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

doi:10.12140/j.issn.1000-7423.2022.04.011

Abstract

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

CLC Number:

R382.3

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