Elucidating the Role of circTIAM1 in Guangling Large-Tailed Sheep Adipocyte Proliferation and Differentiation via the miR-485-3p/PLCB1 Pathway

Fat tissue—a vital energy storage organ—is intricately regulated by various factors, including circular RNA, which plays a significant role in modulating fat development and lipid metabolism. Therefore, this study aims to clarify the regulatory mechanism of sheep adipocyte proliferation and differentiation by investigating the involvement of circTIAM1, miR-485-3p, and its target gene PLCB1. Through previous sequencing data, circTIAM1 was identified in sheep adipocytes, with its circularization mechanism elucidated, confirming its cytoplasmic localization. Experimental evidence from RNase R treatment and transcription inhibitors highlighted that circTIAM1 is more stable than linear RNA. Additionally, circTIAM1 promoted sheep adipocyte proliferation and differentiation. Furthermore, bioinformatic analysis demonstrated a robust interaction between miR-485-3p and circTIAM1. Further experiments revealed that miR-485-3p inhibits fat cell proliferation and differentiation by inhibiting PLCB1, with circTIAM1 alleviating the inhibitory effect via competitive binding. In summary, our findings elucidate the mechanism through which circTIAM1 regulates Guangling Large-Tailed sheep adipocyte proliferation and differentiation via the miR-485-3p–PLCB1 pathway, offering a novel perspective for further exploring fat metabolism regulation.


Introduction
Sheep are economically important livestock that yield various valuable products, including meat, wool, leather, and milk.According to tail type, sheep can be divided into three types: fat-tailed sheep, fat-rumped sheep, and thin-tailed sheep.Fat-tailed sheep include long fat-tailed sheep and short fat-tailed sheep, mainly distributed in the northwest and northern regions of China.Due to human activities, the geographical distribution of sheep has changed [1].The tail type differences resulting from this geographical distribution are mainly adaptations to the environment, a result of natural and artificial selection [2,3].Sheep distributed in high-altitude areas have a higher proportion of energy metabolism-regulating genes [4], further demonstrating that sheep adjust their fat metabolism mechanisms to adapt to the environment.As the tail is the main site of fat storage in fat-tailed sheep, sheep with different tail types have become important animal models for studying fat deposition and energy storage [4].For instance, in fat-tailed sheep, white adipose tissue is mainly found subcutaneously around the tail, with relatively low levels around the viscera and muscle tissues [4].The fat tail serves as a significant energy reservoir, enabling these sheep to adapt to harsh environmental conditions such as droughts, extreme cold, and feed shortages [5].The Guangling Large-Tailed sheep, as a type of fat-tailed sheep, has sparked the interest of researchers due to its significant adaptation to its environment.Despite being primarily bred for meat, the high tail-fat content, that can make up to 20% of the body weight, decreases the market value.This is attributed to consumer preference for leaner meat, thereby significantly affecting its economic value [6].Therefore, studying the mechanism of fat deposition in sheep tails is important for improving production efficiency.
Fat deposition involves complex processes regulated by various transcriptional factors [7].Circular RNAs (circRNAs) are noncoding RNAs characterized by a closed-loop structure, lacking 5 ′ caps and 3 ′ polyA tails.They exhibit structural conservation and stability, making them resistant to degradation [8].CircRNAs are prevalent in eukaryotic organisms, primarily in the cytoplasm and extracellular vesicles, with a minority found in the nucleus [9].Recent studies highlight their role in transcriptional regulation, alternative splicing, and chromatin looping [10][11][12].Additionally, circRNAs function as molecular sponges, binding to miRNAs, influencing target gene expression [13].For example, circSAMD4A serves as a sponge for miR-138-5p, regulating EZH2 expression and adipogenesis in individuals with obesity [14].Similarly, CircBDP1 may regulate SIRT1 expression during bovine fat development by sequestering miR-204 and miR-181b [15].Additionally, CircPAPPA2 acts as a competing endogenous RNA (ceRNA), sequestering miR-2366 to regulate GK expression, thereby inhibiting adipocyte differentiation [16].Collectively, these findings suggest a pivotal role for circRNAs in sheep adipocyte development.
Therefore, this study aims to elucidate the regulatory mechanism involving circTIAM1, miR-485-3p, and its target gene PLCB1 in Guangling Large-Tailed sheep adipocyte proliferation and differentiation.We specifically investigate how circTIAM1 upregulates PLCB1 by competitively binding miR-485-3p, thereby promoting sheep adipocyte proliferation and differentiation.The findings could offer important insights into the circRNA regulatory network in sheep fat deposition and provide valuable references for future investigations into the regulatory mechanisms underlying sheep fat metabolism.

