Abstract
Extensive regulation of microRNAs (miRNAs) that belong to a type of endogenous small noncoding RNAs on gene expression in sheep has been recognised. Meanwhile, utilisation of water extract of Allium mongolicum Regel (WEA) in diet of sheep has been proved to regulate fatty acid composition and flavour in mutton. However, the regulatory mechanism remains unclear. We conducted miRNA and mRNA sequencing in this study. Briefly, 30 male small-tailed Han sheep (4.5 months old) were randomly assigned to two groups. A basal diet with 3.4 g·sheep−1·d−1 WEA supplementation (WEA group) or with no WEA supplementation (CON group) was fed to sheep, respectively. Subcutaneous and visceral adipose tissue samples were collected during slaughter after 75-day feeding experiment. We obtained 30 differentially expressed miRNAs (DE-miRNAs) after application of WEA in diet of sheep in subcutaneous adipose tissue. Likewise, 15 DE-miRNAs in visceral adipose tissue were identified. A total of 746 differentially expressed mRNAs (DE-mRNAs) with 90 lipid-related DE-mRNAs in subcutaneous adipose tissue and 569 DE-mRNAs with 73 lipid-related DE-mRNAs in visceral adipose tissue were identified after WEA supplementation. The intersection genes from predicted target genes of DE-miRNAs by databases and actual DE-mRNAs by mRNA sequencing were enriched in several lipid-related pathways both in subcutaneous adipose and in visceral adipose tissue. We constructed differential miRNAs-mRNAs networks by Cytoscape software. Our results will help to explain regulatory mechanism of fatty acid composition in mutton by dietary WEA and will provide a theoretical basis for rational use of the plant extract in diet of sheep.
Utilisation of Allium mongolicum Regel water extract led to alterations of endogenous miRNA and gene expression profiles in subcutaneous and visceral adipose tissue of sheep.
Allium mongolicum Regel water extract improved nutritional value and flavour of mutton by changing fatty acid composition in adipose tissue.
Establishment of miRNA-mRNA regulatory network with regard to manipulation of fatty acid composition in mutton by Allium mongolicum Regel water extract, and provided a theoretical basis for rational utilisation of this plant extract in diet of sheep.
HIGHLIGHTS
Introduction
Incremental proofs manifest indispensable roles of noncoding RNAs (ncRNAs) in diverse animals or cell lines. We are more inclined to explore microRNA (miRNA) mediated impact on gene expression, since both long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) may exert their regulatory effect on gene expression indirectly by competitive adsorption of miRNAs (Kang et al. Citation2020; Yang et al. Citation2022). From definition, miRNAs are characterised by their shorter and linear sequences, the lengths of which are approximately 22 nt. Despite miniature size of miRNAs, their ubiquitous impact on normal life activities or disease should never be ignored. Inhibition of translation or target mRNA degradation occurs when seed region of miRNAs physically bind to 3′ untranslated region (3′ UTR) of corresponding target mRNAs by base pairing (Sedgeman et al. Citation2019). In addition to suppressive effects of miRNAs on expression of downstream target mRNAs in most cases, Ørom and his co-workers (Orom et al. Citation2008) raised that combination of miRNAs with 5′ untranslated region (5′ UTR) of mRNAs might take enhanced effects on mRNA translation. Any external stimulus (e.g. nutrition) that affect factors implicated in endogenous miRNA biosynthesis in vivo including initial transcription process and modification of specialised enzymes may change miRNA production, in other words, may result in alteration of miRNA expression profiles (Gulyaeva and Kushlinskiy Citation2016).
Considerable efforts have been undertaken to identify miRNAs with regard to lipid metabolism in lipid-related tissues containing adipose tissue (Zhou et al. Citation2017), liver (Dai et al. Citation2019), mammary tissue (Billa et al. Citation2019). Additionally, miRNAs are able to regulate lipid metabolism under special circumstances, for instance, adipose tissue inflammation (Jaiswal et al. Citation2019) and modulate adipocyte-based events (e.g. adipocyte proliferation, differentiation and apoptosis) according to cell transfection or ‘rescue’ experiment (Du et al. Citation2018; Zhang et al. Citation2018). Furthermore, inclusion of specific nutrients (e.g. resveratrol) into diet of pig was reported to influence miRNA expression in muscle and led to changes in expression of downstream target genes related to lipid metabolism (Zhang et al. Citation2019). Several ‘diet-specific’ miRNAs were identified by miRNA-sequencing.
