The 7-day-old Jianzhou Daer male goats (n = 3) were used as an experimental model. The GIPCs were isolated and cultured, as we previously described (Xu et al., 2018; Ma et al., 2021). Briefly, the isolated longissimus dorsi muscles of goats were washed with PBS for three times and minced and then were digested with an equal volume of collagenase type II at 37°C for 2 h in a shaking water bath every 5 min. Steel mesh filters of 200 and 400 µm were utilized to isolate digested cells. The rinsed filtrated cells by DMEM/F12 medium were centrifuged twice at 2000 r/min for 5 min to collect sediment clumps and then the supernatant was discarded. The viable cells were resuspended in DMEM/F12, including 10% fetal bovine serum and plated in 25-cm² flask in a 5% CO₂ atmosphere at 37°C for subsequent culture. Experimentation on goats performed in the present study had been given prior approval by the Ethics Committee of Southwest Minzu University under permit no. SMU20160108, and all of the methods were performed according to the guidelines and regulations.

We induced GIPCs adipogenic differentiation in vitro as before (Xu et al., 2018). Briefly, adipogenesis induction medium [MEM/F12 containing 10% FBS and 50 μmol•L^-1 oleic acid (Sigma)] was used to culture GIPCs in 12-well plates with a density of 1 × 10⁶ cells per well for the required time point. We changed the culture medium every other day. After induction, Oil Red O staining and BODIPY staining were used to distinguish mature adipocytes from preadipocytes during the process of culture.

Adipogenic differentiation of the GIPCs was assessed by Oil Red O or BODIPY staining as previously described (Xu et al., 2018). The GIPCs were washed with PBS and fixed in 10% formaldehyde for 10 min, then washed with PBS, and stained using the Oil Red O or BODIPY working solutions for 20 min. The cells were then observed and photographed after washing. After photographing, the cells were destained in 1 ml 100% isopropanol for 15 min and the Oil Red signal was quantified by measuring the absorbance at 490 nm (OD 490) as a semi-quantitative assessment method to determine the extent of differentiation. The stained area of Oil Red O or BODIPY staining was measured using ImageJ (NIH, Bethesda, MD, United States).

Total RNA from cells was extracted using the TRIzol reagent (TaKaRa) according to the manufacturer’s protocol. The mRNAs were reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (Thermo) according to the protocol. Then, amplification reactions were performed using amplification primers with the SYBR Green PCR Master Mix (TaKaRa); the reaction volumes were 20 μl. Then 1 μl of cDNA was applied in every set of experiment. The mRNA expression levels were standardized to UXT or U6. Information on primers for qPCR is listed in Table 1.

Small interfering RNA (siRNA) against KLF8, 5’-CAGACUCUUGUAGUGUCCACUUCAAdTdT-3’ was synthesized by Invitrogen. KLF8 expression plasmid was constructed by inserting expanded KLF8 cDNA (KX247671) fragments into pcDNA3.1 vector (sense primer sequence: 5’CGG​GGT​ACC​ATG​GAT​GAA​CTC​ATA​AAC​AAC​T-3’, anti-sense primer sequence: 5’-ATA​AGA​ATG​CGG​CCG​CTT​ACA​CGG​TGT​CAT​GGC​GC-3’). The KLF8 interference (designated as siKLF8) or negative control (siNC) GIPCs were constructed using siRNA. The KLF8 overexpression (designated as KLF8) GIPCs were constructed using an expression plasmid, and the control cells for the KLF8 overexpression group were designated as a vector. Cells had been pre-cultured for 2 h in a serum-free medium for transfection. Then plasmid or siRNA was introduced into the cells using a Lipofectamine 3000 transfection reagent, in accordance with the manufacturer’s instruction (Invitrogen, Carlsbad, United States).

