长链非编码RNA NEAT1通过调控IL-8参与肠上皮细胞抗隐孢子虫反应

doi:10.12140/j.issn.1000-7423.2022.04.011

中国寄生虫学与寄生虫病杂志 ›› 2022, Vol. 40 ›› Issue (4): 487-492.doi: 10.12140/j.issn.1000-7423.2022.04.011

长链非编码RNA NEAT1通过调控IL-8参与肠上皮细胞抗隐孢子虫反应

李腾(), 沈玉娟, 崔丽君, 刘华, 胡媛, 姜岩岩, 曹建平^*()

中国疾病预防控制中心寄生虫病预防控制所（国家热带病研究中心）,国家卫生健康委员会寄生虫病原与媒介生物学重点实验室,世界卫生组织热带病合作中心,国家级热带病国际研究中心,上海200025

收稿日期:2022-02-22 修回日期:2022-06-14 出版日期:2022-08-30 发布日期:2022-09-07
通讯作者: 曹建平
作者简介:李腾（1992-）,女,博士研究生,从事寄生虫感染与免疫研究。E-mail： drliteng@163.com
基金资助:
国家自然科学基金(81971969);国家自然科学基金(81772225);上海市公共卫生体系建设三年行动计划重点学科（2020—2022）(GWV-10.1-XK13)

Long non-coding RNA NEAT1 involves in intestinal epithelial cell response against Cryptosporidium parvum infection via regulating IL-8 expression

LI Teng(), SHEN Yu-juan, CUI Li-jun, LIU Hua, HU Yuan, JIANG Yan-yan, CAO Jian-ping^*()

National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Chinese Center for Tropical Diseases Research);NHC Key Laboratory of Parasite and Vector Biology; WHO Collaborating Center for Tropical Diseases, National Center for International Research on Tropical Diseases, Shanghai 200025, China

Received:2022-02-22 Revised:2022-06-14 Online:2022-08-30 Published:2022-09-07
Contact: CAO Jian-ping
Supported by:
National Natural Science Foundation of China(81971969);National Natural Science Foundation of China(81772225);Fifth Round of Three-Year Public Health Action Plan (2020-2022) of Shanghai, China(GWV-10.1-XK13)

摘要/Abstract

摘要：

目的探索长链非编码RNA NEAT1（lncRNA NEAT1）在调控宿主细胞抗隐孢子虫免疫反应的作用及其初步机制。方法采用微小隐孢子虫感染人结直肠癌细胞系HCT-8细胞（卵囊 ∶ 细胞比例为2 ∶ 1）,构建微小隐孢子虫感染肠上皮细胞模型。分别于感染后4 h和36 h观察感染组及未感染对照组的HCT-8细胞生长情况;提取各组细胞总RNA,采用实时荧光定量PCR方法（qRT-PCR）检测炎性细胞因子白细胞介素-8（IL-8）的基因表达水平,并动态检测lncRNA NEAT1及其长转录本lncRNA NEAT1_2的表达。同时利用lncRNA NEAT1抑制剂Smart Silencer体系建立lncRNA NEAT1敲减的细胞模型（lncRNA NEAT1^KD）,比较其与未敲减对照组细胞IL-8 mRNA的表达水平。结果微小隐孢子虫卵囊与HCT-8细胞共孵育4 h后,子孢子贴附并入侵细胞,感染组lncRNA NEAT1和lncRNA NEAT1_2的相对表达量分别为0.467 ± 0.364和0.282 ± 0.230,较对照组（0.960 ± 0.152和0.865 ± 0.219）降低（t = 2.780、3.672,P < 0.05）;IL-8相对表达量为1.273 ± 0.647,与对照组（1.017 ± 0.231）差异无统计学意义（t = 0.828,P > 0.05）;感染后36 h,隐孢子虫成功侵入宿主细胞并繁殖,感染组细胞lncRNA NEAT1及lncRNA NEAT1_2相对表达量分别为2.728 ± 0.897和3.303 ± 2.643,较对照组（1.029 ± 0.237和1.000 ± 0.449）均上调（t = 4.854,P < 0.01;t = 2.577,P < 0.05）;IL-8相对表达量为6.835 ± 2.065,较对照组（1.046 ± 0.293）上调（t = 5.652,P < 0.01）。lncRNA NEAT1^KD组lncRNA NEAT1及lncRNA NEAT1_2的相对表达量分别为0.382 ± 0.126和0.312 ± 0.068,较对照组（1.040 ± 0.446和1.005 ± 0.410）下调（t = 4.481,P < 0.01;t = 5.301,P < 0.01）。LncRNA NEAT1^KD组IL-8 mRNA相对表达量为0.525 ± 0.230,低于对照组（0.959 ± 0.229）（t = 4.688,P < 0.01）。结论随着微小隐孢子虫感染进程,感染细胞lncRNA NEAT1的表达动态上调,并通过调控细胞因子IL-8的转录,参与宿主细胞抗微小隐孢子虫免疫应答。

