CircVPS13C Promotes Intramuscular Adipogenesis via MiR-5606-X-ECHDC3 Axis in Yaks (Bos grunniens)

TL;DR

This study identifies a circular RNA, circVPS13C, that promotes fat formation in yaks by interacting with miR-5606-x and ECHDC3, offering new insights into intramuscular fat regulation in this low-fat animal species.

Contribution

The study reveals a novel circRNA-mediated regulatory axis (circVPS13C/miR-5606-x/ECHDC3) involved in intramuscular adipogenesis in yaks.

Findings

circVPS13C is a cytoplasmic circRNA that regulates intramuscular adipogenesis in yaks.

circVPS13C promotes differentiation and inhibits proliferation of yak preadipocytes via the miR-5606-x/ECHDC3 axis.

miR-5606-x and ECHDC3 have opposing roles in intramuscular adipogenesis.

Abstract

Although large-scale studies and potential pathways of genes on intramuscular fat (IMF) in livestock have been reported, research on circRNAs in yaks—a unique, low-IMF-content animal species that is native to the Qinghai–Tibetan Plateau—is still lacking. Based on previous high-throughput sequencing results on longissimus dorsi with different IMF content, a novel circRNA encoded by the VPS13C gene (designated as circVPS13C) was found to exhibit significant differential expression. Here, we systematically characterized the function and mechanism of circVPS13C on IMF deposition in yaks by adopting a series of experiments. Sequencing, RNase R processing, and nucleoplasmic separation experiments confirmed the circular structure feature of circVPS13C, and it was predominantly distributed in the cytoplasm. Furthermore, these experiments demonstrated that circVPS13C was mainly distributed in the cytoplasm. The circVPS13C/miR-5606-x/ECHDC3 axis was constructed through ceRNA network analysis and validated by dual-luciferase reporter and rescue experiments. Furthermore, the function of these three potential regulators during IMF deposition was investigated through CCK-8, BODIPY, Oil Red O staining, and qRT-PCR analyses, and results showed that both circVPS13C and miR-5606-x promoted the differentiation and inhibited the proliferation of yak intramuscular preadipocytes, while the function of ECHDC3 was the opposite. In conclusion, circVPS13C could act as a competitive endogenous RNA (ceRNA) sponge to sequester miR-5606-x, thereby relieving the inhibitory effect of miR-5606-x on ECHDC3.

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1. Introduction

The yak (Bos grunniens) is mainly found around the Qinghai-Tibetan Plateau and is famous for its higher protein, vitamin, and trace elements, but relatively low IMF content makes the meat rather tough. China has numerous breeds of yaks, but the IMF content of each breed of yaks is relatively low [1,2]. Therefore, it is impossible to improve the IMF content by crossbreeding among different yak breeds. In addition, when yaks are crossed with cattle, the resulting male offspring are sterile [3]. Therefore, delving deeper into the molecular level to explore the reasons for the low IMF content in yaks is of great significance for improving their commercial value from the perspective of molecular breeding.

IMF is a key determinant of meat quality, significantly contributing to sensory characteristics such as tenderness, juiciness, and flavor, which influence consumer preference and the commercial value of meat products [4]. IMF deposition is a multi-factorial controlled process involving genetic, epigenetic, and nutritional factors [5,6]. In recent years, research on IMF deposition in yaks has mainly focused on the regulation by nutritional factors and individual genes, as well as the identification of potential genes and signaling pathways that affect its deposition through high-throughput sequencing [7,8,9,10]. However, research on the regulatory mechanisms is still relatively scarce. In terms of nutritional factors, the supplementary dietary protein significantly increases the IMF content, accompanied by an increase in enzymatic activities of FASN, ACACA, and LPL [11]. A high-energy diet significantly improves the meat quality of yak, and transcriptomics and lipid metabolomics analysis reveal that the differences in IMF deposition mainly originate from the metabolism of triglycerides, phosphatidylserine, and lysophosphatidylcholine, and are influenced by FABP4, DGAT2, RBP4, and others [12]. In terms of genes, previous studies show that SIRT1 expression negatively correlates with IMF content and serves as a negative regulator during IMF deposition [13,14]. Over-expression of ACADS reduces lipid deposition by inhibiting the expression of adipogenic marker genes, including FASN, C/EBPα, PPARγ, and SREBP [15]. Under hypoxic conditions, HIF1α inhibits the formation of lipid droplets and simultaneously down-regulates the expression of C/EBPα [16]. In addition to the genes encoding proteins, non-coding RNAs also play a significant role during IMF deposition.

