Functional regulatory mechanisms of circRNAs in bovine adipogenesis

Abstract

Circular RNAs (circRNAs) represent a distinct category of endogenous non-coding RNAs, distinguished by their covalently closed-loop structure, which imparts remarkable stability and tissue-specific expression. Historically perceived as less prevalent than linear RNAs, recent findings indicate that circRNAs are integral to various biological processes. A well-documented function of circRNAs is their capacity to serve as microRNA (miRNA) sponges; however, recent investigations have revealed their participation in protein interactions and the potential for translation into functional peptides, thereby broadening their functional scope. Researchers have found that circRNAs play a big role in controlling the growth of adipose tissue in cattle, but we still don’t fully understand the complex networks and downstream pathways they affect. This review aims to consolidate existing knowledge and provide new insights into the mechanistic roles of circRNAs in bovine adipogenesis, highlighting their effects on preadipocyte proliferation, differentiation, and lipid metabolism. To address this gap, a systematic literature search was performed utilizing databases such as ScienceDirect, PubMed, Google Scholar, and Web of Science to gather recent advancements in circRNA research pertinent to bovine adipogenesis. Key areas of focus include circRNA biogenesis and function, circRNA-mediated regulation of adipogenesis, molecular signaling pathways, epigenetic modifications, and the functional diversity of adipogenic regulation. This comprehensive review synthesizes current findings, identifies knowledge gaps, and delineates future research trajectories, particularly concerning the regulatory networks and downstream effectors associated with circRNAs. By elucidating the molecular mechanisms that underlie circRNA function in bovine adipogenesis, this review contributes to the fields of molecular biology, agricultural biotechnology, and veterinary science. Additionally, it highlights the potential of circRNAs as innovative biomarkers and therapeutic targets for enhancing cattle adipose tissue composition and meat quality, thereby establishing a foundation for future translational research in livestock genetics and metabolic regulation.

Introduction

The accumulation of adipose tissue plays a crucial role in determining the quality and economic value of beef, with a particular emphasis on intramuscular fat (IMF), commonly referred to as marbling (Abebe et al. 2024). The IMF content significantly influences the quality of beef by enhancing its tenderness, juiciness, and flavor, ultimately making it more appealing to consumers (Du et al. 2022; Baik et al. 2023). Additionally, the composition of IMF, which includes unsaturated fatty acids such as oleic acid, contributes to the perceived health benefits of beef (Zhang et al. 2022a; Picard et al. 2019; Feng et al. 2022a). This underscores the importance of beef in consumer choices and market evaluations (Schumacher et al. 2022). Therefore, it is crucial for economic viability within the beef cattle industry to efficiently manage and optimize fat deposition in cattle (Gao et al. 2022).

Various factors, including genetics, breed, management practices, and nutrition, are essential in regulating fat accumulation in beef cattle (Park et al. 2018; Jesko et al. 2022). A broad understanding of the molecular mechanisms and genetic factors involved in fat deposition can lead to improved production efficiency and sustainability in livestock farming (Abebe et al. 2024; Park et al. 2018). Understanding the molecular mechanisms underlying adipogenesis, the process by which preadipocytes differentiate into mature adipocytes, is essential for optimizing fat deposition and improving beef quality (Abebe et al. 2024).

Unlike linear RNAs, circular RNAs (circRNAs) are depicted by a distinct covalently closed-loop structure resulting from backsplicing, which imparts increased stability and resistance to exonuclease degradation (Sinha et al. 2022; Sun et al. 2022; Vashchenko et al. 2022). These molecules are highly conserved across evolution and often exhibit tissue-specific expression profiles, especially in muscle tissue (Ju et al. 2023). CircRNAs play a pivotal role in gene regulation through various mechanisms, such as aiding as microRNA sponges, modulating RNA-binding proteins, acting as templates for translation, and participating in autophagy processes (Yang et al. 2022a; Huang et al. 2020; Yu and Kuo 2019). Their regulatory functions encompass a wide range of biological processes, including transcription, translation, and splicing events, thereby constituting integral components of gene regulatory networks (Yu and Kuo 2019; Xiao et al. 2021). In addition, circRNAs have garnered attention as promising biomarkers for disease diagnosis and potential therapeutic targets due to their stability, evolutionary conservation, and altered expression in pathological states (Yu et al. 2021a; Verduci et al. 2021; Ma et al. 2021; Zhang et al. 2023).

Moreover, circRNAs, distinguished by their stable circular structure and absence of a 5′-cap and 3′-poly (A) tail, have been identified to have the ability to encode proteins through cap-independent translation processes, play a role in gene transcription, and regulate various cellular functions (Yuan et al. 2023; Sun and Yang 2023). In the realm of bovine adipogenesis, circRNAs have attracted considerable interest as potential regulators of fat tissue development. A few studies have identified specific circRNAs, such as circADAMTS16 (Hu et al. 2023), circFUT10 (Jiang et al. 2020), circPPARγ (Wu et al. 2022a), and circBDP1 (Zhang et al. 2022b), that influence the proliferation, differentiation, and apoptosis of adipocytes by interacting with miRNAs and other regulatory molecules. However, the complex regulatory networks and downstream pathways controlled by circRNAs in bovine adipogenesis are not fully elucidated.

Therefore, the main aim of this paper is to synthesize the current knowledge and provide valuable insights on the specific functions and regulatory mechanisms of circRNAs in the process of adipogenesis in cattle. The review gathered data from a range of secondary sources, such as relevant books, academic articles, and online materials. Non-English-language studies and those with insufficient data were excluded. All the collected information was then presented in line with the objectives of this paper. This exploration is crucial for the development of diagnostic and therapeutic approaches for adipogenesis-related disorders and for enhancing the quality of livestock meat and insights from various studies are imperative to gain a thorough understanding of the intricate functions of circRNAs in bovine adipogenesis.