circTIAM1 Serves as a Molecular Sponge for miR-485-3p
Recent studies suggest that circRNAs can function as molecular sponges, competitively binding and participating in post-transcriptional gene regulation.Using RNAhybrid, we predicted the binding sites of circTIAM1 and miR-485-3p (Figure 7A).To confirm this hypothesis, we constructed wild-type (circTIAM1 WT) and mutant (circTIAM1 MUT) vectors containing the binding sites (Figure 7B).Co-transfection of these vectors with miR-485-3p mimic (or miR-485-3p NC) into 293T cells revealed that co-transfection of the miR-485-3p mimic with circTIAM1 WT significantly decreased luciferase activity compared with transfection with miR-485-3p NC (p < 0.05) (Figure 7C).Conversely, co-transfection of the circTIAM1 MUT with the miR-485-3p mimic demonstrated no effect on luciferase activity (p > 0.05) (Figure 7C).This suggests a target relationship between miR-485-3p and circTIAM1.Additionally, temporal expression analysis during sheep adipocyte differentiation over 10 days demonstrated that circTIAM1 and PLCB1 expression peaked on day 6 of adipocyte differentiation.However, miR-485-3p expression reached its lowest point (Figure 7D).These expression patterns suggest a negative correlation between circTIAM1 and miR-485-3p and between miR-485-3p and PLCB1.In summary, these findings suggest that circTIAM1 functions as a molecular sponge to bind miR-485-3p competitively.

circTIAM1 Serves as a Molecular Sponge for miR-485-3p
Recent studies suggest that circRNAs can function as molecular sponges, competitively binding and participating in post-transcriptional gene regulation.Using RNAhybrid, we predicted the binding sites of circTIAM1 and miR-485-3p (Figure 7A).To confirm this hypothesis, we constructed wild-type (circTIAM1 WT) and mutant (circTIAM1 MUT) vectors containing the binding sites (Figure 7B).Co-transfection of these vectors with miR-485-3p mimic (or miR-485-3p NC) into 293T cells revealed that co-transfection of the miR-485-3p mimic with circTIAM1 WT significantly decreased luciferase activity compared with transfection with miR-485-3p NC (p < 0.05) (Figure 7C).Conversely, co-transfection of the circTIAM1 MUT with the miR-485-3p mimic demonstrated no effect on luciferase activity (p > 0.05) (Figure 7C).This suggests a target relationship between miR-485-3p and circTIAM1.Additionally, temporal expression analysis during sheep adipocyte differentiation over 10 days demonstrated that circTIAM1 and PLCB1 expression peaked on day 6 of adipocyte differentiation.However, miR-485-3p expression reached its lowest point (Figure 7D).These expression patterns suggest a negative correlation between circTIAM1 and miR-485-3p and between miR-485-3p and PLCB1.In summary, these findings suggest that circTIAM1 functions as a molecular sponge to bind miR-485-3p competitively.