Recent studies have shed light on the significant involvement of ncRNAs in the regulation of IMF deposition in yaks [17,18,19]. For example, RNA-seq and ChIP-seq have been performed to reveal the super-enhancer associated miRNAs [20], and revealed that the miR-6529a [21] and miR-2400 [22] as regulators of IMF deposition in yaks. LncFAM200B has been observed to enhance the activity of the Notch signaling pathway during yak intramuscular pre-adipocytes (YIMAs) differentiation [21]. Using the latissimus dorsi muscle from calf and adult yak, sixty-six circRNAs with differential expression patterns were identified, including circ_12686 (an exons-derived circRNA) and ci_106 (an introns-derived circRNA). These circRNAs are significantly enriched in the PPARγ signaling and fatty acid degradation pathways and might serve as potential circRNAs for the regulation of IMF deposition [17]. CircRILPL1 has been demonstrated to enhance the proliferation and differentiation of myoblasts in laboratory settings, while simultaneously inhibiting apoptosis and promoting the growth of bovine myoblasts [23]. Although these multifaceted findings reveal the diverse regulatory roles of ncRNA during IMF deposition, the molecular mechanisms behind this process require further investigation.

Based on our previous study on the expression profile of circRNAs in the longissimus dorsi muscle of yaks with different IMF content [24]. We focused on a circular RNA, circVPS13C (originating from the VPS13C gene on chromosome 11), which exhibited differential expressions in the longissimus dorsi muscle with high- and low-IMF content, and overall high expression levels. Our comprehensive investigation in YIMAs revealed that the circVPS13C/miR-5606-x/ECHDC3 regulatory axis could significantly promote the proliferation and differentiation of YIMAs. Our findings uncover a previously unrecognized regulatory axis governing IMF deposition, which may contribute to improving yak breeding and providing new insights into the genetic mechanism of IMF deposition. The aim of this study was to investigate whether circVPS13C promotes yak intramuscular adipocyte differentiation and lipid accumulation through a ceRNA-mediated mechanism involving the miR-5606-x/ECHDC3 axis.

2. Materials and Methods

2.1. Sample Collection

Yaks of similar body weight were raised on natural pastures until the age of 4 years, and a grazing system was adopted to ensure uniform treatment and nutritional conditions. Subsequently, they were fattened for 6 months under the same nutritional conditions. All yaks were euthanized in accordance with humanitarian principles to minimize animal suffering. The longissimus dorsi muscle samples of the yaks were collected promptly after euthanasia to ensure the freshness of the samples.

2.2. Isolation, Culture, and Differentiation of Yak Intramuscular Preadipocytes

YIMAs were isolated from the longissimus dorsi located between the 12th and 13th ribs (right half carcass). Yak intramuscular preadipocytes were isolated from the longissimus dorsi muscle by enzymatic digestion, followed by filtration and centrifugation to obtain the YIMAs, as described previously [13]. YIMAs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified incubator at 37 °C with 5% CO_2_ [25]. When the cells reached approximately 80% confluence, the complete culture medium was replaced with induction medium containing 50 μmol·L^−1^ oleic acid (Sigma-Aldrich, St. Louis, MO, USA) to induce differentiation for 48 h. All cells used in this experiment were the third-passage cells.

2.3. Establishment of the ceRNA Regulatory Network

CircRNA–miRNA–mRNA interactions were evaluated and visualized using Cytoscape software (v3.6.0; http://www.cytoscape.org/). The differentially expressed miRNAs and mRNAs were obtained from the same samples used in our previous study [24], and the negatively co-expressed miRNA–mRNA pairs were used for network construction. The corresponding sequencing data have been deposited in the NCBI database under accession number PRJNA1014567. The binding sites between circRNAs, miRNAs, and their target mRNAs were predicted using RNAhybrid (V3.6.0; http://www.cytoscape.org/).