As a member of Allium plants, Allium mongolicum Regel has long been recognised to exert a beneficial role in human and animal health and it exists mainly in northwest China. Earlier studies proved that inclusion of water extract of Allium mongolicum Regel (WEA) in diet of sheep could reduce drip loss and cooking loss of mutton and could also improve meat quality by modulating fatty acid composition in adipose tissue and muscle of sheep, on account of bioactive molecules in this plant extract such as flavonoids, polyphenols. And changes in fatty acid composition might partly explain the reduced ‘mutton flavour’ by WEA (Zhang et al. Citation2019; Ding et al. Citation2021; Yaxing et al. Citation2021). Also, application of WEA was an effective way to reduce mutton flavour by decreasing contents of 4-alkyl branched chain fatty acids (BCFAs), which were recognised as main contributors to mutton flavour (Liu W and Ao Citation2021). From a genetic point of view, it was demonstrated that A. mongolicum Regel decreased expression of genes associated with lipogenesis gene in subcutaneous adipose tissue of sheep to prevent excessive fat deposition, thereby indirectly reducing the adverse impact on human health (Zhang et al. Citation2019). Xue et al. (Citation2021) screened and obtained candidate lncRNAs that participated in regulation of fatty acid composition in sheep by dietary WEA.
However, from perspective of miRNA, there is a lack of in-depth studies on the mechanism by which dietary WEA regulates fatty acid composition in mutton. Here, we performed miRNA and mRNA sequencing in subcutaneous and visceral adipose tissue after WEA supplementation in diet of sheep. This study may provide a miRNA-mRNA mechanism for regulation of fatty acid composition in mutton by dietary WEA.
Methods
Acquisition of the plant and its water extract
Freshly picked A. mongolicum Regel was dried to constant weight at 65 °C after ridding yellow leaves and weeds. The dried A. mongolicum Regel was ground to make powder. Thereafter, WEA was obtained based on extraction procedures described in detail in a previous study (Ding et al. Citation2021). After soaking by distilled water, filtration and concentration, finally, WEA was obtained by freeze-drying on a freeze dryer (FDU-2110, Elang technology international trade Co., Ltd., Shanghai, China) for approximately two days. The collected WEA was pulverised, packaged and properly stored until start of animal feeding experiment. The principal chemical constituents of WEA were polyphenols and flavonoids. (Wang et al. Citation2019).
Utilisation of WEA in diet of sheep and sampling
Thirty healthy, male, small-tailed Han sheep (4.5 months old) with similar initial weight (36.30 ± 0.80 kg) were raised in our experiment and some key vaccines had been delivered. The sheep were randomly assigned to two groups with 15 sheep in each group. And sheep from each group were divided into three replicates with five sheep in each replicate. Amongst two groups, sheep in one group were fed basal diet without WEA supplementation (CON group), the other group of sheep were fed a basal diet with WEA (3.4 g·sheep−1 per day) supplementation (WEA group). The dosage of 3.4 g mentioned here was based on a prior study by Du et al. (Du et al. Citation2019). The initial body weight of the sheep in three replicates of CON group was 36.70 ± 0.90 kg, 36.50 ± 0.70 kg, 36.20 ± 0.80 kg. The initial body weight of the sheep in WEA group was 36.10 ± 0.70 kg, 36.40 ± 0.90 kg, 35.90 ± 0.80 kg. The rearing cycle included 15-day adaptation period and 60-day formal experimental period. The experiment was performed at a well-known sheep farming company named Fuchuan in Bayannaoer, Inner Mongolia, China. The composition of basal feedstuff is exhibited in Table . The basal diet met maintenance needs for sheep as described by the National Research Council (NRC 2012). At the end of experimental period, two sheep from each replicate were randomly selected and slaughtered at a local slaughterhouse. Thus, six sheep were slaughtered in CON group and WEA group respectively. Samples of subcutaneous adipose tissue from CON group and WEA group were collected with autoclaved scalpels and forceps and were denoted as sCON and sWEA, respectively. Samples of visceral adipose tissue (perirenal adipose tissue) were denoted as pCON and pWEA, respectively. One portion of samples were aliquoted into well-labelled cryovials and immediately stored in liquid nitrogen. Samples in liquid nitrogen were then for RNA extraction and sequencing. The other portion of samples that were vacuum-packed were stored in a −20 °C refrigerator for analysis and determination of fatty acid composition.
Table 1. Information for constituents in basal diet and nutritional value.
