刚地弓形虫慢性感染小鼠脑组织中lncRNA102796的差异表达及其作用机制

doi:10.12140/j.issn.1000-7423.2022.02.009

中国寄生虫学与寄生虫病杂志 ›› 2022, Vol. 40 ›› Issue (2): 187-193.doi: 10.12140/j.issn.1000-7423.2022.02.009

刚地弓形虫慢性感染小鼠脑组织中lncRNA102796的差异表达及其作用机制

王振勋(), 熊思思, 孙夏慧, 王永亮, 潘格, 何深一, 丛华^*()

山东大学齐鲁医学院基础医学院病原生物系,济南 250012

收稿日期:2021-06-04 修回日期:2022-02-25 出版日期:2022-04-30 发布日期:2022-04-22
通讯作者: 丛华
作者简介:王振勋（1999-）,男,本科生,弓形虫致病机制研究。E-mail： sduwzx@163.com
基金资助:
1 山东省自然科学基金(ZR2021MH44);1 山东省自然科学基金(ZR2017MH043);2 山东大学齐鲁医学院本科教学改革与研究项目(qlyxjy-201903)

Differential expression and action mechanism of lncRNA102796 in the brain of mice with chronic infection of Toxoplasma gondii

WANG Zhen-xun(), XIONG Si-si, SUN Xia-hui, WANG Yong-liang, PAN Ge, HE Shen-yi, CONG Hua^*()

Department of Pathogenic Biology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, China

Received:2021-06-04 Revised:2022-02-25 Online:2022-04-30 Published:2022-04-22
Contact: CONG Hua
Supported by:
Natural Science Foundation of Shandong Province(ZR2021MH44);Natural Science Foundation of Shandong Province(ZR2017MH043);Undergraduate Teaching Reform and Research Project of Qilu Medical College(qlyxjy-201903)

摘要/Abstract

摘要：

目的了解长链非编码RNA102796（lncRNA102796）在刚地弓形虫慢性感染小鼠脑组织中的差异表达及其作用机制。方法取SPF级雌性BALB/c小鼠构建弓形虫慢性感染小鼠模型。感染后2个月,取感染小鼠（10只）和健康对照小鼠（10只）脑组织,提取总RNA以及神经细胞的细胞质和细胞核RNA,采用实时定量PCR（qRT-PCR）检测小鼠脑组织中lncRNA102796的表达和在神经细胞中的定位。生物信息学分析预测lncRNA102796的靶基因为阿片受体1（opioid receptor delta 1,oprd1）基因。取感染小鼠和健康对照小鼠脑组织,提取总RNA和细胞蛋白,qRT-PCR检测脑组织中oprd1 mRNA相对转录水平,蛋白质免疫印迹（Western blotting）检测脑组织中OPRD1的表达情况。构建lncRNA102796的干扰质粒（干扰lncRNA102796）和过表达质粒（过表达lncRNA102796）,分别以未干扰lncRNA102796的质粒pGPU6/GFP/Neo和未过表达lncRNA102796的质粒pcDNA3.1+为干扰对照和过表达对照。将质粒转染至神经小胶质细胞（BV-2）,提取细胞内RNA,qRT-PCR检测lncRNA102796的干扰和过表达对oprd1 mRNA相对转录水平的影响;提取蛋白质,Western blotting检测lncRNA102796的干扰和过表达对OPRD1表达的影响。取干扰和过表达质粒及2种对照质粒转染后的BV-2细胞,细胞计数试剂盒（CCK-8）实验检测lncRNA102796对细胞增殖的调节作用。构建过表达正义和过表达反义lncRNA102796质粒,提取2种质粒转染的BV-2细胞的RNA和蛋白质,RNA-蛋白沉降（RNA pull down）和Western blotting分析与lncRNA102796结合的蛋白。结果 qRT-PCR检测结果显示,弓形虫慢性感染小鼠脑组织中lncRNA102796相对转录水平为0.303 ± 0.054,较健康小鼠下调（69.7 ± 6.7）%（t = 18.12,P < 0.05）;lncRNA102796在细胞核内的表达[（85.04 ± 9.41）%]高于细胞质[（14.95 ± 9.41）%]（t = 7.45,P < 0.05）。qRT-PCR检测结果显示,感染小鼠脑组织中oprd1 mRNA相对转录水平为0.170 ± 0.040,较健康对照小鼠下调了（83.0 ± 5.3）%（t = 27.17,P < 0.05）;Western blotting检测结果显示,感染小鼠脑组织中的OPRD1蛋白表达量较健康对照小鼠减少。干扰lncRNA102796后,lncRNA102796相对转录水平为0.311 ± 0.054,较对照组下调了（68.9 ± 6.6）%（t = 18.00,P < 0.05）,oprd1 mRNA相对转录水平为0.175 ± 0.040,较对照组下调（82.5 ± 5.1）%（t = 28.08,P < 0.05）,OPRD1蛋白的表达量较对照组减少;过表达lncRNA102796后,lncRNA102796相对转录水平为8.220 ± 1.192,较对照组上调（722.0 ± 146.0）%（t = 8.56,P < 0.05）,oprd1 mRNA相对转录水平为2.533 ± 0.365,较对照组上调（153.3 ± 44.7）%（t = 5.95,P < 0.05）,OPRD1蛋白表达量较对照组增加。CCK-8实验结果显示,干扰lncRNA102796可抑制神经小胶质细胞的增殖,培养48和72 h后,A₄₅₀值分别为0.272 ± 0.021、0.508 ± 0.014,低于对照组的0.473 ± 0.024、0.816 ± 0.014（t = 6.35、46.77,P < 0.05）;过表达lncRNA102796可促进小胶质细胞的增殖,培养48、72 h后,A₄₅₀值分别为0.621 ± 0.038、1.026 ± 0.114,高于对照组的0.365 ± 0.010、0.530 ± 0.147（t = 10.55、5.17,P < 0.05）。RNA pull down后,经Western blotting检测结果显示,OPRD1可以与lncRNA102796结合。结论在弓形虫慢性感染的小鼠脑组织中,lncRNA102796通过与靶基因oprd1结合抑制小胶质细胞的增殖,影响细胞周期,从而导致神经细胞的损伤。

