基于RNA-Seq数据分析葡萄糖诱导绵羊卵泡颗粒细胞衰老的差异表达基因

doi:10.16431/j.cnki.1671-7236.2022.02.025

摘要/Abstract

摘要： 【目的】探讨葡萄糖对绵羊卵泡颗粒细胞衰老及其基因表达的影响。【方法】将原代分离培养的绵羊卵泡颗粒细胞分别用含17.5(H组)和2 mmol/L(L组)葡萄糖的培养基进行体外培养, 细胞处理72 h后, 利用β-半乳糖苷酶染色检测细胞衰老, 采用RNA-Seq技术进行转录组测序, 对差异表达基因进行GO功能富集分析和KEGG通路富集分析, 并用实时荧光定量PCR验证锚定蛋白重复域蛋白1(ANKRD1)、白细胞介素8(IL-8)、催产素(OXT)、烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)基因的表达量。【结果】 H组β-半乳糖苷酶染色阳性细胞率极显著高于L组(P<0.01)。转录组测序结果显示, 两组间存在401个差异表达基因, 其中上调基因153个, 下调基因248个, 高糖诱导的细胞异常相关基因9个, 细胞周期相关基因6个。GO功能富集分析发现, 差异表达基因参与了细胞过程、生物调节、代谢过程、多细胞生物过程及细胞运动等生物过程, 在细胞成分上主要富集在胞外区、膜、突触、细胞器及超分子复合体等, 在分子功能主要富集在催化活性、整合、转运活性、分子功能调节剂及结构分子活性等。KEGG信号通路富集分析发现, 差异表达基因主要富集在糖尿病并发症中的晚期糖基化产物(AGE)-晚期糖基化终末产物受体(RAGE)信号通路、肿瘤坏死因子(TNF)信号通路、过氧化物酶体增殖物激活受体(PPAR)信号通路等。实时荧光定量PCR结果表明, 与L组相比, H组IL-8基因表达水平极显著上调(P<0.01), ANKRD1基因表达水平显著上调(P<0.05), OXT基因表达水平极显著下调(P<0.01), NOX4基因表达量呈上升趋势, 但差异不显著(P>0.05), 实时荧光定量PCR结果与转录组测序结果一致。【结论】 17.5 mmol/L葡萄糖可诱导绵羊卵泡颗粒细胞衰老及衰老相关基因表达变化, 为葡萄糖诱导颗粒细胞衰老的功能和分子机制提供了依据。

关键词: RNA-Seq; 葡萄糖; 绵羊; 颗粒细胞; 衰老

Abstract: 【Objective】 This study was to investigate the effect of glucose on the senescence and gene expression of sheep follicular granulosa cells.【Method】 Primary isolated sheep follicular granulosa cells were cultured in vitro with 17.5 (H group) and 2 mmol/L (L group) glucose.Each group was treated for 72 h, and cellular senescence was detected by β-galactoside staining.Transcriptome sequencing was performed on the preadipocytes by RNA-Seq technology, and the differentially expressed genes (DEGs) were analyzed by GO function enrichment analysis and KEGG signal pathway enrichment analysis.The expression levels of ankyrin repeat domain-containing protein 1(ANKRD1), interleukin-8(IL-8), oxytocin(OXT) and NADPH oxidase 4(NOX4) genes were detected by Real-time quantitative PCR.【Result】 The percentage of β-galactoside-positive cells in group H was extremely significantly higher than that in group L (P < 0.01).RNA-Seq results showed that there were 401 DEGs between the two groups, including 153 up-regulated genes and 248 down-regulated genes, including 9 genes related to cell abnormalities induced by high glucose and 6 genes related to cell cycle.The GO function enrichment analysis showed that the DEGs were involved in biological processes such as cellular process, biological regulation, metabolic process, multicellular organismal process and locomotion.The concentration of cellular component mainly included extracellular region, membrane, synapse, organelle and supramolecular complex.The enrichment of molecular function was mainly in catalytic activity, binding, transporter activity, molecular function regulator and structural molecule activity.KEGG signal pathway enrichment analysis showed that the DEGs were mainly concentrated in AGE-RAGE signaling pathway in diabetic complications, TNF signaling pathway, PPAR signaling pathway.The Real-time quantitative results showed that compared with L group, the expression level of IL-8 gene was extremely significantly up-regulated (P < 0.01), the expression level of ANKRD1 gene was significantly up-regulated (P < 0.05), and the expression level of OXT gene was extremely significantly down-regulated (P < 0.01), NOX4 gene expression showed an upward trend, but the difference was not significant (P > 0.05).The result of Real-time quantitative PCR was basically consistent with that of transcriptome sequencing.【Conclusion】 17.5 mmol/L glucose induced senescenc and genes expression changes in sheep follicular granulosa cells.The results provided a basis for the functional and molecular mechanism of glucose induced senescence of granulosa cells.