CircRNA biogenesis and function

Backsplicing, a critical mechanism involved in the formation of circular RNAs (circRNAs), is subject to the influence of various factors (Shen et al. 2022). Factors such as the kinetics of RNA polymerase II (RNAPII), the availability of splicing factors, and the architecture of genes are key determinants in the process of backsplicing (Eleazer et al. 2023). Research has indicated that certain proteins, such as PARP1, can modulate alternative splicing and potentially impact circRNA biogenesis by their localization on chromatin and their PARylation activity (Eleazer et al. 2023). Moreover, RNA-binding proteins like SXL have been found to regulate sex-specific backsplicing in Drosophila, thereby facilitating the production of sex-specific circRNAs (Fan et al. 2023). Additionally, the presence of intronic sequences has been observed to influence circRNA production, with forward splicing promoting the generation of circRNAs containing multiple exons (Fan et al. 2023). A comprehensive understanding of these mechanisms and factors is essential for elucidating the intricate regulatory network that governs circRNA biogenesis and its implications in gene expression and the development of diseases.

CircRNAs possess distinctive characteristics, including remarkable stability attributed to their closed-loop structure, making them more resistant to degradation compared to linear RNAs (Hoffmann et al. 2023; Xu et al. 2023). These molecules are expressed in a cell- and tissue-specific manner and can function as miRNA sponges by sequestering miRNAs and inhibiting their ability to suppress target genes (Sun and Yang 2023). Moreover, circRNAs have the capacity to interact with RNA-binding proteins and facilitate protein synthesis through cap-independent translation mechanisms, highlighting their adaptability in cellular processes and potential therapeutic applications (Wei et al. 2023). Additionally, circRNAs have been associated with the regulation of alternative splicing, transcription, and the cis-regulation of host genes, emphasizing their diverse roles in biological processes and disease development, particularly in tumorigenesis (Gu et al. 2023).

Biogenesis and functions of circRNAs is given in Fig. 1. circRNAs represent a unique category of non-coding RNAs distinguished by their closed-loop structures formed via back-splicing, a mechanism where the 3′ end of one exon is linked to the 5′ end of a preceding exon, deviating from the conventional linear splicing pathway as shown in Fig. 1. The circular configuration provides stability to circRNAs, rendering them resistant to degradation by exonucleases and extending their lifespan. Functionally, circRNAs serve as platforms for RNA-binding proteins (RBPs), facilitating the formation of protein complexes that modulate splicing, transcription, and post-transcriptional modifications. Moreover, circRNAs can function as molecular decoys for miRNAs, sequestering them and impeding their interaction with target mRNAs, thereby diminishing miRNA-mediated gene suppression and promoting the translation of miRNA-targeted mRNAs. By engaging in these interactions, circRNAs partake in the broader network of ceRNAs, influencing gene expression and cellular processes by competing for shared miRNAs with other RNA species and reshaping the regulatory milieu within cells. This intricate involvement in genetic regulation and protein synthesis underscores the pivotal role of circRNAs in cellular functions and homeostasis.

Fig. 1.

Biogenesis and functions of circRNAs: CircRNAs can work as multifunctional devices serving as: (1) Regulate transcription of their parental genes. (2) miRNA sponges affecting genes post-transcriptionally. (3) Translation into proteins. (4) They can affect the stability of other RNA molecules (mRNAs or lncRNAs). (5) Promote functional responses and phenotypic changes. (6–7) Interaction with RBPs and acting as either decoy or scaffold molecules. (8) Directing RBP cellular localization

circRNAs and bovine preadipocyte proliferation and differentiation

Research investigating the role of circRNAs in enhancing preadipocyte proliferation has yielded significant discoveries. Noteworthy findings have identified circRNAs such as circBDP1, circ_0005870, and circ_0000946 as pivotal in the regulation of preadipocyte differentiation and proliferation (Zhang et al. 2022b; Yu et al. 2023). These circRNAs have been observed to interact with miRNAs like miR-204, miR-181b, among others, thereby modulating downstream signaling pathways crucial for adipogenesis. For example, circBDP1 has been demonstrated to stimulate bovine preadipocyte proliferation and differentiation by sequestering miR-204 and miR-181b (Zhang et al. 2022b). Furthermore, the construction of circRNA-miRNA-mRNA interaction networks in these investigations has highlighted essential regulators such as FOXO3, PPP2CA, and SIRT1, which play critical roles in adipocyte differentiation and proliferation (Sakshi et al. 2021). The comprehension of these mechanisms offers valuable insights into potential therapeutic targets for disorders associated with aberrant adipogenesis.

CircRNAs play a crucial role in modulating preadipocyte differentiation into mature adipocytes through various mechanisms. They can regulate post-transcriptional gene expression by sequestering microRNAs and interacting with proteins that are essential in differentiation pathways, such as the PPARγ/C/EBPα signaling axis (Huang and Choo 2023). Furthermore, the differential expression of circRNAs during osteogenic differentiation suggests their involvement in the regulation of adipogenesis, where they competitively bind to microRNAs to control differentiation processes (Sun et al. 2023a). Studies on pediatric obesity have identified specific circRNAs, such as hsa_circ_0046367 and hsa_circ_0000284, which are associated with adipogenesis and neural plasticity mechanisms, thereby influencing susceptibility to obesity (Li et al. 2023). These collective findings indicate that circRNAs play a significant role in preadipocyte differentiation by coordinating complex regulatory networks involving microRNAs and proteins, offering potential therapeutic targets for adipogenesis-related conditions and opportunities to improve meat quality in the livestock sector (Yu et al. 2023; Yan et al. 2023).