Detection of fatty acid in adipose tissue
The method of AOAC (2002) was employed to determine fatty acid contents in homogenised adipose tissue samples (Mossoba et al. Citation2001). Briefly, pre-treatment of samples was based on hydrolytic method. And methyl esterification was achieved by using boron trifluoride in methanol (373-57-9, Mkseal, Shanghai, China). A gas chromatograph (Clarus680, PerkinElmer, Massachusetts, USA) with a hydrogen flame ionisation detector (FID) and a capillary column (SP2560, 100 m × 0.25 mm i.d., and 0.20 μm film thickness, Merck, Shanghai, China) was used to quantify fatty acid methyl esters (FAME) synthesised above. Data of individual fatty acid was ultimately presented as a percentage of total fatty acids. The injection volume was 1 μL. The temperature for injector and detector was 260 °C and nitrogen was used as carrier gas.
Workflow for library construction and sequencing
Two types of sequencing for miRNA and mRNA shared the same method in total RNA extraction and quality analysis. Twenty-four RNA samples of subcutaneous and visceral adipose tissue derived from twelve experimental sheep were obtained by extraction with TRIzol® Reagent (Invitrogen, Carlsbad, CA) from homogenised samples ground in liquid nitrogen. RNA concentration was measured using ND-2000 (NanoDrop Technologies). RNA integrity was assessed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The isolated RNA samples were taken as high-quality samples for subsequent library construction. Then, miRNA sequencing libraries and mRNA sequencing libraries were generated successfully by using NEBNext® Multiplex Small RNA Library preparation Kit Set for Illumina® (NEB, Ipswich, MA) and TruSeqTM RNA sample preparation Kit (Illumina, Inc., San Diego, CA) respectively following the manufacturers’ instructions. After library construction, miRNA and mRNA sequencing were performed with Illumina Hiseq 2500/2000 platform and Illumina HiSeq xten/NovaSeq 6000 sequencer (2 × 150 bp read length), respectively. Finally, 75 bp single-end reads were obtained in miRNA sequencing.
Data filtering, mining and differential expression analysis
Regardless of miRNA sequencing or mRNA sequencing, raw data deposited in form of FASTQ was firstly filtered to remove interfering components (low-quality reads) and finally clean reads were generated. For miRNA sequencing, the mapping of obtained clean reads from miRNA sequencing to designated sheep reference genome was performed by application of Bowtie (http://bowtie-bio.sourceforge.net/index.shtml) software (Griffiths-Jones et al. Citation2006; Culwick et al. Citation2020). Annotation and identification of known miRNAs and other small non-coding RNAs such as siRNAs, piRNAs were realised by miRbase 21.0 (http://www.mirbase.org/) database and Rfam database version 10.1 (http://rfam.sanger.ac.uk/), respectively. For unmapped reads in above two databases, by making use of miRDeep2 software, novel miRNAs were identified according to secondary structures (Friedlander et al. Citation2012). For quality control of mRNA sequencing data, after trimming and elimination of low-quality reads, alignment of clean reads to sheep reference genome (Oar_v3.1) was performed by application of HISAT2 software. Mapped reads in mRNA sequencing underwent assembling process that relied on StringTie. The identified known and novel miRNA expression levels were normalised by transcripts per million (TPM) values. Differentially expressed miRNAs between different groups were screened by edgeR software version 3.14.0 (http://bioconductor.org/packages/stats/bioc/edgeR/). Fragments per kilobases per million (FPKM) values were used to normalise mRNA expression levels. Differential expression analysis of mRNAs between different groups was conducted by DESeq2 (http://bioconductor.org/packages/stats/bioc/DESeq2/). Fold change ≥ 1.3 and p-value < .05 were taken as screening criteria for identification of differentially expressed miRNAs or mRNAs (Lim et al. Citation2021). The Benjamini-Hochberg (BH) methodology was used to control the False Discovery Rate (FDR).
Bioinformatics analysis
Target gene prediction of differentially expressed miRNAs was achieved by utilising miRanda, Targetscan, and RNA hybrid databases. The predicted target genes of differentially expressed miRNAs and actual differentially expressed mRNAs according to RNA sequencing were integrated and analysed. In the meantime, gene ontology (GO) (http://www.geneontology.org) and kyoto encyclopaedia of genes and genomes (KEGG) (https://www.genome.jp/kegg/) enrichment analyses of intersection genes were performed. miRNA-mRNA interactive network diagram was drawn by Cytoscape_v3.9.1.
