Abstract Micro RNA (miR) are recognized for their important roles in biological processes, particularly in regulatory componentization. Among the miR, miR-150 has been the focus of intense scrutiny, mostly due to its role in malignant tumors. A comparison between steer and bull adipose tissues identified bta-miR-150 as one of the nine downregulated miRNAs, although its function remains unknown (GEO:[42]GSE75063). The present study aimed to further characterize the role of bta-miR-150 in cattle. bta-miR-150 has a negative regulatory effect on the differentiation of bovine adipocytes and promotes proliferation. Overexpression of bta-miR-150 can promote mRNA and protein expression of the marker genes CDK1, CDK2, and PCNA, increase the number of EdU-stained cells, promote adipocyte proliferation, inhibit adipocyte differentiation, and reduce lipid droplet formation. Results of RNA-seq and WGCNA analyses showed that the mammalian target of the rapamycin signaling pathway, which plays a major regulatory role, is dysregulated by the overexpression and inhibition of miR-150. We found that the target gene of bta-miR-150 is AKT1 and that bta-miR-150 affects AKT1 phosphorylation levels. These results showed that bta-miR-150 plays a role in adipogenic differentiation and might therefore have applications in the beef industry. Keywords: bta-miR-150, mTOR, RNA-seq, adipocyte differentiation, WGCNA Introduction Adipose tissue that differentiates from mesenchymal stem cells during the embryonic period is responsible for many functions, including energy storage, lipid metabolism, and hormone secretion (Ambele et al., [43]2020; Poklukar et al., [44]2020). Since the amount of adipose tissue in meat impacts the economic value of animals, understanding the molecular mechanisms underlying adipose tissue generation in animals will ensure that production is maximized within an appropriate range. MicroRNA are non-coding RNA with a maturity length of 18–25 nt; they are ubiquitous in the animal kingdom and are produced by RNA polymerase II transcription. MicroRNA are involved in many different physiological processes in animals (Raza et al., [45]2020), including inhibition of the translation of target genes, affecting mRNA degradation, and regulating the transcription of target genes (Twayana et al., [46]2013). Differentiation of adipocytes is regulated by many miRNA, which are the focus of the current investigation. Advancements to high-throughput sequencing technology has allowed deeper exploration of cellular regulatory processes (Katz et al., [47]2010). Previous studies have already revealed the regulatory effects of human miRNA, including miR-204 (Zhang Z. et al., [48]2020), miR-27a (Kim et al., [49]2010), miR-210 (Ren et al., [50]2020), miR-24 (Liu et al., [51]2020), miR-378 (Duarte et al., [52]2020), miR-149-5p (Khan et al., [53]2020), miR-143 (Zhang L. et al., [54]2020), and miR-145 (Wang et al., [55]2020), on adipogenesis. These classic mechanisms also occur in the adipose tissue, where they have a large impact on the surrounding tissue (Arcidiacono et al., [56]2020; Raza et al., [57]2020). For example, miR-143 promotes the differentiation of adipocytes by directly acting on its target gene, MAP2K5. Many biologically significant miRNA have been discovered, with high-throughput sequencing technology continuously improving in parallel with a reduction in cost (An et al., [58]2016). Because of its high expression level in immune-related cells, hsa-miR-150 was one of the first miRNAs to be studied in humans. Human (hsa)-miR-150 plays an important role in the differentiation of hematopoietic cell lines; thus, investigations of hsa-miR-150 