Discussion
Fat tissue in animals functions as an energy storage site and plays a vital role in regulating metabolism [20], hormone secretion [21], immune function [22], and other essential physiological processes.Various non-coding RNAs (ncRNAs) regulate fat tissue formation in animals, with circRNA playing pivotal roles during adipogenesis [23][24][25].Existing studies on circRNAs in sheep have predominantly centered on muscle development [26], hair follicle development [27], and reproductive performance [28].Studies investigating the mechanisms through which circRNAs participate in adipocyte proliferation and differentiation remain limited.In this study, we discovered a circRNA uniquely expressed in sheep adipocytes, named circTIAM1, derived from the reverse splicing of the sixth exon of TIAM1.The identification and characterization findings revealed that the

Discussion
Fat tissue in animals functions as an energy storage site and plays a vital role in regulating metabolism [20], hormone secretion [21], immune function [22], and other essential physiological processes.Various non-coding RNAs (ncRNAs) regulate fat tissue formation in animals, with circRNA playing pivotal roles during adipogenesis [23][24][25].Existing studies on circRNAs in sheep have predominantly centered on muscle development [26], hair follicle development [27], and reproductive performance [28].Studies investigating the mechanisms through which circRNAs participate in adipocyte proliferation and differentiation remain limited.In this study, we discovered a circRNA uniquely expressed in sheep adipocytes, named circTIAM1, derived from the reverse splicing of the sixth exon of TIAM1.The identification and characterization findings revealed that the circularization mechanism of circTIAM1 predominantly occurs in the cytoplasm, aligning with its post-transcriptional regulatory role.ActD, as a transcription inhibitor, blocks new RNA synthesis, leading to the gradual degradation of already synthesized RNA.By collecting samples at different time points and measuring the levels of target RNA, decay curves for the target RNA can be established.Using these data, the half-life of the target RNA can be calculated, which is an important parameter for measuring RNA stability.Similar to other known circular RNAs [13], circTIAM1 exhibits higher stability compared to linear TIAM1 under RNase R and ActD treatments due to its closed-loop structure.This suggests its potential utility as a stable biomarker or therapeutic target for fat-related diseases [29].Previous studies have highlighted the common correlation between the functions of circRNAs and their host genes [30].TIAM1 regulates actin organization and fat cell formation [17].Consequently, we hypothesized that circTIAM1 holds a considerable significance in adipocyte development.Our experimental results confirm that circTIAM1 stimulates the proliferation and differentiation of sheep adipocytes, aligning with previous findings.However, the specific mechanism underlying how circTIAM1 functions as a non-coding RNA remains unclear.
To explore the role of circTIAM1 as a non-coding RNA in regulating sheep adipocyte development, we conducted bioinformatics analysis and speculated on robust binding between miR-485-3p and circTIAM1.Further investigation into the role of miR-485-3p during adipocyte development demonstrated its inhibitory effects on the proliferation and differentiation of sheep adipocytes.Additionally, miR-485-3p inhibits cell proliferation by regulating TRIP6 expression, which is consistent with our findings [18].The contrasting roles of miR-485-3p and circTIAM1 in sheep adipocytes indicate that circTIAM1 functions as a molecular sponge, modulating adipocyte development by sequestering miR-485-3p.However, the pathway through which miR-485-3p regulates adipocyte development remains elusive, highlighting the need for further research to elucidate these mechanisms.
Numerous studies have highlighted the role of miRNAs in gene expression level regulation through 3 ′ UTR targeting of mRNA [31][32][33].However, studies on their binding to the 5 ′ UTR [34] and CDS sequences remain limited [35].In this study, bioinformatics software (version 2.1.2) was utilized to predict a binding site between the seed sequence of miR-485-3p and the 3 ′ UTR of PLCB1.Dual-luciferase reporter assays validated their target relationships, supported by several experiments, including qRT-PCR and Western blotting.These demonstrated a negative regulatory relationship between them.Additionally, PLCB1 has been shown to participate in the cell cycle progression by regulating the levels of various cell cycle proteins, thereby promoting cell proliferation.For example, PLCB1 can significantly increase the expression levels of Cyclin D3 and the percentage of S phase cells in the K562 human erythroleukemia cell line [36].PLCB1 also modulates the expression of Cyclin E, which plays a critical role in the S phase of the cell cycle [37], and it can be involved in G2/M cell cycle progression [38].During adipogenic differentiation, PLCB1 regulates the expression of cell cycle proteins cyclin D3 and cdk 4, which are essential for maintaining the differentiation state during both the early and late stages of mitosis [39].Another study found that PLCB1 can enhance Cyclin D3 expression, activating PPARγ to promote adipogenesis [40].This provides evidence elucidating how miR-485-3p inhibits the proliferation and differentiation of fat cells by suppressing PLCB1 expression.
CircRNAs commonly function as competitive endogenous RNAs, specifically binding to miRNAs and acting as molecular sponges to inhibit the effects of miRNAs on their target genes [41,42].We constructed circTIAM1 WT and circTIAM1 MUT dual-luciferase reporter vectors to determine their target relationships.Co-transfection experiments involving circTIAM1 and miR-485-3p suggested that circTIAM1 mitigated the inhibitory effect of miR-485-3p on downstream PLCB1 through competitive binding, thereby regulating the proliferation and differentiation of sheep adipocytes.The expression patterns of circTIAM1, miR-485-3p, and PLCB1 during differentiation also partially demonstrated the role of circTIAM1 in regulating sheep adipocyte proliferation and differentiation via the miR-485-3p/PLCB1 pathway.However, miR-485-3p is not the sole target of circTIAM1.Moreover, it might regulate other target genes.This highlights the need for further exploration into the regulatory network of adipocyte development.
Advancements in the investigation of sheep adipocyte development regulatory mechanisms will enhance our understanding of circTIAM1, miR-485-3p, and PLCB1 regulatory networks.The integration of diverse experimental techniques and bioinformatics methods will facilitate the identification of more miR-485-3p target genes, potentially elucidating their functions and regulatory networks in adipocyte biology.Furthermore, our investigation into the role of PLCB1 in adipocyte proliferation and differentiation sheds light on its potential interactions with other signaling pathways.This offers a comprehensive understanding of its status and function in regulating fat cell metabolism.These studies contribute valuable insights into adipose tissue biology and disease development.

