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The complete replacement of fresh baits (FB) by formulated artificial feeds (AF) after domestication in mandarin fish (Siniperca chuatsi) has matured in recent years, while the underlying molecular events of long-term AF diet feeding after diet shifts remained unclear. Therefore, mandarin fish, which had been successfully domesticated, were fed with the FB or AF for 180 days to explore the molecular mechanisms underlying the differences due to shifted food habit. Compared to the FB group, slight intestinal histological damage was found in the AF group of mandarin fish. An integrated analysis of miRNA-mRNA sequencing and their interaction networks was conducted between the FB and AF groups. The result showed that a total of 209 differentially expressed mRNAs (DEmRNAs) and 2 differentially expressed miRNAs (DEmiRNAs) with significance were identified. The DEmRNAs were enriched to 39 pathways including tight junction, steroid biosynthesis and protein processing in ER. Oni-Let-7a and aca-Let-7i were the two significantly identified DEmiRNAs, whose target prediction was enriched with GO terms and KEGG pathways including TGFβ signaling pathway, steroid biosynthesis, and retinol metabolism. In general, our research was the first exploration to characterize the interacted miRNA-mRNA sequencing of mandarin fish fed the FB and AF diets, which could reveal the mechanisms of long-term adaptation to new foods in organisms who subjected to a food conversion in their early life. Data Availability Statement: The dataset generated by deep-sequencing platforms was deposited in the NCBI Sequence Read Archive (SRA, http://www.ncbi.nlm.nih.gov/Traces/sra). The NCBI Sequence Read Archive accession number is PRJNA897748.

Florfenicol (FLR), a broad-spectrum antibacterial agent, is commonly used in the breeding and cultivation of aquatic animals. microRNAs (miRNAs) control gene expression by mediating post-transcriptional mRNA repression. However, not much has been reported regarding their functions when crustaceans encounter FLR stress. Herein we observed histological alterations and performed small RNA sequencing to characterize differentially expressed miRNAs in the hepatopancreas of Portunus trituberculatus exposed to FLR (80 mg/kg) stress for 3 h and 12 h. Our results indicated irregularities in the tubular structure of hepatopancreas, separation between epithelium and myoepithelial layer, ruptured epithelial cells, and tubule degeneration. Thirty-five (19 up- and 16 downregulated) and 31 (17 up- and 14 downregulated) differentially expressed miRNAs were detected between Group-C and Group-FLR-3 h and between Group-C and Group-FLR-12 h, respectively. Dual luciferase reporter assay was performed for predicting target genes of miR-34 and miR-263b. We found that mir-263b directly inhibited cytochrome C expression at the cell level in vitro. Functional analyses of predicted target genes of differentially expressed miRNAs revealed that FLR stress induced xenobiotic metabolism, immune toxicity, and cell apoptosis, as well as influenced diverse metabolic processes. To our knowledge, this is the first report on the response of P. trituberculatus to FLR stress. Our findings provide evidence that miRNAs participate in cell apoptosis and immune regulation in crabs.

ABSTRACT: The regulation of skeletal muscle growth and development in chicken is complex. MicroRNAs (miRNAs) have been found to play an important role in the process, and more research is needed to further understand the regulatory mechanism of miRNAs. In this study, leg muscles of Jinghai yellow chickens at 300 d with low body weight (slow-growing group) and high body weight (fast-growing group) were collected for miRNA sequencing (miRNA-seq) and Bioinformatics analysis revealed 12 differentially expressed miRNAs (DEMs) between the two groups. We predicted 150 target genes for the DEMs, and GO and KEGG pathway analysis showed the target genes of miR-24-3p and novel_miR_133 were most enriched in the terms related to growth and development. Moreover, networks of DEMs and target genes showed that miR-24-3p and novel_miR_133 were the 2 core miRNAs. Hence, miR-24-3p was selected for further functional exploration in chicken primary myoblasts (CPMs) with molecular biology technologies including qPCR, cell counting kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU) and immunofluorescence. When proliferating CPMs were transfected with miR-24-3p mimic, the expression of cyclin dependent kinase inhibitor 1A (P21) was up-regulated and both CCK-8 and EdU assays showed that the proliferation of CPMs was inhibited. However, when the inhibitor was transfected into the proliferating CPMs, the opposite results were found. In differentiated CPMs, transfection with miR-24-3p mimic resulted in up regulation of MYOD, MYOG and MYHC after 48 h. Myotube areas also increased significantly compared to the mimic negative control (NC) group. When treated with inhibitor, differentiation CPMs produced the opposite effects. Overall, we revealed 2 miRNAs (novel_miR_133 and miR-24-3p) significantly related with growth and development and further proved that miR-24-3p could suppress the proliferation and promote differentiation of CPMs. The results would facilitate understanding the effects of miRNAs on the growth and development of chickens at the post-transcriptional level and could also have an important guiding role in yellow-feathered chicken breeding.