关键词: 刚地弓形虫, 长链非编码RNA102796, 神经胶质细胞, 阿片受体δ1

Abstract:

Objective To investigate the differential expression and mechanism of long non-coding RNA102796 (lncRNA102796) in the brain of mice with chronic Toxoplasma gondii infection. Methods The model for chronic T. gondii infection was established using specific-pathogen-free female BALB/c mice. Two months after infection, the brains of infected mice (n = 10) and healthy mice (n = 10) were collected two months after infection. The expression of lncRNA102796 in mice chronically infected with T. gondii was detected by qRT-PCR, using the total RNA extracted from the brains of infected mice. The location of lncRNA102796 was determined by qRT-PCR using RNA which is extracted from the nucleus and cytoplasm of mice brains. The target gene of lncRNA102796 was predicted as opioid receptor delta 1 (oprd1) gene by bioinformatics analysis. The expression of oprd1 was further determined by qRT-PCR using the total RNA from the brains of infected mice and healthy mice. The expression of OPRD1 was further determined by Western blotting, using the brains of the infected mice. The interference and overexpression plasmids of lncRNA102796, the uninterfered lncRNA102796 plasmid pGPU6/GFP/Neo and non-overexpressed lncRNA102796 pcDNA3.1+ plasmid were used as control. The plasmids were transfected into BV-2 cells. The regulation of lncRNA102796 expression on oprd1 was investigated by qRT-PCR, using the RNA extracted from BV-2 cells. The regulation of lncRNA102796 expression on OPRD1 was investigated by western blotting, using protein extracted from BV-2 cells. The regulation of lncRNA102796 expression on cell proliferation was investigated by cell counting kit-8 assay, using BV-2 cells transfected with constructed plasmids. Constructed overexpression plasmids of anti-sense lncRNA102796 and sense lncRNA102796. The protein binding to lncRNA102796 was determined by RNA pull down and Western blotting, using the RNA and protein extracted from the overexpression BV-2 cells. Results qRT-PCR results confirmed that the expression of lncRNA102796 in the brain of mice infected chronically with T. gondii was 0.303 ± 0.054, which was down-regulated by (69.7 ± 6.7)% (t = 18.12, P < 0.05) compared to uninfected mice. The expression of lncRNA102796 was higher in the nucleus (85.04 ± 9.41)% than that in the cytoplasm (14.95 ± 9.41)% of BV-2 cells (t = 7.45, P < 0.05). The results from qRT-PCR suggested that the expression of oprd1 mRNA was 0.170 ± 0.040 in the infected mice brain, which was down-regulated (83.0 ± 5.3)% (t = 27.17, P < 0.05) compared to that of the normal mouse brain. Results from Western blotting suggested the expression of OPRD1 protein was down-regulated in the infected mice brain compared to that of normal mouse brain; Upon interfering lncRNA102796, the expression of lncRNA102796 was 0.311 ± 0.054, which was down-regulated by (68.9 ± 6.6)% compared to the control group (t = 18.00, P < 0.05); the expression of oprd1 was 0.175 ± 0.04, which was down-regulated by (82.5 ± 5.1)% compared to the control group (t = 28.08, P < 0.05); the expression of oprd1 protein also reduced. Upon overexpressing lncRNA102796, the expression of lncRNA102796 was 8.220 ± 1.192, which was up-regulated by (722.0 ± 146.0)% compared to the control group (t = 8.56, P < 0.05); the expression of oprd1 was 2.533 ± 0.365, which was up-regulated by (153.3 ± 44.7)% compared to the control group (t = 5.95, P < 0.05); the expression of oprd1 protein also increased. Results from CCK-8 assay revealed that interfered lncRNA102796 inhibited the proliferation of microglia. After 48 and 72 hours, the A₄₅₀ of BV-2 cells were 0.272 ± 0.021, 0.508 ± 0.014, respectively, which are lower than the A₄₅₀ of the control group 0.473 ± 0.024, 0.816 ± 0.014 (t = 6.35, 46.77, P < 0.05). Overexpression of lncRNA102796 can accelerate the proliferation of microglia. After 48, 72 h, the A₄₅₀ of BV-2 cells were 0.621 ± 0.038 and 1.026 ± 0.114, which were higher than the A₄₅₀ of control group 0.365 ± 0.010 and 0.530 ± 0.147 (t=10.55, P < 0.05). Results from Western blotting showed that RNA pull down confirmed that sense lncRNA102796 binds to OPRD1. Conclusion In the brain of mice chronically infected with T. gondii, lncRNA102796 suppresses microglial cell proliferation, affecting the cell cycle through binding to the target gene oprd1, thereby, causing neurological cell impairment.

