The miRNAs let-7b and miR-141 Coordinately Regulate Vitellogenesis by Modulating Methyl Farnesoate Degradation in the Swimming Crab Portunus trituberculatus

Abstract

Methyl farnesoate (MF), a crucial sesquiterpenoid hormone, plays a pivotal role in the reproduction of female crustaceans, particularly in the vitellogenesis process. Despite extensive research on its functions, the molecular mechanisms that regulate MF levels during the vitellogenic phase remain largely elusive. This study investigates the roles of microRNAs (miRNAs), significant post-transcriptional regulators of gene expression, in controlling MF levels in the swimming crab Portunus trituberculatus. Through bioinformatic analysis, four miRNAs were identified as potential regulators targeting two genes encoding Carboxylesterases (CXEs), which are key enzymes in MF degradation. Dual luciferase reporter assays revealed that let-7b and miR-141 suppress CXE1 and CXE2 expression by directly binding to their 3' UTRs. In vivo overexpression of let-7b and miR-141 significantly diminished CXE1 and CXE2 levels, consequently elevating hemolymph MF and enhancing vitellogenin expression. Spatiotemporal expression profile analysis showed that these two miRNAs and their targets exhibited generally opposite patterns during ovarian development. These findings demonstrate that let-7b and miR-141 collaboratively modulate MF levels by targeting CXEs, thus influencing vitellogenesis in P. trituberculatus. Additionally, we found that the expression of let-7b and miR-141 were suppressed by MF, constituting a regulatory loop for the regulation of MF levels. The findings contribute novel insights into miRNA-mediated ovarian development regulation in crustaceans and offer valuable information for developing innovative reproduction manipulation techniques for P. trituberculatus.

Keywords: crab; methyl farnesoate; miRNA; ovary; vitellogenesis.

Figure 1

Assessment of miRNA-target interactions via dual luciferase reporter assay. Panel (A) shows the binding sites of let-7b and miR-92b in CXE1 3′ UTR. Panel (B) shows the binding sites of miR-981 and miR-141 in CXE2 3′ UTR. Panel (C) presents the dual luciferase reporter assay results for the interaction between let-7b and CXE1. Panel (E) shows the assay for miR-92b and CXE1 interactions. Panel (D) illustrates the assay findings for miR-141 and CXE2, while Panel (F) displays the results for miR-92b and CXE2. Statistical significance was determined using one-way ANOVA with subsequent Tukey’s multiple comparison tests. Distinct letters above values denote significant differences (p < 0.05). In these assays, HEK293T cells were co-transfected with either miRNA mimics or control mimics, along with pmirGLO vectors that contain either the wild-type (WT) (blue letters) or mutant (MT) (red letters) miRNA binding sites. Seed sequences were indicated in green. The data are shown as mean ± SD, n = 3.

Figure 2

Temporal expression profiles of miRNAs (let-7b and miR-141), their target genes, and hemolymph MF levels during ovarian maturation. Panels (A–C) chart the expression patterns of let-7b in the hepatopancreas (A), miR-141 in the hepatopancreas (B), and miR-141 in the ovary (C). Panels (D–F) detail the relative expression of CXE1 in the hepatopancreas (D), CXE2 in the hepatopancreas (E), and CXE2 in the ovary (F). Panel (G) quantifies the hemolymph MF concentrations across various stages of ovarian development (G). Statistical significance was determined using one-way ANOVA with subsequent Tukey’s multiple comparison tests. Distinct letters above values denote significant differences (p < 0.05). Data are presented as mean ± standard deviation (SD), n = 5.

Figure 3

Impact of overexpressing let-7b and miR-141 on target gene expression. Panels (A–C) depict the expression levels of let-7b in the hepatopancreas (A), miR-141 in the hepatopancreas (B), and miR-141 in the ovary (C) subsequent to agomir injection. Panels (D–F) illustrate the expression levels of CXE1 in the hepatopancreas (D), CXE2 in the hepatopancreas (E), and CXE2 in the ovary (F) after agomir administration. Data are expressed as the mean ± SD. The means were statistically analyzed using the Student’s t-test, with *, p < 0.05 and ** p < 0.01. n = 5.

Figure 4

Variations in hemolymph MF levels and gene expression post let-7b and miR-141 overexpression. (A) MF concentration in the hemolymph following agomir administration. (B) Quantified Kr-h1 levels. (C,D) Vitellogenin expression profiles in the hepatopancreas (C) and ovary (D). Data are expressed as mean ± SD. Statistical differences were determined via one-way ANOVA and subsequent Tukey’s multiple comparison test. Distinct letters above values denote significant differences (p < 0.05). n = 5.

Figure 5

Expression levels of miRNAs and their target genes after MF injection. (A) Expression profile of let-7b in the hepatopancreas. (B) miR-141 levels in the hepatopancreas. (C) miR-141 expression in the ovary. (D) CXE1 abundance in the hepatopancreas. (E) CXE2 levels in the hepatopancreas, and (F) CXE2 expression in the ovary following MF administration. The data are presented as the mean ± SD. Statistical analysis was performed using the Student’s t-test, with significance denoted by * p < 0.05, and ns indicates that there was not significant difference n = 3.