Diazinon (DZN), a persistent organophosphate insecticide, has been associated with neurotoxic effects, particularly in the hippocampus. However, the specific molecular mechanisms of DZN-induced hippocampal toxicity remain unknown. In this study, we analyzed the mRNA and miRNA expression patterns of HT22 cells following exposure to DZN (125 μM), and the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted subsequently. The integration of miRNA sequencing (miRNA-seq) and mRNA sequencing (mRNA-seq) data identified 33 differentially expressed miRNAs (DEMIs, 15 up-regulated and 18 down-regulated) and 271 differentially expressed mRNAs (DEMs, 69 up-regulated and 202 down-regulated) targeted by the DEMIs. Moreover, the 3 most central mRNAs (ITGAV, FN1, and EGFR) and 7 associated miRNAs (mmu-miR-700-5p, mmu-miR-26a-2-3p, mmu-miR-452-3p, mmu-miR-25-3p, mmu-miR-582-5p, mmu-miR-467a-5p, and mmu-miR-467b-5p) were screened and validated using quantitative real-time PCR. Furthermore, the GO analysis revealed that the identified DEMs were enriched in biological adhesion extracellular matrix, and growth factor binding, while the KEGG analysis suggested that the enriched DEMs were involved in ECM-receptor interaction, mTOR signaling pathway, MAPK signaling pathway, and AMPK signaling pathway. Our results may aid in elucidating the underlying mechanisms associated with DZN-induced hippocampal toxicity and provide valuable insights into the pathogenesis of neurotoxicity triggered by other organophosphorus pesticides., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest!, (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Evidence has shown that miRNAs could play a role in dental fluorosis, but there is no study has investigated the global expression miRNA profiles of fluoride-exposed enamel organ. In this study, we analysed the differentially expressed (DE) miRNAs between fluoride-treated and control enamel organ for the first time and found several candidate miRNAs and signaling pathways worthy of further research. Thirty Wistar rats were randomly distributed into three groups and exposed to drinking water with different fluoride contents for 10 weeks and during the gestation. The three groups were a control group (distilled water), medium fluoride group (75 mg/L NaF), and high fluoride group (150 mg/L NaF). On the embryonic day 19.5, the mandible was dissected for histological analysis, and the enamel organ of the mandibular first molar tooth germ was collected for miRNA sequencing (miRNA-seq) and quantitative real-time PCR analysis (qRT-PCR). Typical dental fluorosis was observed in the incisors of the prepregnant rats. In addition to the disorganized structure of enamel organ cells, 39 DE miRNAs were identified in the fluoride groups compared with the control group, and good agreement between the miRNA-seq data and qRT-PCR data was found. The functional annotation of the target genes of 39 DE miRNAs showed significant enrichment in metabolic process, cell differentiation, calcium signaling pathway, and mitogen-activated protein kinase(MAPK) signaling pathway terms. This study provides a theoretical reference for an extensive understanding of the mechanism of fluorosis and potential valuable miRNAs as therapeutic targets in fluorosis.

