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Front Immunol
2022 Jan 01;13:927796. doi: 10.3389/fimmu.2022.927796.
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MiR-7 Regulates Pathogen-Induced Immune Response via PAK1 in the Sea Cucumber Apostichopus japonicus.
Zhao T
,
Ren L
,
Li C
,
Liu L
,
Zou Y
,
Yan H
,
Zhan Y
,
Chang Y
.
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MicroRNA-7 (miR-7) is a highly conserved short non-coding RNA involved in various bioprocesses via the regulation of multiple target genes. To enrich our knowledge of the functions of miR-7 in innate immune regulation in echinoderms, we first investigated the targeting relationship between miR-7 and PAK1 in the sea cucumber Apostichopus japonicus and then explored the functions of miR-7, the PAK1 gene, and the miR-7/PAK1 axis in the pathogen-induced immune response of A. japonicus. Our results showed that miR-7 can bind to the 3'UTR of PAK1 and negatively regulate the expression of PAK1 in A. japonicus. Overexpression and inhibition of miR-7 and inhibition of the expression of PAK1 can alter phagocytosis, cellular agglutination, and lysozyme contents in A. japonicus. Both miR-7 and the PAK1 gene are involved in immune defense against Vibrio splendidus infection; the miR-7/AjPAK1 axis showed immune regulatory function at 48 to 72 h post-infection (hpi) after V. splendidus infection in A. japonicus. In summary, the results of this study established that miR-7 regulates the pathogen-induced immune response by targeting PAK1 in A. japonicus.
Figure 1. Spatial expression patterns of miR-7 and AjPAK1 (mRNA and protein) in Apostichopus japonicus and identification of miR-7 binding sites in the 3’UTR of AjPAK1. (A) Relative spatial expression patterns of miR-7 and AjPAK1 mRNA. (B) Relative spatial expression pattern of AjPAK1 protein. (C) The predicted target site of miR-7 was found in the 3’UTR of AjPAK1.
(D) Analysis of relative luciferase activities. u, tube foot; Co, coelomocytes; Bo, body wall; In, intestine; Re, respiratory tree; Lo, longitudinal muscle.. ** represents an extremely significant difference (P < 0.01) between AjPAK1 mRNA and miR-7 in the same tissue or between AjPAK1 3’UTR WT + NC and AjPAK1 3’UTR WT + mimics. Different lower-case letters (e.g., a or a’) indicate significant differences between different tissues (P < 0.05).
Figure 2. Relative expression of miR-7 and AjPAK1 (mRNA and protein) and phagocytic capacity, cellular agglutination, and lysozyme (LZM) content analyses in A. japonicus after Vibrio splendidus infection. (A) Relative expression of miR-7 in A. japonicus coelomocytes after V. splendidus infection. (B) Relative expression of AjPAK1 in A. japonicus coelomocytes after V. splendidus infection. (C) Relative expression of AjPAK1 protein in A. japonicus coelomocytes after V. splendidus infection. (D) Relative phagocytic capacity of coelomocytes in A. japonicus after V. splendidus infection. (E) Relative cellular agglutination of coelomocytes in A. japonicus after V. splendidus infection. (F) Relative LZM content of coelomic fluid in A. japonicus after V. splendidus infection. ** represents an extremely significant difference compared with 0 hpi (P < 0.01); * represents a significant difference compared with 0 hpi (P < 0.05).
Figure 3. Relative expression of AjPAK1 in coelomocytes of Apostichopus japonicus. (A) Relative expression levels of AjPAK1 mRNA in A. japonicus coelomocytes after AjPAK1 silencing, miR-7 overexpression, or miR-7 inhibition. (B) Expression levels of AjPAK1 protein in A. japonicus coelomocytes after AjPAK1 silencing, miR-7 overexpression, or miR-7 inhibition. (C) Relative expression levels of AjPAK1 mRNA in A. japonicus coelomocytes after AjPAK1 silencing, miR-7 overexpression, or miR-7 inhibition upon Vibrio splendidus infection. (D) Expression levels of AjPAK1 protein in A. japonicus coelomocytes after AjPAK1 silencing, miR-7 overexpression, or miR-7 inhibition upon V. splendidus infection. NC, negative control; RNAi, AjPAK1 silencing; Agomir, miR-7 overexpressed; Antagomir, miR-7 inhibition. ** represents an extremely significant difference compared with NC or 0 hpi (P < 0.01); * represents significant difference compared with NC or 0 hpi (P < 0.05).
Figure 4. Functional analysis of miR-7 and AjPAK1 on phagocytic capacity and cellular agglutination of coelomocytes in Apostichopus japonicus.
(A) Morphology of phagocytes and the process of phagocytosis of yeast cells of A. japonicus. The solid box indicates that phagocytes extend pseudopods and approach yeast cells; the dotted box represents phagocytes that engulfed yeast cells; the ellipse frame indicates the normal morphology of the phagocytes. (B) Cellular agglutination effect of coelomocytes in A. japonicus. (C) Effects of AjPAK1 silencing, miR-7 overexpression, and miR-7 inhibition on relative phagocytic capacity of coelomocytes of A. japonicus. (D) Effects of AjPAK1 silencing, miR-7 overexpression, and miR-7 inhibition on relative cellular agglutination of coelomocytes of A. japonicus. (E) Effects of AjPAK1 silencing, miR-7 overexpression, and miR-7 inhibition on relative phagocytic capacity of coelomocytes of A. japonicus after Vibrio splendidus infection. (F) Effects of AjPAK1 silencing, miR-7 overexpression, and miR-7 inhibition on relative cellular agglutination of coelomocytes of A. japonicus after V. splendidus infection. NC, negative control; RNAi, AjPAK1 silencing; Agomir, miR-7 overexpressed; Antagomir, miR-7 inhibition ** represents an extremely significant difference compared with NC (P < 0.01); * represents significant difference compared with NC (P < 0.05).
Figure 5. Effects of miR-7 and AjPAK1 on relative lysozyme (LZM) content in coelomic fluid of Apostichopus japonicus.
(A) Relative LZM content in A. japonicus coelomic fluid after AjPAK1 silencing, miR-7 overexpression, or miR-7 inhibition. (B) Relative LZM content in A. japonicus coelomic fluid after AjPAK1 silencing, miR-7 overexpression, or miR-7 inhibition upon Vibrio splendidus infection. NC, negative control; Agomir, miR-7 overexpressed; Antagomir, miR-7 inhibition. ** represents an extremely significant difference compared with NC (P < 0.01); * represents significant difference compared with NC (P < 0.05).
Figure 6. Schematic diagrams of AjPAK1 and miR-7 involved in host-pathogen interaction of Apostichopus japonicus.
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