Validation of miRNA sequencing and mRNA sequencing results
The extracted RNAs from adipose tissue samples were converted to cDNA by Geneseed®IIFirst Strand cDNA Synthesis Kit (Geneseed, Guangzhou, China) for mRNA validation and were converted to cDNA by miRcute Plus miRNAs First-Strand cDNA Kit (TIANGEN, Beijing, China) for miRNA validation. Subsequent quantitative PCR procedure for mRNA was conducted by Geneseed® qPCR SYBR® Green Master Mix (Geneseed, Guangzhou, China) and for miRNA was conducted by miRcute Plus miRNA qPCR Kit (TIANGEN, Beijing, China) with use of ABI PRISM® 7500 sequence detection system (Applied Biosystems, Foster City, CA). Moreover, four differentially expressed miRNAs (novel_20_33380, miR-218a, novel_14_26842, miR-214-3p) and four differentially expressed mRNAs (CIDEA, NDUFS6, ABCA6, JAGN1) between sCON group and sWEA group were randomly selected for validation. Four miRNAs (miR-200a, miR-200b, novel_X_41185, miR-150) and mRNAs (CCND2, INSIG1, FABP1, ACADS) were also selected between pCON group and pWEA group. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin were designated as reference genes for mRNA. U6 snRNA and 5S-rRNA were designated as reference genes for miRNA validation. The relative expression was analysed with method of 2-ΔΔct. The temperature and cycle program for mRNA was 95 °C for five minutes, followed by 40 cycles at 95 °C for 10 s, with a final annealing procedure at 60 °C for 34 s. The temperature and cycle program for miRNA was 95 °C for 15 min, followed by 40 cycles at 94 °C for 15 s, with a final annealing procedure at 60 °C for 34 s. Primer sequences are presented in Supplemental Tables S1–S3.
Statistical analysis
SPSS Statistics 26.0 was utilised for data analysis and significant differences in fatty acid contents between different groups were assessed according to One-way ANOVA. A p < .05 was considered statistically significant, while a p < .10 represented a significantly different trend.
Results
Fatty acid profiles in different groups
To assess variations in phenotype-related indicators after adding WEA to diet of sheep, we conducted fatty acid analysis in adipose tissue. The result revealed that effect of dietary WEA on fatty acid profile in subcutaneous adipose tissue displayed some degree of similar trend with that in visceral adipose tissue. A significant decrease in total saturated fatty acids (SFAs) and a significant increase in monounsaturated fatty acids (MUFAs) were observed (p < .05) in both tissues after inclusion of WEA in diet of sheep (Tables ), indicating the beneficial effects on health of humans who consumed the meat (Chikwanha et al. Citation2018). Additionally, with application of WEA, contents of myristic acid (C14:0), palmitic acid (C16:0) were obviously decreased, while alpha-linolenic acid (C18:3n3), eicosadienoic acid (C20:2n6), docosadienoic acid (C22:2n6), polyunsaturated fatty acids (PUFAs) were remarkably increased (p < .05) compared to control in subcutaneous adipose tissue (Table ). Fatty acid analysis in visceral adipose tissue showed that adding WEA to sheep’s diet significantly decreased pentadecanoic acid (C15:0) content and significantly increased palmitoleic acid (C16:1), C18:1n9c (p < .05) contents (Table ).
Table 2. Fatty acid profile in subcutaneous adipose tissue with inclusion of WEA (%).
Table 3. Fatty acid profile in visceral adipose tissue with inclusion of WEA (%).
Summary of sequencing data for miRNA and mRNA
For miRNA sequencing, to discard low-quality reads and adapter sequences, raw reads were subjected to strict filtering procedure, after which totally 75.23 million, 87.04 million, 90.83 million and 97.69 million clean reads were obtained for sCON group, sWEA group, pCON group and pWEA group, respectively. Also, alignment information of generated clean reads onto sheep genome is displayed in Supplemental Table S4 and S5. Likewise, detailed information for mRNA sequencing was analysed (Supplemental Tables S6–S7).
Of the identified miRNAs, both known miRNAs and novel miRNAs were included. Supplemental Figure S1 and Figure S2 exhibit length characteristic and non-coding small RNAs (sRNAs) classification of four groups, respectively. It was demonstrated from statistics of miRNA lengths that miRNAs with sizes of 21–23 nt occupied the most proportion and the same pattern of size distribution was found among four groups. Also, result from sRNA classification illuminated that miRNA sequences were the most annotated types among all sRNAs (Supplemental Figure S2). Result of chromosome distribution indicated the extensive distribution of known and novel miRNAs across chromosomes (Supplemental Figure S3).