CircRNA-mediated regulation of adipogenesis in bovine

Critical regulators of bovine preadipocyte proliferation and differentiation include diacylglycerol O-acyltransferase 2 (DGAT2), serum/glucocorticoid-inducible kinase 1 (SGK1), and specific microRNAs such as miR-181b and miR-204. DGAT2 plays a pivotal role in lipid metabolism by facilitating lipid droplet formation and adipogenesis (Guo et al. 2023a, b). SGK1 is indispensable for bovine preadipocyte differentiation as it modulates adipogenesis and proliferation via the PI3K/Akt signaling pathway (Lei et al. 2022). Moreover, microRNAs like miR-181b and miR-204 target genes such as SIRT1 and TRARG1, thereby impacting fat development in cattle (Zhang et al. 2022a, b, c). Additionally, various small-molecule compounds like Repsox, VPA, and TTNPB have demonstrated the ability to prompt the transdifferentiation of bovine fibroblasts into adipocyte-like cells by activating crucial pathways such as PPAR and PI3K-Akt signaling during adipogenesis (Sun et al. 2023a, b, c). These discoveries collectively offer valuable insights into the transcription factors, signaling pathways, and regulatory mechanisms that govern bovine preadipocyte proliferation and differentiation.

The maturation process of preadipocytes in cattle is controlled by a variety of mechanisms that impact changes in gene expression as they develop. Research has emphasized the significant roles of DGAT isoforms, specifically DGAT1 and DGAT2, in the regulation of lipid metabolism and adipogenesis in bovine preadipocytes (Guo et al. 2023a, b). Analysis of the entire transcriptome through sequencing has identified differentially expressed mRNAs, circRNAs, lncRNAs, and miRNAs at different stages of differentiation in intramuscular preadipocytes, providing insight into the intricate regulatory networks involved in the deposition of intramuscular fat in cattle (Yang et al. 2022b). This comprehension of these mechanisms offers valuable knowledge about the molecular processes that drive adipogenesis in bovine preadipocytes, presenting potential targets for enhancing beef quality.

CircRNAs have been identified as critical regulators of essential adipogenic transcription factors, including PPARγ, C/EBPα, and SREBP1, functioning as ceRNAs (Wu et al. 2022a) (Fig. 2). By modulating these transcription factors, circRNAs significantly influence adipogenesis and lipid metabolism (Liu et al. 2022a). Research across various species, including sheep, humans, and pigs, has revealed distinct expression patterns of circRNAs during the process of adipogenesis (Zhang et al. 2022c; Shen et al. 2024). These circRNAs engage in complex regulatory networks with miRNAs and mRNAs, thereby affecting downstream signaling pathways such as the PPARγ/C/EBPα axis, fatty acid biosynthesis, and Wnt/TGF-β signaling (Zhang et al. 2022c; Shen et al. 2024). By competitively binding to miRNAs and regulating target genes such as PPARD, circRNAs play a vital role in the intricate regulatory mechanisms that govern adipogenic transcription factors and lipid metabolism within adipose tissues (Wu et al. 2022a; Feng et al. 2022b).

Fig. 2.

The role of circPPARγ in enhancing adipocyte differentiation. A, B present the transcriptional levels of PPARγ, C/EBPα, and C/EBPβ measured six days post-transfection with either the circPPARγ overexpression vector or si-circPPARγ. C, D depict the translational levels of PPARγ, C/EBPα, and C/EBPβ assessed at 3 and 6 days following transfection with the circPPARγ overexpression vector and si-circPPARγ. Furthermore, E, F demonstrate that the circPPARγ overexpression vector and si-circPPARγ facilitate adipogenesis, as evidenced by Oil Red O staining. The scale bars represent 100 μm. Statistical significance is indicated with *p < 0.05 and **p < 0.01

The significant role of circRNAs in modulating miRNAs that target adipogenic transcription factors is evident, as they act as miRNA sponges, preventing the repression of genes essential for adipogenesis (Chen et al. 2021). Through the establishment of ceRNA networks, circRNAs competitively bind to miRNAs, thereby influencing the expression of critical adipogenic transcription factors. Notably, differentially expressed circRNAs in adipose tissues, such as circRNA_06424 and circRNA_08840, have been shown to interact with specific miRNAs, thereby regulating pathways associated with fat deposition (Feng et al. 2022b). Furthermore, circINSR has been found to inhibit the adipogenic differentiation of stromal vascular fractions by sequestering miR-152, which subsequently impacts the miR-152/MEOX2 pathway (Zhao et al. 2023). These findings highlight the complex regulatory role of circRNAs as miRNA sponges in the modulation of adipogenic transcription factors and lipid metabolism.

CircRNAs play a crucial role in the intricate regulatory feedback loops that control adipogenic transcription factors by modulating post-transcriptional gene expression (Wu et al. 2022a). Research indicates that circRNAs interact with miRNAs to regulate mRNA expression, thereby influencing adipogenic signaling pathways (Verduci et al. 2021; Papatsirou et al. 2021). For instance, circPPARγ is a notable circRNA that enhances adipocyte differentiation while suppressing proliferation and apoptosis in primary bovine adipocytes, highlighting its regulatory importance in adipogenesis (Wu et al. 2022a). Similarly, in ovine adipogenic differentiation, circRNAs like circ_004977, circ_006132, and circ_003788 act as ceRNAs, coordinating preadipocyte differentiation and lipid metabolism through complex networks involving circRNAs, miRNAs, and mRNAs (Shen et al. 2024). These discoveries underscore the vital role of circRNAs in managing regulatory feedback loops that affect adipogenic transcription factors and pathways, providing valuable insights into potential therapeutic targets for conditions associated with adipogenesis.

