Novel lncRNA lncRNA001074 participates in the low salinity-induced response in the sea cucumber Apostichopus japonicus by targeting the let-7/NKAα axis

Abstract

Salinity fluctuations have severe impacts on sea cucumbers and therefore important consequences in sea cucumber farming. The responses of sea cucumbers to salinity changes are reflected in the expression profiles of multiple genes and non-coding RNAs (ncRNAs). The microRNA (let-7) which is a developmental regulator, the ion transporter gene sodium potassium ATPase gene (NKAα), and the long ncRNA lncRNA001074 were previously shown to be involved in responses to salinity changes in various marine species. To better understand the relationship between ncRNAs and target genes, the let-7/NKAα/lncRNA001074 predicted interaction was investigated in this study using luciferase reporter assays and gene knockdowns in the sea cucumber Apostichopus japonicus. The results showed that NKAα was the target gene of let-7 and NKAα expression levels were inversely correlated with let-7 expression based on the luciferase reporter assays and western blots. The let-7 abundance was negatively regulated by lncRNA001074 and NKAα both in vitro and in vivo. Knockdown of lncRNA001074 led to let-7 overexpression. These results demonstrated that lncRNA001074 binds to the 3'-UTR binding site of let-7 in a regulatory manner. Furthermore, the expression profiles of let-7, NKAα, and lncRNA001074 were analyzed in sea cucumbers after the knockdown of each of these genes. The results found that lncRNA001074 competitively bound let-7 to suppress NKAα expression under low salinity conditions. The downregulation of let-7, in conjunction with the upregulation of lncRNA001074 and NKAα, may be essential for the response to low salinity change in sea cucumbers. Therefore, the dynamic balance of the lncRNA001074, NKAα, and let-7 network might be a potential response mechanism to salinity change in sea cucumbers.

Keywords: Apostichopus japonicus; Na+/K+-ATPase α; Salinity response; ceRNA; let-7; lncRNA001074.

Fig. 1

The expression levels of long non-coding lncRNA RNA001074, let-7, and NKA in sea cucumbers under salinity stress

Fig. 2

Prediction of the binding sites of let-7 and NKAα, and lncRNA001074, and the WT and MUT sequences for the luciferase reporter assay. a Prediction of the binding sites of let-7 and NKAα, and the WT and MUT sequences for the luciferase reporter assay. b Prediction of the binding sites of let-7 and lncRNA001074, and the WT and MUT sequences for the luciferase reporter assay

Fig. 3

Luciferase assay, using HEK293T cells transfected with vectors containing NKAα seed sequences (wt) or mutant constructs (mut),with vectors containing lncRNA001074 seed sequences (wt) or mutant constructs (mut). a Luciferase assay, using HEK293T cells transfected with vectors containing NKAα seed sequences (wt) or mutant constructs (mut). b Luciferase assay, using HEK293T cells transfected with vectors containing lncRNA001074 seed sequences (wt) or mutant constructs (mut). Data are presented as means ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001

Fig. 4

Expression profiles of let-7, lncRNA001074, and NKAα. a let-7, lncRNA001074 and NKAα were detected in sea cucumber coelomocyte transfected with agomir-NC, Agomir-let-7, Si-lncRNA001074, and Si-NKAα. b let-7, lncRNA001074, and NKAα were detected in sea cucumber transfected with agomir-NC, Agomir-let-7, Si-lncRNA001074, and Si-NKAα. “+” represents added substance or substance exists. Plus if the authors explain the key up front, they do not need to explain the key with each part of that figure. Expression profiles of let-7, lncRNA001074 and NKAα in vivo after transfection. The experiments were repeated 3 times. Data are presented as the mean ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001

Fig. 5

NKAα expression profiles in sea cucumbers coelomocytes transfected with agomir-NC, Agomir-let-7, Si-lncRNA001074, Si-NKAα, agomir-NC + antagomir-NC, Si-lncRNA001074 + antagomir-let-7, and Si-KNAα+antagomir-let-7. Data are presented as the mean ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001

Fig. 6

Relative expression levels of let-7, lncRNA001074, and NKA in each treatment group after salinity stress. a Transfected with agomir-NC. b Transfected with agomir-let-7. c Transfected with si-lncRNA001074. ) Transfected with si-NKAα. e Transfected with agomir-NC + antagomir-NC. f Transfected with si-lncRNA001074 + antagomir-let-7. g Transfected with si-NKA + antagomir-let-7. Data are presented as the means ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001

Fig. 7

The enzyme activity of NKAα, sodium ion concentrations, potassium ions, and chloride ions in cells transfected with different RNAs. a, b, c, d The enzyme activity of NKAα, concentration of sodium, potassium ions, chloride ions with agomir-NC, agomir-let-7, si-lncRNA001074, or si-NKAα; e, f, g, hThe enzyme activity of NK, concentration of sodium ions, potassium ions, chloride ions transfected with agomir-NC + antagomir-NC/si-lncRNA001074 + antagomir-let-7/ si-NKAα+antagomir-let-7. Data are presented as the mean ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001