Assessment of differential expression
So as to clarify miRNA-mRNA mechanism by which dietary WEA modulate lipid metabolism in sheep, we evaluated variations in miRNA and mRNA expression in adipose tissue after adding WEA to diet of sheep. Noticeably, differentially expressed miRNAs (DE-miRNAs) with low expression were ruled out. As a result, thirty DE-miRNAs were found in subcutaneous adipose tissue (p < .05) after inclusion of WEA in diet of sheep. Three DE-miRNAs were upregulated in sWEA group compared with sCON group and upregulated miRNAs were novel_20_33380, miR-136, miR-338-3p, while the remaining 27 DE-miRNAs including miR-218a were down-regulated by WEA. The top 10 expressed DE-miRNAs among the above-mentioned DE-miRNAs are shown in Table . For visceral adipose tissue, fifteen DE-miRNAs were identified between pCON group and pWEA group (p < .05), of which six DE-miRNAs including miR-133, miR-200a, miR-200b, miR-1-3p, miR-340-5p, let-7e-5p were significantly upregulated, while the rest were significantly downregulated after utilisation of WEA. The top 10 expressed DE-miRNAs between pCON group and pWEA group are shown in Table . As anticipated, a greater number of DE-miRNAs were found between two types of adipose tissue with no WEA supplementation. Thereinto, the number of DE-miRNAs screened between sCON group and pCON group was 69 (p < .05). The top 10 expressed DE-miRNAs are shown in Table .
Table 4. Top 10 expressed DE-miRNAs between sCON group and sWEA group.
Table 5. Top 10 expressed DE-miRNAs between pCON group and pWEA group.
Table 6. Top 10 expressed DE-miRNAs between sCON group and pCON group.
Likewise, a total of 746 differentially expressed mRNAs (DE-mRNAs) were found between sCON group and sWEA group with 319 significantly upregulated DE-mRNAs and 427 downregulated DE-mRNAs by WEA addition (p < .05). And dietary WEA resulted in obvious differences in visceral adipose tissue. Among total 569 differential genes, numbers of upregulated DE-mRNAs and downregulated DE-mRNAs by WEA supplementation were 280 and 289, respectively. Herein, ninety lipid-related DE-mRNAs between sCON group and sWEA group and seventy-three lipid-related DE-mRNAs between pCON group and pWEA group are listed in hierarchical cluster heatmaps (Figure ). Also, a greater number of DE-mRNAs were found between two types of adipose tissue with no WEA supplementation. Figure displays the top 100 DE-mRNAs related to lipid metabolism out of a total of 2797 DE-mRNAs between two types of adipose tissue. Detailed information for DE-mRNAs between different groups is listed in Supplemental Tables S8–S10. The consistent trend of differential expression from qRT-PCR and sequencing results could prove the reliability of miRNA and mRNA sequencing (Figure ).
Figure 1. Cluster heatmap of differentially expressed mRNAs (DE-mRNAs) regarding lipid metabolism between different groups. (a) Lipid-related DE-mRNAs in subcutaneous adipose tissue between control (sCON) group and water extract of Allium mongolicum Regel (sWEA) group. (b) Lipid-related DE-mRNAs in visceral adipose tissue between control (pCON) group and water extract of Allium mongolicum Regel (pWEA) group. (c) Lipid-related DE-mRNAs between subcutaneous (sCON) and visceral (pCON) adipose tissue without WEA supplementation.
Figure 2. QRT-PCR results of randomly selected differentially expressed miRNAs (DE-miRNAs) and differentially expressed mRNAs (DE-mRNAs) in contrast to results of sequencing. (a) QRT-PCR and sequencing results in subcutaneous adipose tissue between control (CON) group and water extract of Allium mongolicum Regel (WEA) group. (b) QRT-PCR and sequencing results in visceral adipose tissue between CON group and WEA group, * indicates a significant difference with a p value less than 0.05.