Functional regulatory mechanisms of circRNAs in bovine adipogenesis

Adipocyte differentiation and lipid metabolism are intricate biological processes regulated by complex molecular networks, with circRNAs increasingly recognized as key regulators in bovine species (Liu et al. 2022a). Beyond their traditional roles in transcriptional and translational control, circRNAs modulate adipogenesis and lipid homeostasis through multiple mechanisms, including acting as ceRNAs, interacting with RBPs, forming regulatory complexes, and influencing transcriptional activity or protein synthesis (Jiang et al. 2020; Liu et al. 2022a; Zhang et al. 2022c; Chen et al. 2021). As ceRNAs, circRNAs sequester specific miRNAs, thereby alleviating their suppressive effects on messenger RNAs encoding critical adipogenic transcription factors such as PPARγ and C/EBPs. This regulatory axis controls preadipocyte differentiation, proliferation, and lipid accumulation (Gao et al. 2022; Min et al. 2021; Mitra et al. 2018; Zhou et al. 2021). For example, circADAMTS16 targets miR-10167-3p, resulting in inhibited differentiation coupled with enhanced proliferation, whereas circRNF111 functions as a sponge for miR-27a-3p, thereby promoting PPARγ-driven adipogenesis.

Additionally, circBDP1 and circFUT10 coordinate adipogenic gene expression and lipid droplet formation through competitive miRNA binding and interactions with RBPs, respectively (Hu et al. 2023; Zhang et al. 2022b; Shen et al. 2023). Comparative analyses in other livestock species have identified circRNAs such as CircMARK3 in buffalo and CircCWC22 in yak that regulate fat deposition via miRNA sponging and modulation of downstream targets, underscoring the conserved role of circRNAs in mammalian adipogenesis (Feng et al. 2022c; Qin et al. 2024). These contrasting roles suggest circRNAs function in a finely tuned regulatory balance, where some enhance differentiation and others restrict it, ensuring proper adipose tissue development.

Beyond their role as ceRNAs, circRNAs regulate post-transcriptional and transcriptional networks through interactions with RBPs, formation of complexes with chromatin or mRNA, and recruitment of transcriptional regulators to gene promoters (Zhang et al. 2022c; Shen et al. 2023; Zhong et al. 2018). Additionally, certain circRNAs undergo cap-independent translation mediated by internal ribosome entry sites (IRES) or N6-methyladenosine (m6A) modifications, resulting in the synthesis of functional proteins that modulate cellular processes such as proliferation, differentiation, and tissue remodeling—for example, FBXW7-185aa and circNlgn (Du et al. 2021; Yang et al. 2018). Through these multifaceted mechanisms, circRNAs integrate signals from diverse metabolic and environmental pathways, thereby precisely orchestrating adipocyte differentiation and lipid metabolism in accordance with physiological demands (Zhang et al. 2022c; Zhou et al. 2020; Wu et al. 2022b; Yu et al. 2021b).

CircRNAs also play a critical role in lipid metabolism within bovine adipose tissue. By regulating key genes and enzymes involved in fatty acid synthesis, lipolysis, and oxidation, circRNAs modulate lipid accumulation, storage, and systemic metabolic homeostasis (Zhang et al. 2022c; Shen et al. 2023; Zhong et al. 2018). Specific circRNAs, such as circOgdh and CircRNA-02191, interact with microRNAs (miRNAs) to regulate enzymes including adipose triglyceride lipase (ATGL) and acyl-CoA synthetase long-chain family member 1 (ACSL1), thereby influencing the production of unsaturated fatty acids and mammary lipid metabolism (Chen et al. 2023; Liu et al. 2022b). Furthermore, circRNAs modulate adipogenic signaling pathways, including PPARγ/C/EBPα, ARRB1, PPARD, PI3K-Akt, and adipokine-mediated cascades, highlighting their integral role in coordinating adipogenesis and lipid metabolic processes (Feng et al. 2022c; Shao et al. 2023). Weighted gene co-expression network analysis (WGCNA) has further elucidated circRNA–miRNA–mRNA regulatory networks that underlie intramuscular fat deposition, providing mechanistic insights relevant to beef quality and livestock production (Feng et al. 2022c).

The functional roles of circRNAs extend to the regulation of adipose tissue morphology and remodeling. CircRNAs influence adipocyte proliferation, differentiation, and lipid droplet formation by modulating transcription factors, miRNAs, and adipokine secretion, thereby shaping both the structure and function of adipose tissue (Wu et al. 2022a; Zhang et al. 2022d; Ru et al. 2023). Alterations in circRNA expression have been linked to metabolic disorders such as obesity and insulin resistance, underscoring their critical role in maintaining adipose tissue integrity and functionality (Zeng et al. 2021; Mumtaz et al. 2020; Misir et al. 2022). Moreover, circRNAs exhibit dynamic expression profiles during brown adipogenesis and thermogenesis, highlighting their involvement in energy homeostasis and the regulation of fat depots (Xiao et al. 2022; Panda 2018; Kang et al. 2020).