Key words: RNA-Seq; glucose; sheep; granulosa cells; senescence

中图分类号:

张配颖, 宋鹏琰, 周营, 陈晓勇, 周荣艳. 基于RNA-Seq数据分析葡萄糖诱导绵羊卵泡颗粒细胞衰老的差异表达基因[J]. 中国畜牧兽医, 2022, 49(2): 631-640.

ZHANG Peiying, SONG Pengyan, ZHOU Ying, CHEN Xiaoyong, ZHOU Rongyan. Analysis of Differentially Expressed Genes in Glucose-induced Senescence of Sheep Follicular Granulosa Cells Based on RNA-Seq Data[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 631-640.

参考文献

[1] 李曼曼, 于昊, 薛洋, 等. 生殖激素对山羊颗粒细胞孕酮合成的影响[J]. 中国兽医学报, 2020, 40(8): 1652-1659.
LI M M, YU H, XUE Y, et al. Effects of reproductive hormones on progesterone synthesis in granulocyte cells of goats[J]. Chinese Journal of Veterinary Science, 2020, 40(8): 1652-1659. (in Chinese)
[2] BRAZERT M, KRANC W, CHERMULA B, et al. Human ovarian granulosa cells isolated during an IVF procedure exhibit differential expression of genes regulating cell division and mitotic spindle formation[J]. Journal of Clinical Medicine, 2019, 8(12): 2026.
[3] YU Y C, HAN J M, KIM S. Aminoacyl-tRNA synthetases and amino acid signaling[J]. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2021, 1868(1): 118889.
[4] 郭旭川, 王艳萍, 张强, 等. 不同血清对驴卵泡颗粒细胞体外培养的影响[J]. 石河子大学学报(自然科学版), 2021, 39(4): 440-445.
GUO X C, WANG Y P, ZHANG Q, et al. Effects of different serum on the culture of donkey follicular granulosa cells in vitro[J]. Journal of Shihezi University(Natural Science), 2021, 39(4): 440-445. (in Chinese)
[5] YAO P P, SHENG M J, WENG W H, et al. High glucose causes apoptosis of rabbit corneal epithelial cells involving activation of PERK-eIF2α-CHOP-caspase-12 signaling pathway[J]. International Journal of Ophthalmology, 2019, 12(12): 1815-1822.
[6] 杨吾燕, 高丽, 秦雪梅. 葡萄糖水平与衰老机制相关性研究进展[J]. 中国药理学与毒理学杂志, 2020, 34(11): 873-880.
YANG W Y, GAO L, QIN X M, et al. Research progress in potential mechannisms of glucose-induced aging[J]. Chinese Journal of Pharmacology and Toxicology, 2020, 34(11): 873-880. (in Chinese)
[7] MORRESI C, CIANFRUGLIA L, SARTINI D, et al. Effect of high glucose-induced oxidative stress on paraoxonase 2 expression and activity in caco-2 cells[J]. Cells, 2019, 8(12): 1616-1630.
[8] BURANASIN P, MIZUTANI K, IWASAKI K, et al. High glucose-induced oxidative stress impairs proliferation and migration of human gingival fibroblasts[J]. PLoS One, 2018, 13(8): e0201855.
[9] LI B M, BIAN X W, HU W Z, et al. Regenerative and protective effects of calcium silicate on senescent fibroblasts induced by high glucose[J]. Wound Repair and Regeneration, 2020, 28(3): 315-325.
[10] SONIA K B, EUGENIA B, NOUREDDINE I K, et al. Angiotensin Ⅱ-induced redox-sensitive SGLT1 and 2 expression promotes high glucose-induced endothelial cell senescence[J]. Journal of Cellular and Molecular Medicine, 2020, 24(3): 2109-2122.
[11] ZHANG D Y, LU H F, CHEN Z X, et al. High glucose induces the aging of mesenchymal stem cells via Akt/mTOR signaling[J]. Molecular Medicine Reports, 2017, 16(2): 1685-1690.