Phycocyanin is a type of marine functional food additive, exerting good antitumor property. However, its antineoplastic mechanism in non-small cell lung cancer (NSCLC) remains unclear. In this work, a high-throughput micro-RNA sequencing was employed to analyze key miRNAs regulated by phycocyanin in A549 cells for the first time. Results showed that a total of 61 miRNAs were differentially expressed between control and phycocyanin-treated cells, including 18 up-regulated and 43 down-regulated miRNAs. Subsequently, 483 validated target genes were predicted. Strikingly, protein–protein interaction analysis showed that target genes like EGFR, CDK2, POLR2E, TP53, and MED28 occupied high degrees in networks, which were predicted regulated by miR-1249-5p, miR-1231, miR-547-5p, and miR-93-5p. Furthermore, qRT-PCR showed strong consistent expression trends of differential miRNAs with miRNA-seq results. Consequently, this study significantly advances our understanding of the regulatory mechanisms of phycocyanin, especially with regard to miRNAs, and also lays a theoretical foundation for the treatment of NSCLC.

The specific miRNA regulation triggered by enzyme-treated soybean protein in response to well-known stressors, such as the prophylactic use of the antimicrobial oxytetracycline, remains unknown. Hence, this study aimed to evaluate the regulatory changes of hepatic miRNAs induced by oxytetracycline and enzyme-treated soybean protein in largemouth bass dietary formulations. The experiment was designed with three groups: the normal control (NC), the oxytetracycline exposure treatment group (OTC), and the pre-treatment with enzyme-treated soybean protein before oxytetracycline exposure group (ETSP). miRNA sequencing was employed to characterize the differences between these groups. In conclusion, the NC group exhibited up-regulation of 13 host miRNAs and down-regulation of 1 miRNA compared to the OTC group, whereas the ETSP group showed an increasing trend of 36 host miRNAs and a decreasing trend of 13 host miRNAs compared to the OTC group. Nine miRNAs were identified as prudential targets for enzyme-treated soy protein, protecting the largemouth bass liver from oxytetracycline. Furthermore, gene ontology analysis revealed nine key miRNAs that mediate signaling pathways with significant differences. The cellular lipid metabolic process was identified as the most important biological process, and the propanoate metabolism pathway was highlighted as significant. These results will facilitate further exploration of the mechanism by which enzyme-treated soy protein alleviates the effects of oxytetracycline on largemouth bass in water environments., Competing Interests: Declaration of competing interest There is no conflict of interest that should be disclosed., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Red skin color in Plectropomus leopardus is important to its ornamental and economic value. However, the color of P. leopardus can change during the rearing process, darkening and turning black due to the influence of environmental background color. The underlying molecular mechanisms that regulate this phenomenon remain unclear. MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that play important roles in numerous biological processes, such as skin differentiation and color formation in many animals. Therefore, we performed miRNA sequencing of P. leopardus skin before (initial) and after rearing with three different background colors (white, black, and blue) using Illumina sequencing to identify candidate miRNAs that may contribute to skin color differentiation. In total, 154,271,376 clean reads were obtained, with over 92 % of them successfully mapped to the P. leopardus reference genome. The miRNA length distributions of all samples displayed peaks around a typical length of 22 nt. Within these sequences, 243 known and 287 novel miRNAs were identified. A total of 65 significantly differentially expressed miRNAs (DEMs) were identified (P < 0.05), including 40 known DEMs and 25 novel DEMs. These DEMs included novel&#95;561, miR-141-3p, and miR-129-5p, whose target genes were primarily associated with pigmentation related processes, including tyrosine metabolism, melanogenesis, and the Wnt signaling pathway. These findings shed light on the potential roles of miRNAs in the darkening of skin color in P. leopardus, thus enhancing our understanding of the molecular mechanisms involved in skin pigmentation differentiation in this species., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Temperature is a critical factor that regulates the reproduction processes in teleost. However, the gonadal response mechanism to cold stress in fish remains largely unknown. In the present study, female zebrafish were exposed to different extents of low temperatures at 18 °C and 10 °C for 48 h. The ovarian histology was remarkably damaged after cold stress