Functional analysis of differentially expressed target genes
GO enrichment analysis of intersection genes from predicted target genes of DE-miRNAs and actual DE-mRNAs was performed (Figure , Supplemental Tables S11–S13). No matter in subcutaneous or visceral adipose tissue, after application of WEA, above-mentioned intersection genes, in other words, differentially expressed target genes of DE-miRNAs were mainly enriched in GO terms related to cellular components, molecular functions, biological processes. And the same result of GO enrichment analysis between two types of tissues were found (Figure ). Analysis of the top 20 KEGG enriched pathways for intersection genes from predicted target genes of DE-miRNAs and actual DE-mRNAs (Supplemental Tables S14–S16) indicated that intersection genes between sCON group and sWEA group were enriched in several lipid-related pathways such as glycerophospholipid metabolism, PPAR signalling pathway, phospholipase D signalling pathway, PI3K-Akt signalling pathway (Figure ). It turned out that intersection genes between pCON group and pWEA group were enriched in several lipid-related pathways such as PI3K-Akt signalling pathway, regulation of lipolysis in adipocytes (Figure ). Intersection genes between two types of adipose tissue were enriched in several lipid-related pathways such as phospholipase D signalling pathway, glycerophospholipid metabolism (Figure ). Ultimately, we conducted lipid-related miRNA-mRNA network analysis by Cytoscape software. According to correlation analysis, after WEA supplementation, nine DE-mRNAs related to lipid metabolism in subcutaneous adipose tissue were regulated by seven DE-miRNAs (Figure ). Regarding visceral adipose tissue, five DE-mRNAs related to lipid metabolism were regulated by four DE-miRNAs (Figure ). Five DE-miRNAs regulated expression of 11 lipid metabolism-related DE-mRNAs between two types of adipose tissue (Figure ).
Figure 3. The top 50 enriched GO terms for intersection genes from target genes of differentially expressed miRNAs (DE-miRNAs) and actual differentially expressed mRNAs (DE-mRNAs). (a) GO terms for intersection genes between sCON group and sWEA group. (b) GO terms for intersection genes between pCON group and pWEA group. (c) GO terms for intersection genes between sCON group and pCON group.
Figure 4. The top 20 enriched KEGG pathways for intersection genes from target genes of DE-miRNAs and actual DE-mRNAs. (a) Enriched KEGG pathways for intersection genes between sCON group and sWEA group. (b) Enriched KEGG pathways for intersection genes between pCON group and pWEA group. (c) Enriched KEGG pathways for intersection genes between sCON group and pCON group.
Figure 5. MiRNA-mRNA network with regard to lipid metabolism. (a) Differential miRNA-mRNA network diagram between sCON group and sWEA group. (b) Differential miRNA-mRNA network diagram between pCON group and pWEA group. (c) Differential miRNA-mRNA network diagram between sCON group and pCON group. Pink represents up-regulation, green represents down-regulation.
Discussion
It is known that WEA is of importance in improving mutton quality and fatty acid composition (Zhang et al. Citation2019; Ding et al. Citation2021; Yaxing et al. Citation2021). In current study, the decreased saturated fatty acids (SFAs) and elevated monounsaturated fatty acids (MUFAs) by dietary WEA in two types of adipose tissue were in parallel with a prior study, in which total SFAs were decreased while MUFAs were increased in muscle of WEA-fed sheep (Yaxing et al. Citation2021). We demonstrated that polyunsaturated fatty acids (PUFAs) in subcutaneous adipose tissue were significantly enhanced by dietary WEA, and this result also coincide with change of PUFAs in muscle. Elevation of PUFAs and C18:3n3 contents in subcutaneous adipose tissue of sheep receiving diet supplemented with WEA might partly explain the improved nutritive value of mutton, since the beneficial effect of these components for human (Joris et al. Citation2020; Su et al. Citation2022). Linoleic acid (C18:2n6c) and C18:0 contents are associated with mutton flavour (Sanudo et al. Citation2000), the content of C18:2n6c in our study had a trend of significant increase by dietary WEA, despite the fact that the alteration of C18:0 did not reach a significant level. Combined with previous research by our team, in which meat from WEA-fed sheep had lower concentrations of flavour-related 4-alkyl BCFAs and C18:0 (Liu et al. Citation2019), collectively, no matter from perspective of nutritional value or flavour value, WEA can ultimately improve meat quality by regulating fatty acid composition (Su et al. Citation2022).