[12] YAO Y L, REHEMAN A, XU Y F, et al. miR-125b contributes to ovarian granulosa cell apoptosis through targeting BMPR1B, a major gene for sheep prolificacy[J]. Reproductive Sciences, 2019, 26(2): 295-305.
[13] VAN D, CLOETE S, STORBECK K, et al. The role of cytochrome P450 17-alpha-hydroxylase/17, 20-lyase (CYP17) in the stress coping ability of a divergently selected Merino sheep population[A]. Association for the Advanvment of Animal Breeding & Genetics[C]. 2009.
[14] ALVES H, VAN-GINKEL J, GROEN N, et al. A mesenchymal stromal cell gene signature for donor age[J]. PLoS One, 2012, 7(8): e42908.
[15] PORTERFIELD V, KHAN S S, FOFF E P, et al. A three-dimensional dementia model reveals spontaneous cell cycle re-entry and a senescence-associated secretory phenotype[J]. Neurobiology of Aging, 2020, 90: 125-134.
[16] CHO S Y, KIM A Y, KIM J, et al. Oxytocin alleviates cellular senescence through oxytocin receptor-mediated extracellular signal-regulated kinase/Nrf2 signalling[J]. The British Journal of Dermatology, 2019, 181(6): 1216-1225.
[17] JIAO N, CHEN Y P, ZHU Y H, et al. Protective effects of catalpol on diabetes mellitus-induced male reproductive damage via suppression of the AGEs/RAGE/Nox4 signaling pathway[J]. Life Sciences, 2019, 256: 116736.
[18] TAN F C, HUTCHISON E R, EITAN E, et al. Are there roles for brain cell senescence in aging and neurodegenerative disorders?[J]. Biogemntology, 2014, 15(6): 643-660.
[19] DE-JESUS B B, BLASCO M A. Assessing cell and organ senescence biomarkers[J]. Circulation Research, 2012, 111(1): 97-109.
[20] 李炳旻, 唐浩文, 马奎, 等. 不同浓度葡萄糖诱导皮肤成纤维细胞老化的实验观察[J]. 解放军医学院学报, 2021, 42(1): 94-98.
LI B M, TANG H W, MA K, et al. Senescent effect of different concentrations of glucose on fibroblast[J]. Academic Journal of Chinese PLA Medical School, 2021, 42(1): 94-98. (in Chinese)
[21] SONIA K B, EUGENIA B, NOUREDDINE I K, et al. Angiotensin Ⅱ-induced redox-sensitive SGLT1 and 2 expression promotes high glucose-induced endothelial cell senescence[J]. Journal of Cellular and Molecular Medicine, 2020, 24(3): 2109-2122.
[22] 程冕, 严金华, 阮磊, 等. 晚期糖基化终末产物对内皮细胞衰老及内皮屏障功能的影响[J]. 中华老年医学杂志, 2019, 38(4): 362-366.
CHENG M, YAN J H, RUAN L, et al. Effects of advanced glycation end products on endothelial cell senescence and endothelial barrier function[J]. Chinese Journal of Geriatrics, 2019, 38(4): 362-366. (in Chinese)
[23] FRANCISQUETI-FERRON F V, FERRON A J T, ALTOMARE A, et al. Gamma-oryzanol reduces renal inflammation and oxidative stress by modulating AGEs/RAGE axis in animals submitted to high sugar-fat diet[J]. Brazilian Journal of Nephrology, 2021, 32(5): 358-361.
[24] 赵丽东, 丛淋淋, 徐瑶, 等. IGF-1通过PI3K/Akt通路对大鼠软骨细胞衰老的影响[J]. 动物医学进展, 2019, 40(10): 64-68.
ZHAO L D, CONG L L, XU Y, et al. Effect of IGF-1 on chondrocytes senescence in rat through PI3K/Akt pathway[J]. Progress in Veterinary Medicine, 2019, 40(10): 64-68. (in Chinese)