Collectively, circRNAs serve as pivotal regulators of bovine adipogenesis by orchestrating adipocyte-specific gene networks through ceRNA interactions, RBP modulation, transcriptional regulation, and signaling pathway control. Notable circRNAs, including circADAMTS16, circPPARγ, and circMARK3, interact with miRNAs such as miR-10167-3p, miR-92a-3p, miR-181b, and miR-204 to regulate key genes like PPARγ, SIRT1, and TRARG1. These interactions govern processes including proliferation, differentiation, lipid metabolism, and tissue morphology (Hu et al. 2023; Wu et al. 2022a; Zhang et al. 2022b; Feng et al. 2022b). Elucidating these molecular mechanisms not only advances the understanding of bovine fat deposition and adipose tissue development but also identifies promising targets for enhancing meat quality, metabolic health, and livestock productivity through molecular breeding and circRNA-targeted therapeutic strategies (Verduci et al. 2021; Ulshöfer et al. 2021).

Functional regulatory mechanism of circRNAs in bovine adipogenesis is illustrated in Fig. 3. This detailed illustration reveals the complex relationship between circRNA biogenesis and diverse functions in bovine adipogenesis. The process begins with the genetic information stored in DNA, which contains exonic regions separated by introns. CircRNA biogenesis progresses as precursor messenger RNA (pre-mRNA) transcripts are formed, and circularization events occur within the nucleus. Assisted by nuclear pore complexes, circRNAs move into the cytoplasm where they play various regulatory roles crucial for adipogenic processes. In this active environment, circRNAs act as key regulators, participating in miRNA sponging to influence gene expression by binding to microRNAs such as let-7, miR-92a-3p, miR-181b, and miR-204. Moreover, circRNAs stabilize protein complexes, as demonstrated by the interaction between circFUT10 and AMP kinase, which promotes adipocyte growth and maintains metabolic balance. Apart from their traditional functions, circRNAs exhibit a diverse range of activities, such as promoting cell proliferation, underscoring their substantial role in shaping the intricate network of bovine adipogenesis.

Fig. 3.

Regulatory mechanisms of circRNAs functions

Moreover, the abundance of Alu repeats, which are widely distributed throughout the genome, could potentially enhance the generation of circRNAs by facilitating complementary base pairing and promoting crucial back-splicing events that lead to circularization. Besides, RNA-binding proteins (RBPs) are essential in the regulation of circRNA metabolism and functionality, overseeing various processes such as splicing, transportation, and stability, thus impacting the regulation of adipogenesis. Apart from their conventional functions, circRNAs demonstrate a diverse range of roles, including the facilitation of cellular proliferation, highlighting their significant contribution to shaping the complex landscape of bovine adipogenesis.

Functional diversity of circRNAs in bovine adipogenesis, regulatory networks and downstream signaling pathways

High-throughput RNA sequencing combined with bioinformatics tools such as CIRI and CIRCexplorer has enabled advanced profiling of circRNAs during bovine adipogenesis (Zhang et al. 2022d; Nguyen et al. 2021; Zeng et al. 2017). These studies have revealed dynamic changes in circRNA expression throughout adipocyte differentiation, particularly emphasizing circRNAs derived from genes involved in adipogenic transcriptional regulation and lipid metabolism. Functional analyses indicate that circRNAs regulate adipocyte proliferation, differentiation, and lipid droplet formation primarily through miRNA sponging and interactions with RNA-binding proteins (Zhang et al. 2022c; Zaiou 2020). Integrated analyses have uncovered complex circRNA–miRNA–mRNA regulatory networks, revealing novel pathways implicated in bovine adipogenesis (Dehghanian Reyhan et al. 2023).

Several key circRNAs, including circADAMTS16, circBDP1, and circPPARγ, have been identified as critical regulators of bovine adipocyte biology. Specifically, circADAMTS16 inhibits differentiation while promoting proliferation through miR-10167-3p (Hu et al. 2023); circBDP1 influences preadipocyte fate via miR-204 and miR-181b (Zhang et al. 2022b); and circPPARγ enhances differentiation while suppressing proliferation and apoptosis by targeting miR-92a-3p (Wu et al. 2022a). Additional circRNAs, such as circ-CRLF1 and circOgdh, have been implicated in regulating adipogenesis and lipolysis, underscoring the diverse functional roles of circRNAs in bovine fat development (Liu et al. 2022a; Huang et al. 2022).

Experimental validation of circRNA functions has employed methodologies including dual-luciferase reporter assays, gain- and loss-of-function experiments, reverse transcription quantitative PCR (RT-qPCR), Oil Red O staining, and assays assessing proliferation and apoptosis (Hu et al. 2023; Zhang et al. 2022b; Huang and Choo 2023)(Table 1). Furthermore, innovative approaches such as CRISPR/Cas9 genome editing, single-cell transcriptomics, and high-throughput screening have been utilized to further elucidate circRNA regulatory networks and their downstream effectors (Bhattacharjee et al. 2022; Aslan et al. 2023).

Table 1.