The miR-10a-5p mimics (designated as mimics: UAC​CCU​GUA​GAU​CCG​AAU​UUG​U), an inhibitor (designated as inhibitor: ACA​AAU​UCG​GAU​CUA​CAG​GGU​A), and a respective negative control (designated as mock: UUG​UAC​UAC​ACA​AAA​GUA​CUG, NC: CAG​UAC​UUU​UGU​GUA​GUA​CAA) (Genepharma, Shanghai, China) as needed were transfected into the GIPCs by Lipofectamine 3000 (Invitrogen, Carlsbad, United States) and opti-MEM (Gibco BRL Co., LTD) culture medium according to the manufacturer’s instruction.

After 12-h transfection, the original medium was replaced by a fresh differentiation medium to induce GIPC differentiation. After 48-h induction, the cells were used for Oil red O or BODIPY staining or collected to extract RNA for qPCR detection.

For the luciferase reporter assay, the 3’UTR of KLF8 containing the wild or mutant miR-10a-5p target sites was cloned using primers with NotI and XhoI (Thermo, MA, United States) cleavage sites. The wild or mutant type 3’UTR fragment was inserted into the corresponding site of the psiCHECK vector and then co-transfected into 293T cells with miR-10a-5p mimics/mock. After 48 h transfection, the cells were harvested and the Dual-Luciferase Reporter Assay System Kit (Promega, Madison, WI, United States) was used for detecting dual-luciferase activity, according to the manufacturer's instructions.

All data were presented as “mean ± SD.” The variance of data was analyzed by SPSS 17.0, followed by Duncan’s multiple comparisons test. * indicates the p values were <  0.05, ≥  0.01, whereas ** indicates p values <  0.01. All experiments in our study were carried out for three times at least.

MicroRNAs have been reported to regulate adipogenic differentiation (Hamam et al., 2015; Tang et al., 2017; Ai et al., 2019; Li et al., 2019); however, the role of miR-10a-5p in goat intramuscular adipogenesis has not been reported. The differential expression of miR-10a-5p after GIPC differentiation has been observed by miRNA sequencing technology in our previous study (data not shown). In order to clarify the role of miR-10a-5p in the differentiation of GIPCs, we first isolated intramuscular preadipocytes from goat longissimus dorsi and induced them to adipogenic differentiation. Oil red O staining was used to ascertain the extent of differentiation; our obtained results showed that the lipid droplets accumulation increased with the extension of induction time, and the GIPCs were differentiated completely after 60-h induction (Figure 1A). Then qRT-PCR was implemented to research the role of miR-10a-5p in adipogenic differentiation of GIPCs. The test result showed an obvious alteration in the expression of miR-10a-5p during differentiation when compared to 0 h (Figure 1B). All above indicate that miR-10a-5p may regulate the adipogenic differentiation of GIPCs.

To clarify the effect of miR-10a-5p on GIPC adipogenic differentiation, miR-10a-5p mimics (named as miR-10a-5p) or its control (named as mock) was transfected to GIPCs to overexpress miR-10a-5p. The expression of miR-10a-5p in GIPCs increased ∼ 10,000 times caused by mimic transfection than the same amount of control vector transfected cells (Figure 2A). Preadipocyte differentiation is associated with lipid droplets accumulation in the cells. Oil red O and BODIPY stainings were utilized as lipid droplet detection methods. In our study, after miR-10a-5p overexpression, the lipid droplet accumulation was significantly decreased in the group of mimic-transfected cells than the control group (Figures 2B–E). Oil red O and BODIPY staining results showed about 20–30% reduction of lipid droplets with miR-10a-5p overexpression (Figures 2B–E). Additionally, there are representative of the genes upregulated during the differentiation process from preadipocytes to mature adipocytes. Indeed, the mRNA expression levels of representative genes AP2, DGAT2, FASN, HSL, LPL, and Pref1 were obviously inhibited due to the overexpression of miR-10a-5p in GIPCs (Figure 2F). All these results implied that miR-10a-5p may weaken the adipogenic differentiation of GIPCs.