Key words: Toxoplasma gondii, lncRNA102796, Neurogliocyte, Opioid receptor delta 1

中图分类号:

R531.8

王振勋, 熊思思, 孙夏慧, 王永亮, 潘格, 何深一, 丛华. 刚地弓形虫慢性感染小鼠脑组织中lncRNA102796的差异表达及其作用机制[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(2): 187-193.

WANG Zhen-xun, XIONG Si-si, SUN Xia-hui, WANG Yong-liang, PAN Ge, HE Shen-yi, CONG Hua. Differential expression and action mechanism of lncRNA102796 in the brain of mice with chronic infection of Toxoplasma gondii[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(2): 187-193.

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参考文献 23

[1]	Dubey JP,, Tiao N,, Gebreyes WA, et al. A review of toxoplasmosis in humans and animals in Ethiopia[J]. Epidemiol Infect, 2012, 140(11): 1935-1938. pmid: 22874099
[2]	Robert-Gangneux F,, Dardé ML. Epidemiology of and diagnostic strategies for toxoplasmosis[J]. Clin Microbiol Rev, 2012, 25(2): 264-296. doi: 10.1128/CMR.05013-11 pmid: 22491772
[3]	Smith NC,, Goulart C,, Hayward JA, et al. Control of human toxoplasmosis[J]. Int J Parasitol, 2021, 51(2/3): 95-121. doi: 10.1016/j.ijpara.2020.11.001
[4]	Rostami A,, Riahi SM,, Esfandyari S, et al. Geo-climatic factors and prevalence of chronic toxoplasmosis in pregnant women: a meta-analysis and meta-regression[J]. Environ Pollut, 2021, 288: 117790. doi: 10.1016/j.envpol.2021.117790
[5]	Ouyang J,, Chen YK. Progress in research on the correlation between Toxoplasma gondii infection and schizophrenia[J]. Chin J Parasitol Parasit Dis, 2019, 37(1): 102-106. (in Chinese)
	(欧阳净,, 陈耀凯. 弓形虫感染与精神分裂症相关性的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(1): 102-106.)
[6]	Wang JL,, Zhang NZ,, Li TT, et al. Advances in the development of anti-Toxoplasma gondii vaccines: challenges, opportunities, and perspectives[J]. Trends Parasitol, 2019, 35(3): 239-253. doi: 10.1016/j.pt.2019.01.005
[7]	Yang J,, Yuan WY. Toxoplasma infection on nerve tissue damage and its mechanism through the blood-brain barrier[J]. Acta Neuropharmacol, 2019, 9(5): 40-43. (in Chinese)
	(杨靖,, 苑文英. 弓形虫感染对神经组织损伤及通过血脑屏障机制[J]. 神经药理学报, 2019, 9(5): 40-43.)
[8]	Yang F,, Yi F,, Cao HQ, et al. The emerging landscape of long non-coding RNAs[J]. Hereditas, 2014, 36(5): 456-468. (in Chinese)
	(杨峰,, 易凡,, 曹慧青, 等. 长链非编码RNA研究进展[J]. 遗传, 2014, 36(5): 456-468.)
[9]	Batista PJ,, Chang HY. Long noncoding RNAs: cellular address codes in development and disease[J]. Cell, 2013, 152(6): 1298-1307. doi: 10.1016/j.cell.2013.02.012 pmid: 23498938
[10]	Yang LQ,, Lin CR,, Jin CY, et al. lncRNA-dependent mechanisms of androgen-receptor-regulated gene activation programs[J]. Nature, 2013, 500(7464): 598-602. doi: 10.1038/nature12451
[11]	Sun XH,, Wang T,, Wang YL, et al. Downregulation of lncRNA-11496 in the brain contributes to microglia apoptosis via regulation of Mef2c in chronic T. gondii infection mice[J]. Front Mol Neurosci, 2020, 13: 77. doi: 10.3389/fnmol.2020.00077
[12]	Möhle L,, Parlog A,, Pahnke J, et al. Spinal cord pathology in chronic experimental Toxoplasma gondii infection[J]. Eur J Microbiol Immunol, 2014, 4(1): 65-75. doi: 10.1556/EuJMI.4.2014.1.6