The key circRNAs experimental techniques, mechanistic insights, and their significance in bovine adipogenesis

Study	CircRNA	Targets	Functions	Methods	Outcomes
Jiang et al. 2020)	CircFUT10	let-7	Promotes adipocyte proliferation; inhibits adipocyte differentiation	RNA-seq, qPCR, luciferase assays	Identified circFUT10 as a crucial regulator of adipogenesis, shedding light on post-transcriptional regulation in adipocytes
Ma et al. 2023)	CircBTBD7	miR-183	Inhibits adipogenesis	RNA pull-down, RIP, gain-of-function assays	Uncovered a novel circRNA-mediated mechanism regulating adipogenesis through the miR-183/SMAD4 signaling axis
Wu et al. 2022a)	CircPPARγ	miR-92a-3p	Modulates adipogenic differentiation	RNA immunoprecipitation, FISH, transgenic mice	Demonstrated the regulatory role of CircPPARγ in adipogenesis and its potential as a therapeutic target for obesity-related disorders
Hu et al. 2023)	CircADAMTS16	miR-10167-3p	Inhibits adipocyte differentiation; promotes proliferation	CRISPR/Cas9 knockout, Seahorse metabolic analysis	Identified CircADAMTS16 as a key regulator of adipocyte function, providing insights into adipose tissue homeostasis
Kang et al. 2020)	CircFLT1	miR-93	Regulate proliferation and differentiation of adipocytes	RNA interference, reporter gene assays	Revealed the intricate regulatory network involving circFLT1, lncCCPG1, and miR-93 in adipocyte biology
Zhu et al. 2024)	PPARG exonic circRNAs	-	Enhance intramuscular fat deposition	CRISPR/Cas9-mediated mutagenesis, metabolomics	Unveiled a novel mechanism involving PPARG exonic circRNAs in regulating adipogenesis and fat deposition
Zhang et al. 2022b)	CircBDP1	miR-181b/miR-204	Regulates bovine fat development	RNA-seq, Western blot, adipocyte culture models	Uncovered the regulatory role of CircBDP1 in bovine adipogenesis and its implications for fat metabolism
Shen et al. 2023)	CircRNF111	miR-27a-3p	Enhances adipocyte differentiation	Knockdown or exogenous expression of circRNF111, dual fluorescein reporter system, immunoprecipitation	CircRNF111 positively correlates with adipocyte differentiation, functioning as a miR-27a-3p sponge to promote adipogenesis by rescuing the inhibitory effect of miR-27a-3p on PPARγ expression
Qin et al. 2024)	CircCWC22	miR-3059-x	Promotes fat deposition	RNA-seq, qPCR, luciferase assays, bioinformatics analysis, in vivo yak models	Identified CircCWC22 as a key regulator of fat deposition in yaks, highlighting its role in post-transcriptional regulation through miRNA sponging
Mumtaz et al. 2020; Misir et al. 2022)	N/A	-	Provide general insights into circRNA biogenesis and functions	Bioinformatic analysis, literature review	Offered comprehensive reviews on circRNA biology, highlighting their diverse functions and therapeutic potential
Kristensen et al. 2019)	N/A	-	Reviewing circRNA biogenesis, biology, and characterization	Literature review, bioinformatics analysis	Provided a comprehensive overview of circRNA research, emphasizing their emerging roles in various biological processes

Beyond adipogenesis, circRNAs expressed in bovine metabolic tissues also regulate liver lipid metabolism, skeletal muscle development, and mammary gland function, thereby influencing systemic metabolic regulation, meat quality, and dairy production (Gao et al. 2022; Chen et al. 2021; Yue et al. 2019). Their roles as miRNA sponges, RNA-binding protein interactors, and gene expression modulators position circRNAs as promising targets for enhancing livestock productivity and meat quality through precision breeding strategies and potential therapeutic interventions (Raza et al. 2022; Sun et al. 2023c; Berry et al. 2017).

The current body of literature on circRNAs in bovine adipogenesis employs a range of high-throughput sequencing and functional assays, yet methodological variability may influence reported outcomes (Table 1). Most studies utilized RNA-seq to profile circRNA expression, though sequencing depth and platform differed, with some studies achieving > 100 million reads per sample (Hu et al. 2023; Yang et al. 2022b), while others relied on more moderate depths. Higher sequencing coverage enhances detection of low-abundance circRNAs such as CircBDP1 and CircADAMTS16, whereas lower-depth studies may underestimate circRNA diversity. Sample sizes also varied; in vitro adipocyte culture studies typically included 3–5 biological replicates, potentially limiting reproducibility, whereas in vivo experiments, such as those involving transgenic mice or yak models, provided more robust validation of circRNA function (Qin et al. 2024). Statistical approaches were generally consistent, employing tools such as DESeq2 or edgeR for differential expression analysis, although thresholds for significance and correction for multiple testing were not uniform, which may contribute to false-positive or false-negative findings.

Discrepancies in circRNA functions across studies may also arise from biological and experimental variables. Breed-specific differences can influence circRNA expression, as observed in yak versus Holstein cattle models. Culture conditions, including differentiation media composition and inducers, affect adipocyte responses and circRNA regulation. Additionally, the age and sex of donor animals may modulate circRNA expression in adipose tissues, while in vitro versus in vivo models can yield divergent functional outcomes. These factors likely underlie some of the observed inconsistencies in circRNA-mediated regulation of adipogenesis, including differences in proliferation, differentiation, and lipid deposition effects reported for circADAMTS16, CircPPARγ, and CircBDP1. Collectively, these considerations emphasize the need for standardized experimental protocols, adequate biological replication, and cross-validation in both in vitro and in vivo models. Future studies integrating multi-omics approaches and comparative analyses across breeds and developmental stages will strengthen the reliability of circRNA functional characterization and clarify their regulatory roles in bovine adipogenesis.

Table 1 presents a comprehensive summary of key circRNAs associated with adipogenic processes, detailing their functions, mechanisms, experimental approaches/models, and the significance of their implications. Each entry in the table corresponds to a unique research study exploring the involvement of circRNAs in adipogenesis. It provides information on the identified circRNAs, their roles in adipocyte biology, the molecular pathways involved, the experimental techniques used for characterization, and the significance of the study’s outcomes. Advanced experimental methods such as RNA immunoprecipitation (RIP), CRISPR/Cas9-mediated mutagenesis, and metabolomics are utilized to elucidate the functions and mechanisms of circRNAs. The table highlights the various regulatory functions of circRNAs in adipogenesis and underscores their potential as targets for therapeutic interventions in metabolic disorders.