2.4. Total RNA Extraction, cDNA Synthesis, Real-Time PCR, and Genomic DNA Extraction

Total RNA was extracted from cells (1 × 10^6^) using TRIzol reagent (TaKaRa, Dalian, China). RNA concentration and purity were measured using a NanoDrop, and samples with A260/A280 ratios between 1.8 and 2.0 were considered acceptable. RNA integrity was verified by 1% agarose gel electrophoresis. For reverse transcription, 1 μg RNA was reverse transcribed into cDNA by PrimeScript RT (Takara, Shiga, Japan) for mRNA/circRNA or the Mir-X miRNA First-Strand Synthesis Kit (TaKaRa) for miRNA. Quantitative PCR (qPCR) was then performed using SYBR Premix Ex Taq (TaKaRa, Japan) on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). GAPDH served as the internal control for mRNA and circRNA, and U6 was used for miRNA normalization. Relative expression levels were calculated using the 2^^−ΔΔCt^ method [23]. Primers used are listed in Supplementary Table S1. For genomic DNA extraction, 1 × 10^6^ YIMAs were collected, and genomic DNA (gDNA) was isolated using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions.

2.5. RNase R Assay

To confirm the circular structure of the candidate RNA, total RNA was treated with GSPure^®^ RNase R (Geneseed Biotech Co., Ltd., Guangzhou, China) to selectively degrade linear RNA. Briefly, 1–3 units of RNase R per microgram of RNA were added with 10× Reaction Buffer, and the final reaction volume was adjusted to 20–50 μL with RNase-free water according to RNA concentration. The mixture was incubated at 37 °C for 15 min to ensure complete digestion of linear RNA, followed by enzyme inactivation at 70 °C for 10 min. The resulting RNA was immediately used for downstream analyses.

2.6. Cytoplasmic and Nuclear RNA Isolation

Nuclear and cytoplasmic RNAs were isolated from YIMAs using the PARIS Kit (Invitrogen, Carlsbad, CA, USA) [26]. Briefly, 1 × 10^7^ YIMAs were collected by centrifugation to obtain a cell pellet. The cytoplasmic fraction was obtained from the supernatant after adding Cell Fraction Buffer, and the nuclear fraction was subsequently extracted from the pellet using Cell Disruption Buffer. RNA from each fraction was purified using the column-based RNA cleanup protocol provided with the kit. After reverse transcription, the abundance of circRNAs, U6, and GAPDH in each fraction was quantified by RT-qPCR.

2.7. Plasmid Construction, siRNA Synthesis and Cell Transfection

To construct overexpression vectors, the full-length circVPS13C sequence with EcoRI and BamHI sites was cloned into the pCD25-ciR vector (Geneseed, Guangzhou, China), and the coding sequence of ECHDC3 was inserted into the pcDNA3.1 vector between HindIII and EcoRI sites. siRNAs targeting circVPS13C and ECHDC3, along with miR-5606-x agomirs/inhibitors and their corresponding negative controls (NCs), were synthesized by GenePharma (Shanghai, China) (Table S2). Adipocytes were transfected with vectors, siRNAs, or miR-5606-x agomirs/inhibitors using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s protocol. The transfected cells were harvested at 24 h to 72 h after transfection and used for subsequent experiments.

2.8. Dual-Luciferase Assay

YIMAs were cultured in 24-well plates until ~80% confluence. For the luciferase reporter assay, cells were transfected with either the PGL3-circVPS13C wild-type vector (circVPS13C-wt) or the PGL3-circVPS13C mutant vector (circVPS13C-mut) along with miR-5606-x mimic or mimic negative control (NC). Similarly, cells were transfected with the PGL3-ECHDC3-3′UTR wild-type vector (ECHDC3-wt) or mutant vector (ECHDC3-mut) together with miR-5606-x mimic or mimic NC. After 48 h of induced differentiation, Renilla and firefly luciferase activities were measured using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions [27].

