Abstract：Thyroid hormone responsive spot 14 (THRSP) is a transcriptional regulator involved in the regulation of lipid metabolism in tissues. In order to understand the role of THRSP gene in lipid metabolism and its molecular mechanism, this study determined the expression of THRSP gene in 12 kinds of chicken (Gallus gallus) tissues, amplified the full-length CDS sequence of THRSP gene using the reverse transcription product of total RNA from chicken adipose tissue as a template, and constructed the eukaryotic expression vector of THRSP gene. After the THRSP gene was overexpressed and down-regulated in LMH (Leghorn male hepatoma) cells, the effect of lipid drop deposition was detected by oil red O staining, the content changes of total cholesterol (TC) and total triglyceride (TG) were determined using colorimetry method, and the expression changes of lipid metabolism-related genes were determined by qRT-PCR. The results showed that THRSP gene was highly expressed in adipose tissues and liver. Western blot analysis showed that THRSP-Myc fusion protein was successfully expressed in LMH. It was found that overexpression of THRSP gene could promote lipid drop deposition in LMH cells (P<0.001), TC (P<0.05) and TG (P<0.01) content significantly increased, the gene expression of peroxisome proliferator activated receptor γ (PPARγ)(P<0.01) and diacylglycerol acyltransferase 2 (DGAT2)(P<0.05) were up-regulated, and the gene expression of propionyl-CoA carboxylase subunit α (PCCA)(P<0.05) and phosphoenolpyruvate carboxykinase 1 (PCK1)(P<0.001) were down-regulated. When THRSP gene was knocked down in LMH cells, lipid drop deposition were inhibited (P<0.05), TC and TG contents were decreased (P=0.06), DGAT2 gene expression was down-regulated (P<0.05), while PCCA (P<0.01) and PCK1 (P<0.001) gene expression were up-regulated. The above results indicate that THRSP gene might play a role in promoting cellular lipid deposition by regulating the gene expression of PPARγ, DGAT2, PCCA, and PCK1. This study provides a new reference for further exploring the function of chicken THRSP gene.
[1] 曹志平, 李辉. 2008. Spot14基因研究进展[J]. 东北农业大学学报: 自然科学版, 39(2): 285-288. (Cao Z P, Li H.2008. Research progress of Spot14 gene[J]. Journal of Northeast Agricultural University: Natural Science Edition, 39(2): 285-288.) [2] 李辉. 1995. 鸡体脂肪及其控制的研究进展[J]. 新疆农业科学, (05): 228-232. (Li H. 1995. Research progress of chicken body fat and its control[J]. Xinjiang Agricultural Sciences, (05): 228-232.) [3] 王鹏, 殷晓航, 谢垚垚, 等. 2021. 鸡Chemerin基因的真核表达载体构建与启动子转录效率分析[J]. 农业生物技术学报, 29(11): 2149-2158. (Wang P, Yin X H, Xie Y Y, et al.2021. Eukaryotic expression vector construction and promoter transcription efficiency analysis of chicken (Gallus gallus) Chemerin gene[J]. Journal of Agricultural Biotechnology, 29(11): 2149-2158.) [4] 闫文龙, 何超, 崔建勋, 等. 2007. 鸡THRSPα基因多态性与脂肪性状的相关性研究[J]. 西南大学学报(自然科学版), (02): 57-60. (Yan W L, He C, Cui J X, et al.2007. Correlation between polymorphisms of THRSPα gene and fat-related traits in chickens[J]. Journal of Southwest University (Natural Science Edition), (02): 57-60.) [5] 詹凯, 杨宁, 徐桂云, 等. 2009. 鸡、鸭甲状腺激素应答基因(THRSP)的研究进展[J]. 遗传, 31(02): 131-136. (Zhan K, Yang N, Xu G Y, et al.2009. Progresses on thyroid hormone responsive spot 14 (THRSP) gene in chickens and ducks[J]. Hereditas (Beijing), 31(02): 131-136.) [6] Ahn J, Woodfint R M, Lee J, et al.2019. Comparative identification, nutritional, and physiological regulation of chicken liver-enriched genes[J]. Poultry Science, 98(7): 3007-3013. [7] Ahonen M A, Höring M, Nguyen V D, et al.2022. Insulin-inducible THRSP maintains mitochondrial function and regulates sphingolipid metabolism in human adipocytes[J]. Molecular Medicine, 28(1): 68. [8] Beale E G, Harvey B J, Forest C.2007. PCK1 and PCK2 as candidate diabetes and obesity genes[J]. Cell Biochemstry Biophysics, 48(2-3): 89-95. [9] Bessone F, Razori M V, Roma M G.2019. Molecular pathways of nonalcoholic fatty liver disease development and progression[J]. Cell Molecular Life Science, 76(1): 99-128. [10] Carré W, Diot C, Fillon V, et al.2001, Development of 112 unique expressed sequence tags from chicken liver using an arbitrarily primed reverse transcriptase-polymerase chain reaction and single strand conformation gel purification method[J]. Animal Genetics, 32(5): 289-297. [11] Chou W Y, Cheng Y S, Ho C L, et al.2007. Human spot14 protein interacts physically and functionally with the thyroid receptor[J]. Biochemical and Biophysical Research Communications, 357(1): 133-138. [12] Cogburn L A, Trakooljul N, Wang X, et al.2020. Transcriptome analyses of liver in newly-hatched chicks during the metabolic perturbation of fasting and re-feeding reveals THRSPA as the key lipogenic transcription factor[J]. BMC Genomics. 