CircRNA-mediated epigenetic regulation of adipogenesis

CircRNAs play a crucial role in adipogenesis by influencing the epigenetic landscape through interactions with chromatin modifiers and mechanisms of DNA methylation (Zhang et al. 2020a) (Fig. 4). These interactions significantly impact gene expression regulation during adipocyte differentiation and lipid metabolism (Zhang et al. 2020a; Chen et al. 2020). Moreover, circRNAs are implicated in the modulation of DNA methylation patterns within adipocytes, contributing to a complex regulatory network that governs gene expression (Li et al. 2021). This underscores their critical function in the regulation of gene expression and cellular processes across diverse tissues, including adipose tissue. Additionally, circRNAs play a role in the plasticity of adipocytes by mediating epigenetic modifications and responding to environmental stimuli (Arcinas et al. 2019).

Fig. 4.

Epigenetic factors influencing circRNA expression in various cellular contexts

CircRNAs are regulated by epigenetic factors that significantly influence their expression and functional roles. Key epigenetic modifications, such as H3K79me2, play a crucial role in the biogenesis of circRNAs. H3K79me2 modulates circRNA expression through two main mechanisms. Firstly, it affects alternative splicing, which is essential for RNA circularization (Chen et al. 2020). By changing the elongation rate of RNA polymerase II, H3K79me2 influences splice site selection and the inclusion or retention of exons, thereby impacting circRNA production (Li et al. 2018; Zhang et al. 2016). Studies suggest that genes producing circRNAs have increased elongation rates of Pol II, with H3K79me2 linked to exon skipping and transcription start-site switching (Li et al. 2018).

Secondly, considering that circRNA levels often correlate with the expression of their host genes, H3K79me2 may also indirectly influence circRNA expression by modulating the transcriptional activity of these host genes (Barrett and Salzman 2016).

Additional epigenetic marks, such as H3K27me3 and H2A.Z, also play a role in the regulation of circRNAs. H3K27me3, which is present at elevated levels in exons relative to introns, may contribute to splicing and circRNA production (Luco et al. 2010) (Fig. 4). H2A.Z, enriched in genes containing introns and known to affect splicing efficiency under stress conditions, may influence circRNA expression through its impact on chromatin structure and splicing dynamics (Nissen et al. 2017). In summary, epigenetic modifications such as H3K79me2, H3K27me3, and H2A.Z are crucial for understanding the mechanisms behind circRNA expression and their role in various cellular processes. These modifications shape the epigenetic environment that regulates circRNA formation and function in different cell types and biological scenarios.

Therapeutic application of circRNAs

CircRNAs are increasingly recognized as promising targets for cancer therapy due to their roles in tumorigenesis and metastasis (Pisignano et al. 2023). An updated examination of circRNAs that exhibit promise as clinical biomarkers (represented in the outer circle) and/or as therapeutic targets (indicated by syringe icons directed towards the chart), associated with different types of cancer (Pisignano et al. 2023). A variety of therapeutic strategies are currently being investigated to modulate circRNA expression. Techniques involving RNA interference (RNAi), such as small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) have been employed to downregulate oncogenic circRNAs. For instance, shRNAs designed to target circCUX1, which is overexpressed in neuroblastoma and facilitates cell proliferation and invasion, have demonstrated significant efficacy in reducing tumor growth in murine models (Zhang et al. 2020b). Likewise, the silencing of circAGO2, which is linked to unfavorable prognoses in multiple cancers, has been shown to inhibit tumorigenesis by influencing interactions with the HuR protein and regulating mRNA (Chen et al. 2019).

Single-stranded DNA antisense oligonucleotides (AONs) are used to selectively inhibit oncogenic circular RNAs (circRNAs). AONs targeting circLONP2, which is known to promote metastasis and invasiveness in colorectal cancer by interacting with miR-7, have been proven to significantly reduce metastasis to distant organs in in vivo studies (Han et al. 2020). Additionally, the utilization of gold nanoparticles (AuNPs) conjugated with small interfering RNAs (siRNAs) or AONs has been shown to improve delivery efficiency. For instance, AuNPs loaded with siRNAs targeting circDNMT1 have effectively suppressed breast tumor growth and enhanced survival rates in murine models (Du et al. 2018). Similarly, AuNPs containing AONs aimed at circCcnb1 have shown the ability to inhibit tumor growth and improve survival (Fang et al. 2019).

In addition, specific pharmacological inhibitors can target circRNAs that play oncogenic roles. Pertuzumab, which targets the HER2 CR1 domain, has been shown to significantly diminish the tumorigenicity of cells expressing HER2-103aa, a product of circHER2 (Li et al. 2020). Moreover, the direct delivery of circRNA expression cassettes into cells can leverage the circRNA’s function as competing endogenous RNAs (ceRNAs) or microRNA sponges. For instance, the use of plasmid-PEG-AuNPs to deliver circFoxo3 has been associated with the induction of stress-related apoptosis and the inhibition of tumor xenograft growth in vivo (Du et al. 2017).