2.9. Cell Proliferation Assay

For the CCK-8 assay, cells were seeded in 96-well plates at 5 × 10^3^ cells per well and cultured in growth medium for 24 h after transfection. Subsequently, 10 μL of CCK-8 solution (Solarbio, China) was added to each well, and the cells were incubated at 37 °C for 2 h. Absorbance at 450 nm was measured using a microplate reader (BioTek, Winooski, VT, USA).

For the EdU assay, cells were incubated with 10 μM EdU solution (BeyoClick™ EdU-488 Cell Proliferation Kit) in complete medium for 2 h at 37 °C. Cells were then fixed with 4% paraformaldehyde for 30 min at room temperature and processed according to the manufacturer’s Click-iT reaction protocol. Nuclei were counterstained with Hoechst 33342, and images were acquired using a fluorescence microscope. EdU-positive cells were quantified using ImageJ software (version 1.53; National Institutes of Health, Bethesda, MD, USA), and differences in Edu percentages were analyzed using t-tests.

2.10. Oil Red O, BODIPY Staining

YIMAs were transfected and seeded in 12-well plates at 1 × 10^5^ cells/well, followed by induction of differentiation for 48 h. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Lipid droplets were visualized using Oil Red O staining (Sigma-Aldrich, USA) or BODIPY staining (Thermo Fisher Scientific, USA) for 20 min and observed under a light microscope. For quantification, Oil Red O was extracted with isopropanol, and absorbance was measured at 490 nm.

2.11. Statistical Analysis

Spearman’s rank correlation coefficient (SCC) and Pearson’s correlation coefficient (PCC) analyses were performed to evaluate circRNA-miRNA and miRNA-mRNA expression correlations for ceRNA network construction. Student’s t-test analyses were performed using SPSS (version 26.0, IBM Corp, Armonk, NY, USA). All experiments were performed in duplicates or triplicates and repeated 3 times (n = 6 or 9). Data are presented as mean ± standard error of the mean (SEM). Statistical significance was determined using two-tailed tests; p < 0.05 was considered statistically significant and represented by * p < 0.05, ** p < 0.01. Charting was performed using GraphPad 8.0 (GraphPad Software, La Jolla, CA, USA).

3. Results

3.1. Identification and Subcellular Localization of circVPS13C

According to the expression profile of circRNAs in longissimus dorsi tissues between high- and low-IMF content, a potential IMF-associated circRNA, the circ030456, with differential and high expression level in yak longissimus dorsi tissues was screened (Figure 1A; Table S3). Through bioinformatics analysis, we identified that circ030456 originated from the yak VPS13C gene (henceforth termed circVPS13C) on chromosome 11 and was generated through the reverse-splicing process of exons 49 to 52 of its source gene, with a total length of 682 bp (Figure 1B,C; Table S4). Convergent and divergent primers for circVPS13C were designed and synthesized based on its structural characteristics, and yak cDNA and gDNA were used for PCR amplification. The results showed that only the divergent primers could amplify circVPS13C from cDNA. However, the source gene VPS13C could be amplified from both cDNA and genomic DNA (Figure 1D). The RNase R treatment experiment further confirmed the circular structure of circVPS13C (Figure 1E). Furthermore, nucleoplasmic separation experiments demonstrated that circVPS13C was mainly distributed in the cytoplasm rather than in the nucleus (Figure 1F), indicating that it may exert its function by competitively binding miRNAs.

Based on the competing endogenous RNAs (ceRNAs) hypothesis, upon Figure 1A, the ceRNA network (Figure 1G; Table S5) involving circVPS13C and the enriched pathways were constructed (Figure 1H; Table S6), respectively. Notably, we found that circVPS13C had a strong targeting relationship with miR-5606-x and showed a significant enrichment in pathways related to lipid metabolism, including regulation of lipolysis in adipocytes. These results suggest that circVPS13C may indeed play a role as a microRNA sponge during the process of IMF deposition.