关键词: 微小隐孢子虫, 长链非编码RNA, lncRNA NEAT1, IL-8, 宿主免疫

Abstract:

Objective To investigate the role and mechanism of long non-coding RNA NEAT1 (lncRNA NEAT1) in regulating host immune response against Cryptosporidium parvum infection. Methods The human colon adenocarcinoma cell line (HCT-8, oocysts/cell = 2 ∶ 1) was used to the establish C. parvum-infection model. The cell growth was evaluated for the infected group and the uninfected group at 4 h and 36 h post-infection of C. parvum oocysts. The total RNA was extracted from the cells at 4 h and 36 h, respectively. qRT-PCR was used to detect the gene expression level of IL-8, the dynamic expression of lncRNA NEAT1 and its long isoform lncRNA NEAT1_2. The knockdown model of lncRNA NEAT1 （lncRNA NEAT1^KD group） was established by the Smart Silencer system, and the IL-8 mRNA expression in the lncRNA NEAT1^KD group was compared with the control group （without knockdown）. Results After 4 h incubation with C. parvum, the sporozoites adhered and invaded the cells. The relative expression levels of lncRNA NEAT1 and lncRNA NEAT1_2 in the infection group were 0.467 ± 0.364 and 0.282 ± 0.230, respectively, which were decreased compared with the control group (0.960 ± 0.152 and 0.865 ± 0.219) (t = 2.780, 3.672; P < 0.05). The relative mRNA level of inflammatory cytokine IL-8 in HCT-8 cells was 1.273 ± 0.647, which was not significantly increased compared with 1.017 ± 0.231 of the control group (t = 0.828, P > 0.05). After 36 h of incubation, C. parvum successfully invades host cells and reproduces. The relative expression levels of IL-8, lncRNA NEAT1 and lncRNA NEAT1_2 were 1.046 ± 0.293, 1.029 ± 0.237 and 1.000 ± 0.449 in control group, while in the infected group the relative expression levels of IL-8, lncRNA NEAT1 and lncRNA NEAT1_2 were 6.835 ± 2.065, 2.728 ± 0.897 and 3.303 ± 2.643, which were significantly up-regulated compared with the control group (t = 5.652, P < 0.01; t = 4.854, P < 0.01; t = 2.577, P < 0.05). In negative control group, the relative transcription levels of lncRNA NEAT1 and lncRNA NEAT1_2 were 1.040 ± 0.446 and 1.005 ± 0.410. After the knockdown, the relative transcription levels of lncRNA NEAT1 and lncRNA NEAT1_2 in the lncRNA NEAT1^KD group were 0.382 ± 0.126 and 0.312 ± 0.068, respectively. The difference was statistically significant compared with negative control group (t = 4.481, P < 0.01; t = 5.301, P < 0.01). Moreover, the relative transcription level of IL-8 mRNA in the lncRNA NEAT1^KD group (0.525 ± 0.230) was significantly lower than that of the control group (0.959 ± 0.229) (t = 4.688, P < 0.01) after inhibiting the expression of lncRNA NEAT1. Conclusion During C. parvum infection, lncRNA NEAT1 was dynamically expressed and was up-regulated during the infection. Moreover, lncRNA NEAT1 can modulate the host immune defence against C. parvum by regulating the transcription of IL-8 in infected host cells.

Key words: Cryptosporidium parvum, Long non-coding RNA, LncRNA NEAT1, IL-8, Host immune response

中图分类号:

R382.3

李腾, 沈玉娟, 崔丽君, 刘华, 胡媛, 姜岩岩, 曹建平. 长链非编码RNA NEAT1通过调控IL-8参与肠上皮细胞抗隐孢子虫反应[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(4): 487-492.

LI Teng, SHEN Yu-juan, CUI Li-jun, LIU Hua, HU Yuan, JIANG Yan-yan, CAO Jian-ping. Long non-coding RNA NEAT1 involves in intestinal epithelial cell response against Cryptosporidium parvum infection via regulating IL-8 expression[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(4): 487-492.

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基因名称 Gene name	引物序列（5′→ 3′） Primer sequence （5′→ 3′）
白细胞介素-8 IL-8	F: ATGACTTCCAAGCTGGCCGTGGCT R: TCTCAGCCCTCTTCAAAAACTTCTC
β-肌动蛋白 β-actin	F: CACCATTGGCAATGAGCGGTTC R: AGGTCTTTGCGGATGTCCACGT
长链非编码RNA NEAT1 lncRNA NEAT1	F: CTTCCTCCCTTTAACTTATCCATTCAC R: CTCTTCCTCCACCATTACCAACAATAC
长链非编码RNA NEAT1_2 lncRNA NEAT1_2	F: CAGTTAGTTTATCAGTTCTCCCATCCA R: GTTGTTGTCGTCACCTTTCAACTCT

长链非编码RNA NEAT1通过调控IL-8参与肠上皮细胞抗隐孢子虫反应

Long non-coding RNA NEAT1 involves in intestinal epithelial cell response against Cryptosporidium parvum infection via regulating IL-8 expression

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