Previously, studies on miRNome and mRNome between animal breeds or diverse tissues occupied the vast majority. Researches concerning differential miRNA and mRNA profiles caused by dietary nutrients are deficient. As well investigated by precedent studies, dietary polyphenols from fruits and vegetables or other plant-derived compounds could modulate lipid-related miRNA expression in obesity (Filardi et al. Citation2020). Accordingly, active flavonoids and polyphenols contained in WEA may also alter miRNA expression levels in this study by affecting factors involved in miRNA biosynthesis process (transcription stage or processing stage) in vivo (Wang et al. Citation2019). MiR-218a, miR-214-3p, miR-877-3p are three conserved miRNAs among mammals. In subcutaneous adipose tissue, these three miRNAs were differentially expressed after utilisation of WEA in diet of sheep. Manipulation of adipogenesis differentiation by miR-218a, miR-214-3p was previously validated in porcine preadipocyte and 3T3-L1 preadipocyte respectively, while miR-218a had an inhibitory effect and miR-214-3p had an enhanced effect on adipogenesis (Xi et al. Citation2019; Shan et al. Citation2022). miR-877-3p was implicated in regulation of adipose tissue inflammation and obesity in a research (Brettfeld et al. Citation2017), while miR-877-3p expression was decreased by dietary WEA. From miRNA-mRNA network, as a target gene of miR-218a, fatty acid elongase 5(ELOVL5)was increased after administration of WEA. Dual-luciferase validation of this targeting relationship between two molecules was presented in an earlier study (Zhang et al. Citation2020). Elevation of ELOVL5 expression in subcutaneous adipose tissue by feeding WEA to sheep might provide an explanation for increased PUFAs and C18:3n3 by dietary WEA. Because ELOVL5 has been widely accepted to regulate biosynthesis of PUFAs (Zhang et al. Citation2020). Additionally, Schettini et al. (Citation2022) found that expression of ELOVL5 was correlated with C18:3n3 content in meat (Schettini et al. Citation2022). Meanwhile, flavonoids from Allium mongolicum Regel were reported to change eicosapentaenoic acid (EPA) in mutton significantly (Liu et al. Citation2019). A study in mammary epithelial cells of cow showed that ELOVL5 gene was upregulated by miR-218a to improve milk fatty acid composition (Chen et al. Citation2021). In addition, target genes of DE-miRNAs between sCON group and sWEA group were mainly involved in fatty acid composition, adipocyte differentiation, fatty acid transport, mitochondrial function and adipocyte viability according to miRNA-mRNA network map. Loss of Cell death-inducing DNA fragmentation factor α-like effector A (CIDEA) gene strengthened lipolysis in human adipocytes (Puri et al. Citation2008). Inhibin beta B (INHBB) was proved to promote the differentiation of preadipocytes into mature adipocytes and delta-like 4 (DLL4) was thought to regulate fatty acid uptake. Our data in this study indicated that expression levels of INHBB and DLL4 were downregulated by WEA supplementation. Thus, altered expression of INHBB and DLL4 genes might imply altered lipogenesis and fatty acid uptake (Zambonelli et al. Citation2016; Aupetit et al. Citation2023). Phospholipase C, delta-1 (PLCD1) was thought to be related to linoleic acid (C18:2n6c) content in intramuscular fat of chicken according to a prior study (Liu et al. Citation2023). And PLCD1 was downregulated by dietary WEA in our findings, while C18:2n6c content in subcutaneous adipose tissue had an increased trend after utilisation of WEA. Cellular retinoic acid binding protein 2 (CRABP2), meteorin-like (METRNL) are inhibitory genes of adipogenic differentiation (Berry et al., Citation2012; Loffler et al. Citation2017). WEA-fed sheep had higher levels of CRABP2 and METRNL expression, indicating that subcutaneous adipogenesis was inhibited by WEA to control excessive fat deposition and ensure reasonable distribution of fat in body of sheep. Furthermore, upregulation of NADH-dehydrogenase iron-sulfur protein 6 (NDUFS6) gene by dietary WEA could facilitate development and functional maintenance of adipocytes through modulation of mitochondrial function and adipocyte viability (Li et al. Citation2022). ATP-binding cassette A transporter 6(ABCA6)is a member of ABCA subset that is responsible for transportation of lipid and cholesterol (Imperio et al. Citation2019). The difference of ABCA6 gene expression in our study indicated alteration of lipid transportation ability by WEA supplementation.