3.2. CircVPS13C Promotes Differentiation and Inhibits Proliferation of YIMAs

In order to investigate function, an over-expressed vector, pCD25-circVPS13C (Figure 2A), was transfected into YIMAs. RT-qPCR results showed that the expression of lipid synthesis marker genes, PPARγ (approximately 4-fold, p < 0.01) and C/EBPα (approximately 3-fold, p < 0.01), was significantly increased after the overexpression of circVPS13C (Figure 2B). BODIPY (Figure 2C) and Oil Red O (Figure 2D,E) experiments demonstrated that overexpression of circVPS13C remarkably promoted the lipid deposition in YIMAs. However, overexpression of circVPS13C suppressed the mRNA expression of proliferation marker genes (CCNB1, CCND1, and PCNA) (Figure 2F). In addition, EdU incorporation assays showed a reduced proportion of EdU-positive cells (Figure 2G) and lower EdU-positive rates upon ImageJ-based quantification (Figure 2H). These findings were further supported by the CCK-8 assay, which indicated reduced proliferative capacity (Figure 2I). Collectively, these results suggest that circVPS13C promotes YIMA differentiation while inhibiting YIMA proliferation.

To further clarify the role of circVPS13C in adipogenic differentiation and proliferation of yak intramuscular preadipocytes (in vitro), siRNA-mediated interference was performed to suppress its expression in YIMAs (Figure 3A). The results from RT-qPCR indicate that inhibition of circVPS13C significantly downregulated the mRNA expression of differentiation marker genes (PPARγ, FASN, and SREBP1, p < 0.01) (Figure 3B). Consistently, circVPS13C inhibition significantly suppressed the cellular content of triacylglycerol (p < 0.01, Figure 3C–E). We further explored the effects of circVPS13C inhibition on regulating the proliferation of YIMAs. The RT-qPCR analysis showed that the proliferation-related markers (CCNB1, CCND1, and CCNE1) were significantly up-regulated (p < 0.01, Figure 3F). Additionally, the EdU and CCK-8 assays also indicated that cell proliferation was enhanced (p < 0.01, Figure 3G–I). Collectively, we conclude that circVPS13C promotes the differentiation of YIMA cells while inhibiting their proliferation, ultimately facilitating intramuscular fat deposition.

3.3. CircVPS13C Regulates the Differentiation of YIMAs via the miR-5606-x/ECHDC3 Axis

Analysis of the ceRNA network indicated that circVPS13C may promote adipogenesis through competitive binding to miR-5606-x, thereby modulating its downstream target ECHDC3 (Figure 1G). To validate this, dual-luciferase reporter vectors containing the miR-5606-x binding sites in circVPS13C or the 3′ UTR of ECHDC3 were constructed (Figure 4A). The results showed that miR-5606-x significantly reduced the activity of circVPS13C-WT, whereas no change was observed in the circVPS13C-MUT group (Figure 4B), confirming direct binding between circVPS13C and miR-5606-x. To further support the functional relevance of this interaction, inhibition of miR-5606-x increased circVPS13C expression (Figure 4C) and rescued the downregulation of PPARγ induced by circVPS13C knockdown (Figure 4D), demonstrating that circVPS13C can act as a molecular sponge for miR-5606-x to regulate adipogenic differentiation.

Moreover, to confirm that ECHDC3 is a direct downstream target of miR-5606-x, we co-transfected miR-5606-x mimics with ECHDC3-WT or ECHDC3-MUT into YIMAs. Luciferase activity was significantly inhibited in the ECHDC3-WT group but remained largely unaffected in the ECHDC3-MUT group (Figure 4E), demonstrating that miR-5606-x directly interacts with the 3′-UTR of ECHDC3. Collectively, these results reveal that circVPS13C promotes intramuscular fat deposition in yaks by sponging miR-5606-x, thereby regulating ECHDC3 expression via the circVPS13C/miR-5606-x/ECHDC3 axis.

3.4. miR-5606-x Promotes Differentiation and Inhibits Proliferation of YIMAs

To investigate the role of miR-5606-x in YIMAs, we modulated its expression using mimics or inhibitors. qRT–PCR confirmed an approximately 450-fold increase in miR-5606-x expression 48 h after mimic transfection and a significant reduction following inhibitor transfection, demonstrating efficient overexpression and knockdown (Figure 5A,J). Overexpression of miR-5606-x significantly upregulated the mRNA levels of adipogenic markers C/EBPα, PPARγ, and SREBF1 (p < 0.01) (Figure 5B), and promoted lipid droplet accumulation, as shown by BODIPY and Oil Red O staining (Figure 5C–E), indicating that miR-5606-x promotes adipogenic differentiation.