For further validating the suppression effect of miR-10a-5p on GIPC adipogenic differentiation, miR-10a-5p inhibitor (named as inhibitor) or its control (named as NC) was transfected to GIPCs to silence miR-10a-5p expression. About 80% of the interference efficiency was caused by inhibitor transfection compared to the NC group (Figure 3A). Oil red O and BODIPY staining results showed that compared with the control group, miR-10a-5p knockdown significantly promoted the about 10–20% of lipid droplet accumulation (Figures 3B–E). Coincidently, the mRNA levels of important markers of adipocyte differentiation like ACC, ATGL, CEBPβ, GLUT4, HSL, PPARγ, and Pref1 were upregulated due to the knockdown of miR-10a-5p in GIPCs (Figure 3F). Therefore, miR-10a-5p inhibits adipogenic differentiation of GIPCs.

It has been well known that miRNA binds to the 3’-UTR of target mRNA’s complementary sequences, so the target gene’s mRNA expression can be inhibited (Redis and Calin, 2017; Rouleau et al., 2017; Sun et al., 2017). TargetScan (Garcia et al., 2011), microRNAseq (Yan et al., 2018), miRDB (Wong and Wang, 2015), and DIANA-microT (Maragkakis et al., 2009) were the tools used for predicting the miR-10a-5p possible target genes. Among all the potential target genes predicted in both databases, we chose KLF8, as it is a positive regulator of 3T3-L1 differentiation and knocking down its expression can reduce RXRα overexpression-caused GIPC differentiation (Lee et al., 2012; Xu et al., 2020). Also, its family members are important regulators for adipogenic differentiation (Kinoshita et al., 2010; Pei et al., 2011; Tahmasebi et al., 2013; Shen et al., 2018; Chen J. et al., 2020). By sequence analysis, there is potential binding site of miR-10a-5p in the 3'UTR of KLF8 in goat (Figure 4A). First, we measured the mRNA level of KLF8 in miR-10a-5p mimics or inhibitor-transfected cells. As expected, the mimics of miR-10a-5p significantly downregulated the mRNA level of KLF8, while miR-10a-5p inhibitor upregulated its mRNA expression when compared with each control group, respectively (Figure 4B). In order to make sure whether miR-10a-5p can directly target KLF8 3'UTR, we mutated miR-10a-5p–binding sites in the KLF8 3’UTR area (Figure 4C). Luciferase report vectors were constructed with wild-type KLF8 3’UTR (KLF8 3’UTR WT) and mutated KLF8 3’UTR (KLF8 3’UTR MT). The KLF8 luciferase activity was measured for describing miR-10a-5p function on luciferase translation. The results showed that luciferase activity of wild-type KLF8 3’UTR was significantly inhibited by miR-10a-5p overexpression, yet mutated KLF8 3’UTR terminated this effect (Figure 4D). Taken together, we confirmed that KLF8 is the direct target of miR-10a-5p. Then, we got the conclusion that miR-10a-5p targets KLF8 and regulates KLF8 expression.

Since KLF8 is the target of miR-10a-5p and plenty of studies have shown that KLF8 and its family members are regulators of adipogenic differentiation, we further explored the relationship between KLF8 expression and GIPCs differentiation. We first synthesized KLF8 siRNA (named as siKLF8 and its control named as siNC) and constructed its expression plasmid (named as KLF8 and its control named as vector). Both the expression plasmid and siRNA were effective that cells transfected with the KLF8 expression plasmid can upregulate its expression about 5,000 times, and KLF8 siRNA can downregulate its expression about 60 times (Figure 5A). Oil red O and BODIPY staining results showed that KLF8 overexpression could contribute to the lipid droplet accumulation; nevertheless, KLF8 knockdown can inhibit the lipid droplet accumulation when compared with each control group, respectively (Figures 5B–E). qPCR was used to detect relative adipogenic gene expression, and the crucial adipocyte differentiation markers DGAT2, ACC, and LPL were markedly promoted by KLF8 overexpression (Figure 5F). Also, LPL, PPARγ, and C/EBPβ as adipocyte differentiation genes were obviously inhibited by KLF8 interference (Figure 5F). In summary, our results indicated that KLF8 confers GIPCs with more properties of adipogenic differentiation.