Ethical Approval Declarations and Sample Collection
The methodologies employed in this study were conducted following the guidelines of the College of Animal Science and Veterinary Medicine at Shanxi Agricultural University (Jinzhong, China).All experimental Protocols were reviewed and approved by the institution (The Ethics Committee's approval reference: SXAU-EAW-2022S.UV.010009).
This study employed Guangling Large-Tailed sheep as experimental animals, provided by Guangling County Sheep Farm in Datong, Shanxi.Three one-month-old Guangling Large-Tailed sheep were selected and euthanized by electric shock followed by exsanguination.Subsequently, tail fat cells were collected.

Vector Construction and Cell Transfection
The complete circTIAM1 sequence was integrated into the pCD2.1-ciRvector (Geneseed, Guangzhou, China), while a vector lacking the circTIAM1 sequence served as a control.siRNAs targeting circTIAM1 splicing sites were synthesized by RiboBio (Guangzhou, China) (Table 1).Mimics and inhibitors of miR-485-3p were sourced from Sangon (Shanghai, China).The working concentration of mimics and inhibitors is 20 pmol/L.PLCB1 3 ′ UTR WT, PLCB1 3 ′ UTR MUT, circTIAM1 WT, and circTIAM1 MUT vectors were procured from Sangon (Table 2).When the density of sheep preadipocytes cultured in six-well plates reached 70%, cells were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instruction.After transfection for 6 h, the complete culture medium was replaced, and the cells continued to be cultured.

RNase R Treatment
The cells underwent treatment with 5 U/µg RNase R (R0301; Geneseed, Guangzhou, China), following the manufacturer's instruction, and were then incubated at 37 • C for 15 min.Subsequently, the expression levels of circular and linear RNAs were assessed using qRT-PCR.