To further assess the effect of miR-5606-x on YIMA proliferation, cell viability, and DNA synthesis were evaluated. CCK-8 assays showed that miR-5606-x overexpression significantly reduced cell viability after 12 h compared with the NC (Figure 5F). Consistently, EdU staining revealed a marked decrease in EdU-positive cells in the mimic group (Figure 5G,H). Furthermore, qRT–PCR analysis demonstrated significant downregulation of proliferation markers CCNB1 and PCNA (p < 0.01) (Figure 5I), indicating that miR-5606-x suppressed YIMA proliferation.

Conversely, inhibition of miR-5606-x significantly decreased the expression of adipogenic markers C/EBPα, PPARγ, and FASN (p < 0.01) (Figure 5K), and inhibited lipid droplet formation (Figure 5L–N). In addition, miR-5606-x inhibition enhanced cell proliferation as evidenced by CCK-8 (Figure 5O), EdU assays (Figure 5P,Q), and upregulation of proliferation markers (Figure 5R). These effects were opposite to those observed with miR-5606-x overexpression. Collectively, these results indicate that miR-5606-x positively regulates adipogenic differentiation while suppressing YIMAs proliferation.

3.5. ECHDC3 Inhibits Adipogenic Differentiation While Promoting Proliferation in YIMAs

To investigate the role of ECHDC3 as a target of miR-5606-x in YIMAs, we modulated its expression via overexpression and siRNA-mediated knockdown. Compared with the control, overexpression of ECHDC3 increased mRNA levels by approximately 8000-fold (Figure 6A), whereas siRNA knockdown reduced expression by about 50% (Figure 6B). Functional assays revealed that ECHDC3 overexpression suppressed adipogenic differentiation as evidenced by decreased expression of PPARγ, C/EBPα, and FASN (p < 0.01) (Figure 6C), and reduced lipid droplet formation (Figure 6D,E). Concurrently, proliferation was enhanced, indicated by upregulation of CCND1, KI67, and PCNA (p < 0.01) (Figure 6F), along with increased cell viability in CCK-8 and EdU assays (Figure 6G,H).

In contrast, ECHDC3 knockdown promoted differentiation, with increased PPARγ, C/EBPα, and FASN expression (p < 0.01) (Figure 6I), enhanced lipid accumulation (Figure 6J,K), and suppressed proliferation, reflected by downregulation of CCND1, KI67, and PCNA (p < 0.01) (Figure 6L) and fewer EdU-positive cells (Figure 6M). Similarly, CCK-8 also indicates a decrease in cell numbers (Figure 6N). Collectively, these results demonstrate that ECHDC3 inhibits adipogenic differentiation while promoting proliferation in YIMAs, highlighting its critical role in IMF deposition.

4. Discussion

Circular RNAs are covalently closed in non-coding RNAs derived from pre-mRNAs that can regulate their host genes at multiple levels [28]. Emerging evidence indicates that circRNAs often function in concert with their host genes [29,30]. For instance, Zhang et al. [31] identified 41 circRNAs differentially expressed during adipogenesis, most of which function alongside their host genes. Building on this concept, and our previous profiling of circRNAs in the longissimus dorsi muscle of yaks with differing intramuscular fat content [24], we focused on circVPS13C, which is derived from exons 49 to 52 of the VPS13C gene, and exhibits a high and differential expression in high-IMF tissues. As previously reported, VPS13C is a key regulator of adipocyte lipid homeostasis, promoting differentiation and lipid accumulation by stabilizing galectin-12, maintaining lysosomal lipid degradation, and limiting ATGL-mediated lipolysis in brown and white adipocytes [32,33]. This suggests that circVPS13C may play a potential role in regulating adipogenic differentiation and IMF deposition.