21(1): 109. [13] Cogburn L A, Wang X, Carre W, et al.2003. Systems-wide chicken DNA microarrays, gene expression profiling, and discovery of functional genes[J]. Poultry Science, 82: 6378-6383. [14] Ding Y, Liu X, Yuan Y, et al.2022. THRSP identified as a potential hepatocellular carcinoma marker by integrated bioinformatics analysis and experimental validation[J]. Aging, 14(4): 1743-1766. [15] Gong X, Zheng M, Zhang J, et al.2022. Transcriptomics-based study of differentially expressed genes related to fat deposition in Tibetan and Yorkshire pigs[J]. Frontier in Veterinary Science, 9: 919904. [16] Gu T, Duan M, Liu J, et al.2022. Effects of tributyrin supplementation liver fat deposition, lipid levels and lipid metabolism-related gene expression in broiler chickens[J]. Genes (Basel), 13(12): 2219. [17] Janani C, Ranjitha Kumari B D.2015. PPAR gamma gene-A review[J]. Diabetes Metabolism Syndrome, 9(1): 46-50. [18] Kim M, Voy B H.2021. Fighting fat with fat: N-3 polyunsaturated fatty acids and adipose deposition in broiler chickens[J]. Frontier in Physiology, 12: 755317. [19] Li H, Yu X H, Ou X, et al.2021. Hepatic cholesterol transport and its role in non-alcoholic fatty liver disease and atherosclerosis[J]. Progress in Lipid Research, 83: 101109. [20] Luo N, Shu J, Yuan X, et al.2022. Differential regulation of intramuscular fat and abdominal fat deposition in chickens[J]. BMC Genomics, 23(1): 308. [21] Ma S, Zhou B, Yang Q, et al.2021. A transcriptional regulatory loop of master regulator transcription factors, PPARG, and fatty acid synthesis promotes esophageal adenocarcinoma[J]. Cancer Research, 81(5): 1216-1229. [22] Montaigne D, Butruille L, Staels B.2021. PPAR control of metabolism and cardiovascular functions[J]. Nature Reviews: Cardiology, 18(12): 809-823. [23] Nematbakhsh S, Chong P P, Selamat J, et al.2021. Molecular regulation of lipogenesis, adipogenesis and fat deposition in chicken[J]. Genes (Basel), 12(3): 414. [24] Okeudo N J, Moss B W.2005. Interrelationships amongst carcass and meat quality characteristics of sheep[J]. Meat Science, 62: 1-8. [25] Ren J, Xu N, Zheng H, et al.2017. Expression of thyroid hormone responsive SPOT 14 gene is regulated by estrogen in chicken (Gallus gallus)[J]. Scientific Reports, 7(1):10243. [26] Seelig S, Liaw C, Towle H C, et al.1981. Thyroid hormone attenuates and augments hepatic gene expression at a pretranslational level[J]. Proceedings of the National Academy of Sciences of the USA, 78(8): 4733-4737. [27] Suzuki K, Irie M, Kadowaki H, et al.2005. Genetic parameter estimates of meat quality traits in Duroc pigs selected for average daily gain, longissimus muscle area, backfat thickness, and intramuscular fat content[J]. Journal of Animal Science, 83: 2058-2065. [28] Wang X F, Carre W, Zhou H J, et al.2004. Duplicated Spot14 genes in the chicken: Characterization and identification of polymorphisms associated with abdominal fat traits[J]. Gene, 332: 79-88. [29] Wongkittichote P, Ah Mew N, Chapman K A.2017. Propionyl-CoA carboxylase - A review[J]. Molecular Genetics and Metabolism, 122(4): 145-152. [30] Xu D, Wang Z, Xia Y, et al.2020. The gluconeogenic enzyme PCK1 phosphorylates INSIG1/2 for lipogenesis[J]. Nature, 580(7804): 530-535. [31] Yu Z X, Xiang C, Xu S G, et al.2022. The clinical significance of thyroid hormone-responsive in thyroid carcinoma and its potential regulatory pathway[J]. Medicine (Baltimore), 101(31): e29972. [32] Zhang J F, Choi S H, Li Q, et al.2022. Overexpression of DGAT2 stimulates lipid droplet formation and triacylglycerol accumulation in bovine satellite cells[J]. Animals (Basel), 12(14): 1847.