Actinomycin D Assay
Cells were exposed to 2 µg/mL ActD (MilliporeSigma, Burlington, MA, USA) and incubated for 4, 8, 12, and 24 h, respectively.The stability of the circular and linear RNAs was evaluated through qRT-PCR.4.7.RNA and Genomic DNA Extraction and Quantitative Real-Time PCR RNA was extracted from cells using RNAiso Plus (9108; Takara, Otsu, Japan) according to the instructions of the manufacturer.However, gDNA was extracted using DNAiso Reagent (9770; Takara, Otsu, Japan).Nuclear and cytoplasmic fractions were isolated using the PARIS kit (AM1921; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA).RNA quality was evaluated using the RNA 6000 Nano LabChip Kit and Bioanalyzer 2100 (Agilent Technologies, Beijing, China), while RNA integrity was confirmed through 1% agarose gel electrophoresis.Subsequently, RNA was reverse transcribed following the instructions of the manufacturer, using either the PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time; RR047A; Takara, Otsu, Japan) or miRcute Plus miRNA First-Strand cDNA Kit (KR211; Tiangen, Beijing, China).The qRT-PCR analysis was performed using TB Green Premix Ex Taq II Master Mix (RR820A; Takara, Otsu, Japan) or the miRcute Plus miRNA qPCR kit (FP411; Tiangen, Beijing, China) on a Bio-Rad CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA).Tables 3 and 4 list the primer sequences employed in this study.The adipocytes were evenly seeded in 96-well plates.Cells in the logarithmic growth phase were assessed using the Cell-LightTM EdU DNA Cell Proliferation Kit (C10310-1; RiboBio, Guangzhou, China) in accordance with the instructions of the manufacturer.Furthermore, when cell confluency reached 60%, cells transfected at 0, 12, 24, 36, and 48 h received 10 µL of CCK-8 solution per well.Following a 2-h incubation, the absorbance at 450 nm was measured using a multifunctional microplate reader (Bio-Rad, Hercules, CA, USA) to generate cell proliferation curves.

Oil Red Staining
Differentiated adipocytes underwent three washes with pre-chilled PBS, and they were subsequently fixed with 4% paraformaldehyde at room temperature for 2 days.After fixation, the cells were stained with Oil Red O working solution for 30 min following the instructions of the manufacturer.Following destaining with 60% isopropanol for 60 s, the cells underwent three washes with PBS and were imaged under a microscope (Leica, Wetzlar, Germany).

Statistical Analysis
Each experiment was performed with three samples, with each assessed thrice.Data analysis and visualization were conducted using GraphPad Prism software (version 8.0; GraphPad, San Diego, CA, USA).Student's t-test was employed to assess the differences between the experimental and control groups, while a one-way analysis of variance was used for comparison between multiple groups.Data are presented as mean ± SEM, with a p < 0.05 considered statistically significant.

Conclusions
This study demonstrates that circTIAM1 promotes the proliferation and differentiation of Guangling Large-Tailed sheep adipocytes.Mechanistic studies have shown that circTIAM1 enhances PLCB1 expression by competitively binding to miR-485-3p, consequently promoting the proliferation and differentiation of Guangling Large-Tailed sheep adipocytes.The findings provide valuable insights into circRNA regulatory networks in sheep fat deposition, offering important references for future investigations into sheep fat metabolism regulation.

Figure 1 .
Figure 1.Characterization and identification of sheep circTIAM1.(A) CircTIAM1 formation via circular splicing of TIAM1.(B) Confirmation of circTIAM1 presence through Sanger sequencing.(C) PCR analysis using divergent and convergent primers in cDNA and gDNA.(D) CircTIAM1 and TIAM1 expression after RNase R treatment.(E) CircTIAM1 and TIAM1 expression level in sheep adipocytes following ActD treatment.(F) CircTIAM1 expression in nucleus and cytoplasm after nuclear−cytoplasmic fractionation.***: p < 0.001.

Figure 1 .
Figure 1.Characterization and identification of sheep circTIAM1.(A) CircTIAM1 formation via circular splicing of TIAM1.(B) Confirmation of circTIAM1 presence through Sanger sequencing.(C) PCR analysis using divergent and convergent primers in cDNA and gDNA.(D) CircTIAM1 and TIAM1 expression after RNase R treatment.(E) CircTIAM1 and TIAM1 expression level in sheep adipocytes following ActD treatment.(F) CircTIAM1 expression in nucleus and cytoplasm after nuclear-cytoplasmic fractionation.***: p < 0.001.

Table 3 .
qPCR primers for mRNA application.

Table 4 .
Sequences of divergent and convergent primers for circTIAM1.