In this study, we report the role of circVPS13C on lipogenesis in YIMAs. The circular structure of circVPS13C was first confirmed by amplification from cDNA using divergent primers, the absence of amplification from genomic DNA, and resistance to RNase R digestion, whereas the linear VPS13C transcripts were degraded. These results verified that circVPS13C is a stable circRNA. Functional assays revealed that circVPS13C significantly promotes YIMA differentiation, as evidenced by the increased formation of lipid droplets in cells and the upregulation of adipogenic marker genes, which is consistent with other circRNAs promoting adipocyte differentiation [24,34].

CircVPS13C was identified as an exonic circular RNA (ecircRNA). Subcellular fractionation analysis revealed that circVPS13C is predominantly localized in the cytoplasm, consistent with the typical distribution pattern of ecircRNAs, which mainly reside in the cytoplasm and often function as competing endogenous RNAs (ceRNAs) [35,36]. By constructing a ceRNA regulatory network, we found that circVPS13C may regulate the expression of ECHDC3 by acting as a miR-5606-x sponge, thereby influencing IMF deposition. These findings are consistent with previous studies highlighting the regulatory roles of cytoplasmic circRNAs [34,37].

To further investigate the mechanism by which circVPS13C regulates YIMA differentiation, we overexpressed and inhibited miR-5606-x in YIMAs. The results showed that transfection with miR-5606-x mimics significantly promoted intramuscular adipocyte differentiation, whereas inhibition of miR-5606-x expression suppressed differentiation, indicating that miR-5606-x acts as a positive regulator of YIMA differentiation. Studies on miR-5606-x are currently limited. However, these findings are consistent with our previous reports on YIMA-related miRNAs, such as miR-129 and miR-3059-x, which also promote lipid accumulation [24,38]. This study further expands the functional landscape of miRNAs involved in fat metabolism in yak.

Numerous studies have established that miRNAs regulate target genes post-transcriptionally through inhibition or degradation [38,39]. There was no prior research on the regulation of gene expression by miR-5606-x. In this study, we first predicted ECHDC3 as a potential target gene of goat miR-5606-x and confirmed that miR-5606-x negatively regulates the expression of ECHDC3. Notably, ECHDC3 has been implicated not only in adipose tissue metabolism, where it regulates insulin sensitivity [40], but also in brain white-matter integrity and cognitive function in T2DM patients [41], highlighting its broader physiological relevance. Additionally, our study showed that ECHDC3 suppresses YIMA differentiation, highlighting the role of ECHDC3 in suppressing adipogenesis. Together, these findings reveal that circVPS13C promotes YIMA differentiation and intramuscular fat accumulation by modulating the miR-5606-x–ECHDC3 axis.

In summary, circRNA-seq analysis identified circVPS13C as a novel circRNA associated with intramuscular fat deposition in yaks. Functional and mechanistic analyses revealed that circVPS13C promotes intramuscular adipocyte differentiation and lipid accumulation through the miR-5606-x/ECHDC3 axis as a ceRNA in vitro. A limitation of this study is that all functional analyses were conducted using cultured intramuscular preadipocytes and lacked protein-level validation; therefore, the role of circVPS13C in intramuscular fat deposition at the organismal level requires further in vivo validation. Future studies that utilize animal models or targeted gene manipulation to conduct in vivo functional validation of circVPS13C are crucial for confirming its regulatory role in intramuscular fat deposition. Additionally, circVPS13C and its downstream miR-5606-x/ECHDC3 axis may serve as potential molecular markers or targets for genetic selection and nutritional intervention strategies aimed at improving the quality of yak meat.

5. Conclusions

This work demonstrates that CircVPS13C inhibits the expression of miR-5606-x by acting as a competitive endogenous RNA, thereby promoting the differentiation and lipid accumulation of intramuscular adipocytes, and subsequently relieving its inhibitory effect on ECHDC3. These findings provide new mechanistic insights into ceRNA-mediated regulation of adipogenic differentiation in yak intramuscular preadipocytes, highlighting the importance of circVPS13C as a potential molecular target for improving the quality of yak meat and in molecular breeding (Figure 7).