have mainly focused on malignant tumors (Yugawa et al., [59]2020). In animal, miR-150 has been implicated in the mTOR pathway, where it regulates the expression of leptin in adipocytes (Scrutinio et al., [60]2017) and the production of lipids such as triglycerides (TGs) and free fatty acids (FFAs). The master metabolic factor PGC-1a mediates the reciprocating cycle between TG and FFA to achieve physiologically stable energy consumption (Kang et al., [61]2018). In pigs, ssc-miR-150 is differentially regulated in the adipose tissue miRNA of lean, rather than fatty pig breeds. Ssc-miR-150 is thought to act directly on the 3′UTR of CYP3A4 to promote FFA-induced adipose tissue degeneration (Ma et al., [62]2020). Since miRNA is highly conserved in the animal kingdom, bta-miR-150 might also function in the production of adipose tissue in cattle. Importantly, bta-miR-150 is one of the nine downregulated miRNA among differential expression profiles of steer and bull fat tissues (Liang et al., [63]2019). With the global increase in human population and improvements in living standards, beef fat is increasingly being valued by breeders for the flavor it imparts, as well as its high energy value as a food source (Lillehammer et al., [64]2011). The 480-amino-acid enzyme, RAC-alpha serine/threonine-protein kinase (AKT1), located downstream of the mammalian target of the rapamycin signaling (mTOR) pathway regulates the growth of mammalian cells and is an important protein for regulating fat deposition (Zhang et al., [65]2017; Song et al., [66]2018). In mice, miR-150 inhibits AKT1. Bovine chromosome 21 encodes AKT1, which is an important member of the PI3K/AKT/mTOR pathway. The activity of AKT requires phosphorylation at Thr308 and is further enhanced by phosphorylation at Ser473. When Ser473 is simultaneously phosphorylated by PDK1 and mTORc2, AKT1 further regulates cell proliferation through downstream FOXO transcription factors and p53 regulatory factors (Cai et al., [67]2019; Sanchez-Gurmaches et al., [68]2019; Du et al., [69]2020) The present study aimed to determine the relationship between bta-miR-150 and AKT1 by examining the effects and mechanisms of bta-miR-150 in regulating the proliferation and differentiation of Qinchuan beef cattle pre-adipocytes, thus providing a basis for targeted breeding and genetic improvement based on bta-miR-150. In beef cattle, the process of breeding to increase the intramuscular fat content would be supported by an understanding of the genetic basis of adipogenesis. Our research further clarifies the relationship between bta-miR-150 and adipocyte differentiation in cattle. This information paves the way for further research to expand our understanding of the complex process of adipocyte differentiation and adipose tissue generation. Materials and Methods Isolation of Primary Bovine Pre-adipocytes and Cell Culture The tissue and cell samples used in the experiment in this study were collected from three 1-day-old healthy Qinchuan cattle bulls with consistent growth bred from the National Beef Cattle Improvement Center of Northwest Agriculture and Forestry University (Yangling, China). Bacteria surgical instruments collected tissue samples such as heart, liver, spleen, lung, and muscle. The sample was placed in a sterile, DNase- and RNase-free cell cryotube. Then the sample was immediately put in liquid nitrogen for freezing, and finally stored in a refrigerator at −80°C for later use. We used 1-day-old healthy bulls to isolate the original bovine pre-adipocytes, removed the adipose tissue from different parts under aseptic conditions, washed with PBS supplemented with 10% antibiotics (penicillin/streptomycin) 3 times, and then added I Collagenase digestion for 1–2 h in a 37°C water bath shaker. After digestion, the cells were filtered with a cell sieve and the supernatant was discarded after centrifugation. The red blood cell lysate was added for lysis, and then an appropriate amount of DMEM-F12 (Gibco, Grand Island, NY) medium containing 10% fetal bovine serum (FBS, Invitrogen) was added to resuspend the cells for seeding for subsequent experiments. The cells used in this experiment were all the 3rd generation cells after passage. Construction and Transfection of Plasmid and RNA Oligonucleotides The cells were inoculated in DMEM-F12 supplemented with 10% FBS and 1% antibiotics and kept continuously at 37°C and 5% CO[2]. According to the manufacturer's instructions, the miR-150 mimic (50 nM), miR-150 mimic NC (50 nm), miR-150 inhibitor (100 nM), miR-150 inhibitor NC (100 nM), si-AKT1 (100 nM), and si-NC were transfected into the cells using Lipofectamine^TM 3000 (Invitrogen, San Diego, GA, LUSA). We induced differentiation of adipocytes 48 h after transfection. After 2 days of induction with DMI (0.5 mm IBMX, 1 μm DXMS, and 2 μm insulin) induction solution, we changed to a maintenance medium containing 5 μg/mL insulin. After differentiation induction, adipocytes were harvested at d0, d2, d4, d6, d8, and d10. (We recorded the day of adding DMI as day 0 of induction of differentiation). Two restriction enzymes (Takara, Beijing, China), XhoI and NotI, were used to construct the vector psiCHECK-2(Laboratory retention). The detailed sequence is shown in [70]Table 1. The Bta-miR-150 mimic, mimic NC, inhibitor, and inhibitor NC used in this stage were purchased from RiboBio, Guangzhou, China; the si-AKT1 and si-NC used in this stage were purchased from GenePharma, Shanghai, China. The detailed sequence in is shown [71]Table 1. Table 1. The sequence of RNA oligonucleotides and plasmid. Name Sequence (5′-3′) miR-150 mimic sense: UCUCCCAACCCUUGUACCAGUGU antisense: ACACUGGTACAAGGGUUGGGAGA mimic NC UUGUACUACACAAAAGUACUG miR-150 inhibitor ACACUGGUACAAGGGUUGGGAGA inhibitor NC CAGUACUUUUGUGUAGUACAA si-AKT1 sense: GCCAUGAAGAUCCUAAAGATT antisense: UCUUUAGGAUCUUCAUGGCTT si-NC sense: UUCUCCGAACGUGCACGUTT antisense: ACGUGACACGUUCGGAGAATT psiCHECK-2 5′… GAGCAGTAATTCTAGGCGATCGCTCGAG CCCGGGAATTCGTTTAAACCTAGAGCGGCCGCTGGCCGC AATAAAATA… 3′ [72]Open in a new tab Differential Expression and Pathway Enrichment Analyses Transfected miR-150 mimic, mimic NC, inhibitor, inhibitor NC, and bovine adipocytes were used to induce differentiation, and RNA on day 2 and day 6 were collected. A control was set for each treatment, with three technical replicates per group and a total of eight groups (24 samples). After RNA extraction, the HiSeq-PE150 platform (Illumina, Sandiego, CA, USA) was used for RNA sequence analysis. After filtering the original data, the reads were matched to the bovine genome using HiSat2 (Bos-taurusUMD3.1, release94, Ensembl database), and feature counts were used to count gene expression. The DESeq2 R software package was used to screen DEGs, with Log[2] fold change (log[2]FC) > 1 and false discovery rate (FDR) < 0.05 as the screening criteria. ClusterProfiler was used to realize KEGG pathway analysis and FDR < 0.05 was considered significant. Extraction of RNA, Quantitative Real-Time PCR (qRT-PCR) After 48 h of transfection, we used the RNAiso Plus Kit (Trizol, Takara, Beijing, China) to extract RNA. The specific extraction method can refer to the reference (Junjvlieke et al., [73]2020). After we removed the culture medium from the cells, we washed the cells with PBS 2~3 times in each well, then added 1 mL