains from Guanzhong Dairy Goats [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 3967-3974.
[13]	DAI Yuxing,SHI Yinyin,WEN Zuochen,LUO Yunyan,ZHU Xueli,ZHENG Chunting,LI Shuying,HONG Liang,ZHANG Jianbin,GUO Liang,PU Lei. The Effect of Hlcs Interference on Glycolytic Gene Expression in C2C12 Cells after Myogenic Lipogenic Differentiation [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3391-3402.
[14]	JIA Yanyan, ZHAO Yingying, YU Zuhua, HE Lei, LIAO Chengshui, LI Jing, YU Chuan, ZHANG Chunjie, LI Yinju. Construction and Influence of Beclin-1 Gene shRNA Lentiviral Vector on Autophagy and Virability of B16F10 Cell [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(9): 2609-2616.
[15]	GENG Yanan, WENG Yunan, LUO Jun, WANG Ping, LI Cong. Study on Change Regularity of Milk Yield, Milk Composition and Blood Physiological and Biochemical Indexes in Xinong Saanen Dairy Goat during Lactation Period [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(1): 28-41.