[25] LI P, GAN Y, XU Y, et al. The inflammatory cytokine TNF-α promotes the premature senescence of rat nucleus pulposus cells via the PI3K/Akt signaling pathway[J]. Scientific Reports, 2017, 7: 42938.
[26] JIN H, LIAN N, ZHANG F, et al. Activation of PPARγ/p53 signaling is required for curcumin to induce hepatic stellate cell senescence[J]. Cell Death & Disease, 2016, 7(4): e2189.
[27] CHEN Y X, WANG Y T, HUANG Y Y, et al. PPARα regulates tumor cell proliferation and senescence via a novel target gene carnitine palmitoyltransferase 1C[J]. Carcinogenesis, 2017, 38(4): 474-483.
[28] BRIOT A, DECAUNES P, VOLAT F, et al. Senescence alters PPARγ (peroxisome proliferator-activated receptor gamma)-dependent fatty acid handling in human adipose tissue microvascular endothelial cells and favors inflammation[J]. Arteriosclerosis, Thrombosis and Vascular Biology, 2018, 38(5): 1134-1146.
[29] KIM H J, HAM S A, KIM M Y, et al. PPARδ coordinates angiotensin Ⅱ-induced senescence in vascular smooth muscle cells through PTEN-mediated inhibition of superoxide generation[J]. Journal of Biological Chemistry, 2011, 286(52): 44585-44593.
[30] SPEECKAERT M M, VANFRAECHEM C, SPEECKAERT R, et al. Peroxisome proliferator-activated receptor agonists in a battle against the aging kidney[J]. Ageing Research Reviews, 2014, 14: 1-18.
[31] WU D M, HONG X W, WEN X, et al. MCL1 gene silencing promotes senescence and apoptosis of glioma cells via inhibition of the PI3K/Akt signaling pathway[J]. IUBMB Life, 2019, 71(1): 81-92.
[32] HSU S Y, YU H Y, LEE W C, et al. A novel CXCL8 analog is effective in inhibiting the growth via cell cycle arrest and attenuating invasion of Lewis lung carcinoma[J]. OncoTargets and Therapy, 2019, 12: 7611-7621.
[33] ZHANG S Q, WANG L L, LI Y T, et al. microRNA-126 attenuates the effect of chemokine CXCL8 on proliferation, migration, apoptosis, and MAPK-dependent signaling activity of vascular endothelial cells cultured in a medium with high glucose concentration[J]. Bulletin of Experimental Biology and Medicine, 2021, 171(2): 202-207.
[34] ZHANG H, WU C G, CHEN Q F, et al. Treatment of obesity and diabetes using oxytocin or analogs in patients and mouse models[J]. PLoS One, 2013, 8(5): e61477.
[35] LAWSON E A, OLSZEWSKI P K, WELLER A, et al. The role of oxytocin in regulation of appetitive behaviour, body weight and glucose homeostasis[J]. Journal of Neuroendocrinology, 2020, 32(4): e12805.
[36] FUKUI U, MAKIMURA N, SHIBAZAKI T, et al. Effects of oxytocin and staurosporine on the cell cycle in mouse cumulus cell[J]. International Journal of Gynecology and Obstetrics, 2000, 3(70): C141.
[37] JAFARZADEH N, JAVERI A, KHALEGHI M, et al. Oxytocin improves proliferation and neural differentiation of adipose tissue-derived stem cells[J]. Neuroscience Letters, 2014, 564: 105-110.