Currently, research is being conducted on therapeutic strategies that target circRNAs associated with therapy resistance. For instance, circRNA-SORE, which contributes to the resistance of hepatocellular carcinoma (HCC) cells to sorafenib, can be effectively targeted using small interfering RNAs (siRNAs) to reinstate treatment sensitivity (Xu et al. 2020). Similarly, the peptide AKT3-174aa derived from circAKT3 has demonstrated the ability to restore sensitivity to radiotherapy in glioblastoma (Xia et al. 2019). Furthermore, the overexpression of circ0025202 has been found to enhance the efficacy of tamoxifen in hormone receptor-positive breast cancer (Sang et al. 2019), while circRNA17 has been shown to restore sensitivity to enzalutamide in prostate cancer (Wu et al. 2019). These findings underscore the potential of circRNAs in the development of innovative cancer therapies aimed at overcoming resistance to treatment.

Future perspectives and research directions

To strengthen causal inference in bovine adipogenesis, future research should prioritize in vivo functional validation of candidate circRNAs. Practical strategies include: (i) adipose tissue–targeted knockdown or overexpression using antisense oligonucleotides (ASOs) or adeno-associated virus (AAV)-mediated delivery of back-splice–blocking oligonucleotides in cattle or validated bovine model systems; and (ii) CRISPR/Cas-based perturbations, such as CRISPR interference/activation (CRISPRi/CRISPRa) targeting flanking intronic motifs or precise genome editing designed to disrupt circRNA circularization without affecting linear mRNA transcripts. When full-scale cattle trials are impractical, intermediate validation can be conducted using preclinical ruminant models and ex vivo adipose tissue explants.

Secondly, the development of adipose tissue–specific, gene-edited cattle for example, by crossing adiponectin- or FABP4-Cre lines with animals harboring floxed circRNA circularization elements should be pursued to investigate tissue-specific effects on intramuscular fat deposition, backfat thickness, and meat quality traits under controlled dietary conditions. Concurrent longitudinal field studies involving multiple breeds and sexes are also recommended to capture biological variability and assess the durability of observed effects in real-world settings.

Thirdly, the implementation of integrative multi-omics approaches is essential. Combining single-nucleus RNA sequencing (snRNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) will facilitate cell type–specific regulatory mapping. Ribosome profiling (Ribo-seq) can be employed to exclude unintended translation of circRNAs. Lipidomics and stable-isotope fluxomics will link circRNA perturbations to lipid metabolism, while spatial transcriptomics will enable localization of circRNA regulatory networks within muscle and adipose tissues. Methodological standardization—including RNase R treatment, validation with reverse transcription using divergent primers, confirmation via long-read nanopore sequencing, and consistent differential expression analysis pipelines with false discovery rate (FDR) control—will enhance data comparability across studies.

Finally, establishing breed-stratified cohorts, shared data and analysis repositories, and standardized reporting checklists that detail sequencing depth, biological replicates, statistical methods, and culture conditions is imperative. Generally, these strategies will transform descriptive circRNA catalogs into mechanistic, reproducible evidence, thereby accelerating their translation into precision breeding programs and improvements in carcass quality.

Conclusion

This review aimed to consolidate existing knowledge and provide new insights into the specific functions and regulatory mechanisms of circRNAs during the process of adipogenesis in cattle. By synthesizing current research findings and introducing innovative perspectives, the paper has shed light on the multifaceted roles and underlying molecular mechanisms through which circRNAs influence adipogenesis in bovines. The exploration of intricate regulatory networks and downstream pathways affected by circRNAs has enhanced our understanding of their impact on bovine adipogenesis at a molecular level. These insights not only contribute to advancing our knowledge of circRNA-mediated regulation in adipogenesis but also have significant implications for fields such as molecular biology, agriculture, and veterinary science. By elucidating the complex regulatory roles of circRNAs in bovine adipogenesis, this review sets a high standard for future research efforts aimed at unraveling the intricacies of adipose tissue development. It also underscores the potential of circRNAs as essential regulators in this biological process. In summary, this review has provided an in-depth analysis of the functional regulatory roles and molecular mechanisms of circRNAs in bovine adipogenesis. Through intricate regulatory networks, circRNAs play a significant role in influencing adipocyte differentiation, lipid metabolism, and the morphology of adipose tissue in cattle. These regulatory functions extend to the modulation of adipogenic transcription factors, interactions with microRNAs, and essential signaling pathways involved in adipogenesis. The comprehensive investigation underscores the significance of circRNAs as key regulators in adipose tissue biology, with implications for enhancing livestock production efficiency and meat quality. The utilization of advanced methodologies, such as high-throughput RNA sequencing and sophisticated functional assays has facilitated the elucidation of circRNA-mediated regulatory networks in bovine adipogenesis. The insights derived from this review lay the groundwork for further exploration of circRNA species and their regulatory mechanisms in adipogenesis. By uncovering the intricate molecular mechanisms governing circRNA function, future research endeavors hold promise for identifying new therapeutic targets for metabolic disorders and obesity-related conditions in both cattle and humans. Overall, this review contributes to the current understanding of circRNA biology and its relevance to adipogenesis, paving the way for future advancements in agricultural biotechnology and human health research.

Author contributions

B. K. Abebe: Writing – original draft, Writing – review & editing. D. D. Jilo: Wrote a table. J. Guo: Visualization and Editing. J. Wang: Visualization and Editing. G. Cheng: Visualization and Editing. L. Zan: Visualization and Editing. All authors have read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (grant number 31972994), National Beef and Yak Industrial Technology System (grant number CARS-37), Special Project for the Central Government to Guide Local Science and Technology Development (2060404-51301), Science and Technology Special Project of the Ministry of Agriculture and Rural Affairs (19211178) and Key Research and Development Program of Shaanxi Province (2022NY-050, 2022ZDLNY01-01).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.