[13]	Gao DJ,, Chang S,, Shan XM, et al. Toxoplasma gondii ROP16 causes apoptosis of mouse brain nerve cells[J]. Chin J Parasitol Parasit Dis, 2017, 35(6): 570-574. (in Chinese)
	(高德俊,, 常爽,, 单秀梅, 等. 刚地弓形虫棒状体蛋白ROP16对小鼠脑神经细胞凋亡的影响[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(6): 570-574.)
[14]	Liao WZ,, Xu LQ,, Yao LJ, et al. Characterization of ubiquitinated protein prolife change in host cells caused by Toxoplasma gondii infection[J]. Chin J Parasitol Parasit Dis, 2021, 39(4): 487-493. (in Chinese)
	(廖文中,, 徐李清,, 姚礼捷, 等. 弓形虫感染后宿主细胞泛素化蛋白谱变化的特征分析[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(4): 487-493.)
[15]	Zhang YH,, Wang L,, Wang XL, et al. Microglial activation and inflammatory cytokine expression in the brain of chronic Toxoplasma gondii-infected mice[J]. Chin J Parasitol Parasit Dis, 2013, 31(3): 176-179, 184. (in Chinese)
	(张义华,, 王璐,, 汪学龙, 等. 慢性弓形虫感染小鼠小胶质细胞的活化与炎症因子的表达[J]. 中国寄生虫学与寄生虫病杂志, 2013, 31(3): 176-179, 184)
[16]	Luo T,, Liu P,, Wang XY,, Li LZ, et al. Effect of the autism-associated lncRNA Shank2-AS on architecture and growth of neurons[J]. J Cell Biochem, 2018, 120(2): 1754-1762. doi: 10.1002/jcb.27471
[17]	Wang YL,, Han RX,, Xu ZJ, et al. Upregulation of lncRNA147410.3 in the brain of mice with chronic Toxoplasma infection promoted microglia apoptosis by regulating Hoxb3[J]. Front Cell Neurosci, 2021, 15: 648047. doi: 10.3389/fncel.2021.648047
[18]	Dong RQ,, Liu YM,, Yang YX, et al. MSC-derived exosomes-based therapy for peripheral nerve injury: a novel therapeutic strategy[J]. Biomed Res Int, 2019, 2019: 6458237.
[19]	Li YH,, Fu H. Long noncoding RNAs in gene expression control[J]. J Central South Univ Med Sci, 2014, 39(11): 1204-1210. (in Chinese)
	李莹辉,, 符辉. 长链非编码RNA对基因表达的调控[J]. 中南大学学报(医学版), 2014, 39(11): 1204-1210.)
[20]	Jiang KR,, Ma Z,, Zheng H, et al. Review on the application of integrated transcriptome and proteome analysis in biology[J]. Biotechnol Bull, 2018, 34(12): 50-55. (in Chinese)
	(蒋可人,, 马峥,, 郑航, 等. 转录组与蛋白质组整合分析在生物学研究中的应用[J]. 生物技术通报, 2018, 34(12): 50-55.) doi: 10.13560/j.cnki.biotech.bull.1985.2017-0929
[21]	Al-Hasani R,, Bruchas MR. Molecular mechanisms of opioid receptor-dependent signaling and behavior[J]. Anesthesiology, 2011, 115(6): 1363-1381. doi: 10.1097/ALN.0b013e318238bba6 pmid: 22020140
[22]	Wu B,, Hand W,, Alexov E. Opioid addiction and opioid receptor dimerization: structural modeling of the OPRD1 and OPRM1 heterodimer and its signaling pathways[J]. Int J Mol Sci, 2021, 22(19): 10290. doi: 10.3390/ijms221910290
[23]	Crist RC,, Clarke TK. OPRD1 genetic variation and human disease[J]. Handb Exp Pharmacol, 2018, 247: 131-145.

刚地弓形虫慢性感染小鼠脑组织中lncRNA102796的差异表达及其作用机制

Differential expression and action mechanism of lncRNA102796 in the brain of mice with chronic infection of Toxoplasma gondii

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[15]	侯永恒, 吕芳丽. 弓形虫感染与宿主细胞自噬的相互作用[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(4): 537-542.