Peng et al. (Citation2017) elucidated that miR-377 deficiency was able to improve inflammatory response and insulin sensitivity in adipocyte. In current study, miR-377-5p expression was downregulated by WEA in visceral adipose tissue, suggesting possibly beneficial effects of WEA on visceral adipose tissue. Studies on miR-133 have focussed on its regulation of brown adipose tissue function. A study proved that miR-133 had an inhibitory function towards brown adipocyte differentiation (Trajkovski et al. Citation2012), while its expression was upregulated by dietary WEA in our study. Results concerning effects of WEA on target genes of DE-miRNAs in visceral adipose tissue are shown as follows. Short-chain specific acyl-CoA dehydrogenase (ACADS) was revealed to regulate mitochondrial fatty acid β-oxidation (Zha et al. Citation2005), whereas altered expression of ACADS by dietary WEA in current study might lead to change of short chain fatty acid β-oxidation. Excessive fat deposition in sheep may cause a decline in meat quality and it is not good for health of human consuming such meat. Glycerol-3-phosphate dehydrogenase 1 (GPD1) is a determinant gene for triglyceride synthesis (Gao et al. Citation2011). Descent of this gene expression in our study indicated reduction of triglyceride synthesis in visceral adipose tissue. This inhibition of adipogenesis was in agreement with an earlier research, in which application of polysaccharide of A. mongolicum Regel in diet of sheep resulted in significant reduction of gene expression with regard to fatty acid synthesis in adipose tissue (Zhang et al. Citation2019). Retinol cannot be synthesised in mammalian and the absorbed retinol must be stored in cytoplasmic lipid droplets in form of retinyl ester. Dehydrogenase/reductase member3 (DHRS3) was shown to regulate production of retinyl ester and express in adipocytes that store retinol. And DHRS3 expression was reported to positively correlate with number of lipid droplets (Deisenroth et al. Citation2011). The lower DHRS3 in our study possibly indicated a reduced number of lipid droplets in visceral adipose tissue after WEA supplementation. Except for regulation of fatty acid composition by lipid-related genes, rumen microbial action such as hydrogenation also has an impact on fatty acid profiles in meat and dairy products of ruminants (Frutos et al. Citation2020). A. mongolicum Regel extracts were also reported to affect rumen microbiome in sheep (Du et al. Citation2019), however, we mainly focussed on miRNA-mRNA regulatory mechanism of fatty acid composition in this study. Of note, most researches on flavour-related candidate genes in mutton have so far focussed on metabolism of skatole and androsterone in liver (Zhao et al. Citation2022). Studies on lipid-related candidate genes with regard to mutton flavour are deficient. Further studies on target miRNAs of genes related to skatole and androsterone metabolism need to be carried out and functional verification experiments for identified DE-miRNAs and DE-mRNAs in adipocytes should also be conducted. Finally, we conducted exploration of DE-miRNAs and differentially expressed target genes between sCON group and pCON group. As expected, more DE-miRNAs and DE-mRNAs were found between different types of adipose tissue. Perirenal adipose tissue belongs to deep adipose tissue and is relatively active in fat metabolism. In contrast, subcutaneous adipose tissue is superficial adipose tissue that mainly promotes adipogenesis and stores fat. Our study claimed that significantly upregulated miRNAs (miR-376c-3p (Zehavi et al. Citation2012), miR-433-3p (Byrne et al. Citation2010), miR-1468 (Hao et al. Citation2016), miR-432 (Huang et al. Citation2018)) in perirenal adipose tissue in contrast to subcutaneous adipose tissue were implicated in inhibition of adipogenesis. From miRNA-mRNA network, downregulated expression of acetyl-CoA synthetase 2 (ACSS2), enoyl-CoA hydratase (ECHS1), fatty acid synthase (FASN), annexin A2 (ANXA2) in perirenal adipose tissue compared to subcutaneous adipose tissue could also possibly suppress adipogenesis, as these genes were all confirmed to promote adipogenesis (Kamal et al. Citation2013; Salameh et al. Citation2016; Huang et al. Citation2018).
Conclusions
To sum up, two types of RNA-sequencing identified differential miRNA and differential mRNA candidates associated with lipid metabolism in adipose tissue after utilisation of WEA in diet of sheep. Several differential miRNAs and differential mRNAs involved in lipogenesis, fatty acid oxidation, fatty acid transportation and other processes were identified, which may provide a reasonable explanation for regulation of fatty acid composition in mutton by dietary WEA. By constructing differential miRNA-mRNA regulatory networks, our findings may reveal miRNA-mRNA mechanism for regulation of lipid metabolism in sheep by dietary WEA. This may provide a theoretical basis for rational use of WEA in diet of sheep.
Ethical approval
Our experiment was performed following the recommendations of the Instructive Notions concerning Caring for Experimental Animals, Ministry of Technology of China. The experimental procedure and animal material used acquired the approval of guidelines of the Animal Care and Use Committee of Inner Mongolia Agricultural University ([2020]101, 23 November 2020).
Supplemental Material
Download (1.8 MB)Acknowledgments
We express our gratitude to Fuchuan Sheep Breeding Technology Co., Ltd. of Bayannaoer City, Inner Mongolia Autonomous Region, for providing experimental animals and sites.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Data availability statement
Raw data for sequencing can be accessed by NCBI BioProject database with identifer PRJNA894801.
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References
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