exposure. Integrated analysis of miRNA-mRNA was used to investigate the ovarian response to acute cold stress. A large number of mRNAs and miRNAs were altered by cold stress, which are involved in extensive biological processes. It is indicated that the signal transduction of MAPK and Calcium signaling pathway is highly engaged in zebrafish ovary to adapt to cold stress. The immune system was dysregulated by cold stress while the ovarian autophagy was activated. Remarkably increased gene number related to reproductive functions was identified in the cold stress at 10 °C compared to the control. The cold stress-induced dysregulated reproductive genes include star, hsd3b1, hsd17b1, inha, insl3, amh, nanos1 and foxl2. Combined with the dysregulated insulin, IGF and progesterone signaling, it is suggested that cold stress affects ovarian function in multiple aspects, including oocyte meiosis, folliculogenesis, final maturation and ovarian maintenance. On the other hand, the ovarian miRNA-mRNA regulatory network response to cold stress was also constructed. Overall, our result revealed the ovarian response to cold stress in zebrafish and provided insight into the fish adaptation mechanism to acute temperature change., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The regulation of skeletal muscle growth and development in chicken is complex. MicroRNAs (miRNAs) have been found to play an important role in the process, and more research is needed to further understand the regulatory mechanism of miRNAs. In this study, leg muscles of Jinghai yellow chickens at 300 d with low body weight (slow-growing group) and high body weight (fast-growing group) were collected for miRNA sequencing (miRNA-seq) and Bioinformatics analysis revealed 12 differentially expressed miRNAs (DEMs) between the two groups. We predicted 150 target genes for the DEMs, and GO and KEGG pathway analysis showed the target genes of miR-24-3p and novel&#95;miR&#95;133 were most enriched in the terms related to growth and development. Moreover, networks of DEMs and target genes showed that miR-24-3p and novel&#95;miR&#95;133 were the 2 core miRNAs. Hence, miR-24-3p was selected for further functional exploration in chicken primary myoblasts (CPMs) with molecular biology technologies including qPCR, cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) and immunofluorescence. When proliferating CPMs were transfected with miR-24-3p mimic, the expression of cyclin dependent kinase inhibitor 1A (P21) was up-regulated and both CCK-8 and EdU assays showed that the proliferation of CPMs was inhibited. However, when the inhibitor was transfected into the proliferating CPMs, the opposite results were found. In differentiated CPMs, transfection with miR-24-3p mimic resulted in up regulation of MYOD, MYOG and MYHC after 48 h. Myotube areas also increased significantly compared to the mimic negative control (NC) group. When treated with inhibitor, differentiation CPMs produced the opposite effects. Overall, we revealed 2 miRNAs (novel&#95;miR&#95;133 and miR-24-3p) significantly related with growth and development and further proved that miR-24-3p could suppress the proliferation and promote differentiation of CPMs. The results would facilitate understanding the effects of miRNAs on the growth and development of chickens at the post-transcriptional level and could also have an important guiding role in yellow-feathered chicken breeding., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Rainbow trout (Oncorhynchus mykiss) is an important economic fish in China. Skin color affects the economic value of trout. However, the molecular mechanism of the skin color variation between wild-type (WR) and yellow mutant rainbow trout (YR) is unclear. We sequenced mRNAs and miRNAs of dorsal skin to identify key color variation-associated mRNAs and miRNAs between WR and YR. Overall, 2060 out of 3625 differentially expressed genes were upregulated in YR, and 196 out of 275 differentially expressed miRNAs were downregulated in WR. We identified three key YR-upregulated genes related to the formation of xanthophores (GCH1, SLC2A11, and SOX10). Interestingly, several genes related to melanogenesis (TYR, TYRP1, TYRP2, MC1R, MITF, PMEL, SLC45A2, and OCA2) were downregulated in WR. Integrated analysis identified five miRNAs that target at least two skin color-related genes (miR-495-y, miR-543-y, miR-665-z, miR-433-y, and miR-382-x). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of target genes identified noncoding RNA metabolic process as the most significantly enriched GO term, and several metabolic pathways associated with skin color were enriched significantly, such as tyrosine metabolism, histidine metabolism, and vitamin B6 metabolism. Quantitative real-time PCR of selected mRNAs and miRNAs validated the reliability of the integrated analysis. This study provides in-depth insights into the molecular mechanism of skin color variation between WR and YR, which will accelerate the genetic selection and breeding of rainbow trout with consumer-favored traits., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Evidence has shown that miRNAs could play a role in dental fluorosis, but there is no study has investigated the global expression miRNA profiles of fluoride-exposed enamel organ. In this study, we analysed the differentially expressed (DE) miRNAs between fluoride-treated and control enamel organ for the first time and found several candidate miRNAs and signaling pathways worthy of further research. Thirty Wistar rats were randomly distributed into three groups and exposed to drinking water with different fluoride contents for 10 weeks and during the gestation. The three groups were a control group (distilled water), medium fluoride group (75 mg/L NaF), and high fluoride group (150 mg/L NaF). On the embryonic day 19.5, the mandible was dissected for histological analysis, and the enamel organ of the mandibular first molar tooth germ was collected for miRNA sequencing (miRNA-seq) and quantitative real-time PCR analysis (qRT-PCR). Typical dental fluorosis was observed in the incisors of the prepregnant rats. In addition to the disorganized structure of enamel organ cells, 39 DE miRNAs were identified in the fluoride groups compared with the control group, and good agreement between the miRNA-seq data and qRT-PCR data was found. The functional annotation of the target genes of 39 DE miRNAs showed significant enrichment in metabolic process, cell differentiation, calcium signaling pathway, and mitogen-activated protein kinase(MAPK) signaling pathway terms. This study provides a theoretical reference for an extensive understanding of the mechanism of fluorosis and potential valuable miRNAs as therapeutic targets in fluorosis., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs), a family of non-coding RNAs, have recently gained a considerable attention in neuropsychiatric disorders. Being a pleiotropic modulator of target gene(s), miRNA has been recognized as central to downstream gene regulatory networks. In the recent past, reports have suggested their role in changing the epigenetic landscape in brain of subjects with major depressive disorder (MDD). Anterior cingulate cortex (ACC) is a brain area implicated in several complex cognitive functions, such as impulse control, emotion, and decision-making and is associated with psychopathology associated with mood regulation. In this study, we examined whether MDD is associated with altered miRNA transcriptome in ACC and whether altered miRNA landscape is associated with modifications in specific gene network(s) at the functional level. Using next generation sequencing (NGS), it was observed that 117 miRNAs (4.61%) were significantly upregulated and 54 (2.13%) were downregulated in MDD subjects (n = 22) compared with non-psychiatric controls (n = 25). Using 24 most significantly upregulated miRNAs in the MDD group, we determined functional enrichment of target genes and found them to be associated with long-term potentiation, neurotrophin signaling, and axon guidance. Intra- and inter-cluster similarities of enriched terms based on overrepresented gene list showed neurobiological functions associated with neuronal growth and survival. Web centric parameters and ontology enrichment functions identified two major domains related to phosphatidyl signaling, GTPase signaling, neuronal migration, and neurotrophin signaling. Our findings of altered miRNA landscape along with a shift in targetome relate to previously reported morphometric changes and neuronal atrophy in ACC of MDD subjects., Competing Interests: Declaration of Competing Interest Authors report no biomedical financial interests or potential conflicts of interest., (Copyright © 2020 Elsevier B.V. and ECNP. All rights reserved.)

Although the accuracy of detecting the expression of miRNAs by quantitative real-time polymerase chain reaction (qRT-PCR) is highly dependent on reliable reference miRNAs, many commonly used reference miRNAs are not stably expressed and as such are not suitable for quantification and normalization of qRT-PCR data. To solve this problem, we analyzed the global expression profiles of thousands of samples in 14 types of common human tumors released by The Cancer Genome Atlas (TCGA), and identified the most stably and highly expressed miRNAs as candidate reference miRNAs in each type of tumor. We found that miR-361-5p and let-7i-5p were the most recommended candidate reference miRNAs in nine and eight types of tumors, respectively, followed by let-7a-5p, mir-28-5p and miR-99b-5p. Our results are of important value to those researchers focused on miRNA; however, these candidate reference miRNAs still need to be validated prior to their use in qRT-PCR studies., (Copyright © 2014 Elsevier Inc. All rights reserved.)