Trizol, placed the mixture at 4° for 4 min, and transferred the mixture to a 1.5 mL centrifuge tube. Two hundred microliters of chloroform was added to the centrifuge tube, shaken vigorously for 30 s, left to stand for 5 min, and centrifuged for 15 min. The supernatant was then taken (3 layers in total), isopropanol was added, mixed upside down, left to stand on ice for 10 min, and then centrifuged for 10 min. After centrifugation, add 1 mL of 75% cold ethanol was slowly added to a small amount of precipitation at the bottom, centrifuged at 4°C for 5 min, and then the ethanol was discarded. The cells were dried for 2–5 min at room temperature, then an appropriate amount of RNase-free DEPC water was added to dissolve the precipitate. NanoQuant plateTM (TECAN, Infinite M200 PRO) was used to determine the concentration and purity of RNA, and it was stored at −80°C for later use. The method of extracting total RNA in tissues was to take out about 0.5 g tissue sample in a refrigerator at −80°C, add it to liquid nitrogen for pre-cooling, and then place it in a mortar cooled with liquid nitrogen for grinding. After grinding, the sample is transferred to a 1.5 mL enzyme-free centrifuge tube with 1 mL Trizol, and then placed on ice for 15 min. The next step is the same as that of extracting cellular RNA. Reverse transcription of total RNA was performed following Prime Script ™ RT reagent Kit with gDNA Eraser (Takara, China), TB Green^®Premix Ex TaqTM II(TAKARA, Beijing, China) instructions. The reverse transcription and qPCR of miRNA were performed with miRcute Plus miRNA First-Strand cDNA Kit and miRNA Plus miRNA qPCR Kit (SYBR Green) (Tiangen, Beijing, China). U6 and β – actin was used as the internal control for miRNA quantification and mRNA quantification, respectively. The sequence of all PCR primers is shown in [74]Table 2. All experiments were performed with three biological replicates and three technical replicates. The 2-ΔΔCt method was used to analyze the relative expression levels of different qPT-PCR data. Table 2. mRNA and miRNA Real-time quantitative PCR primer sequences. Genes Primer Sequence (5′-3′) Annealing temperature β-actin F: CATCGGCAATGAGCGGTTCC R: CCGTGTTGGCGTAGAGGTC 60°C PCNA F: CCTTGGTGCAGCTAACCCTT R: TTGGACATGCTGGTGAGGTT 60°C CDK1 F:AGTGGAAACCAGGAAGCTTAG R:ATTCGTTTGGCAGGATCATAGA 60°C CDK2 F:GGGTCCCTGTTCGTACTTATAC R:CCACTGCTGTGGAGTAGTATTT 60°C AKT-1 F:AAGGAGATCATGCAGCACCGATTC R: GTCTTGGTCAGGTGGCGTAATGG 60°C CEBPα F: ATCTGCGAACACGAGACG R: CCAGGAACTCGTCGTTGAA 60°C PPARγ F: GACGACAGACAAATCACCGT R: CTTCCACGGAGCGAAACTGA 60°C FABP4 F:TGAGATTTCCTTCAAATTGGG R: CTTGTACCAGAGCACCTTCATC 60°C RRAGD F: TAAGAAGGAGCGGCAAGT R: GGAAGTCCCAAATCTGAAA 60°C RPS6KA3 F:ACCTAGCAACATTCTTTATGTGGAT R: GCATCATAGCCTTGTCGT 60°C RPS6KB1 F: ATCACCAAGGTCACGTCAAAC R: TGCTCCCAAACTCCACCAAT 60°C LPIN1 F: AGTGAATCTTCAGATGCGTTTA R: CAGGTGTCGGCTTCGTTT 60°C NFKB1 F: GAGAACTTCGAGCCTCTGTAC R: CTCATAGGGTTTCCCATTTA 60°C EIF4E F: TCTAATCAGGAGGTTGCT R: CCCACATAGGCTCAATAC 60°C PIK3CA F:TGGGTTTCTCGGTCCTAA R:CAGTCCGTCCAGTCATCC 60°C TNFRSF1B F: CCAGCACAGCTCCAAGCA R:CAACTATCAGAAGCAGACCCAAT 60°C INSR F: GGAAGGCGAGAAGACCAT R: TGACACCAGGGCATAGGA 60°C EIF4EBP2 F: GTTCCTGATGGAGTGTCGGA R: AACTGTGACTCTTCACCGCCT 60°C PIK3R1 F: GAGCGGGAAGAGGACATTGA R: TCTCCCCTGTCGTCTCGTTA 60°C mTOR F: TGATGCAGAAGGTCGAGGTG R: GATGGGTGTCTATCGCCCAG 60°C PIK3R1 F: CTATCTCCTGGACTTACCG R: GGCGACCTAATGAGTTTC 60°C U6 F:TAGCCACCCTCAAGTATGTTCG R:CGAGGTAGGAGGACAGGAGT 60°C miR-150 UCUCCCAACCCUUGUACCAGUGU 60°C [75]Open in a new tab Western Blot Analysis The methods of extraction and Western blot analysis of total protein have been introduced in references (Khan et al., [76]2019; Wang et al.,