MicroRNA-106a-5p Inhibited C2C12 Myogenesis via Targeting PIK3R1 and Modulating the PI3K/AKT Signaling

The microRNA (miR)-17 family is widely expressed in mammalian tissues and play important roles in various physiological and pathological processes. Here, the functions of miR-106a-5p, a member of miR-17 family, were explored during myogenic differentiation in C2C12 cell line. First, miR-106a-5p was found to be relatively lower expressed in two-month skeletal muscle tissues and gradually decreased upon myogenic stimuli. Forced expression of miR-106a-5p significantly reduced the differentiation index, fusion index as well as the expression of myogenic markers (MyoD, MyoG, MyHC, Myomixer, Myomarker). Meanwhile, the levels of phosphorylated AKT were reduced by overexpression of miR-106a-5p, and administration of insulin-like growth factor 1 (IGF1), a booster of myogenic differentiation, could recover all the inhibitory effects above of miR-106a-5p. Furthermore, miR-106a-5p was elevated in aged muscles and dexamethasone (DEX)-treated myotubes, and up-regulation of miR-106a-5p significantly reduced the diameters of myotubes accompanied with increased levels of muscular atrophy genes and decreased PI3K/AKT activities. Finally, miR-106a-5p was demonstrated to directly bind to the 3’-UTR of PIK3R1, thus, repress the PI3K/AKT signaling.


Introduction
Skeletal muscle comprises approximately 50% of the body's weight and is an endocrine and paracrine organ that plays a key role in the maintenance of the internal environment and homeostasis [1]. Myogenesis is an ordered and extremely complicated process, including myoblasts proliferation, withdrawal from the cell cycle, fusion into multinucleated myotubes, and hypotrophy/atrophy [2]. During muscle development, Myf5, MyoD and MRF4 are myogenic determination factors, and Myogenin is a downstream effector of MyoD and MRF4, and activate the myogenic differentiation program [3,4]. It has been reported that many pathways are involved in myogenesis, such as Wnt/β-Catenin Signaling [5], the TGFβ signaling pathway [6,7], JAK/STAT signaling pathway [8], and PI3K/AKT signaling pathway [9,10]. Recently, more and more microRNAs (miRs) have been reported to participate in myogenesis, such as miR-127 [11], miR-133 [12], miR-1/206 [13], and miR-432 [14]. However, there are still many miRs that remain to be discovered in myoblasts differentiation and muscle formation.
MiR is a class of evolutionarily conserved, regulatory noncoding RNAs of 18-24 nucleotides that participate in the fine-tuning of many, if not all, fundamental biological processes, including skeletal muscle development and muscle-related diseases [15,16]. MiRs have been exquisitely described to control gene expression by binding to mRNA targets using the seed sequences, and miRs with the same or similar seed sequences and origin are grouped into one family or cluster [15]. The miR-17 family consists of the miR-17-92 cluster, miR-106a-363 cluster and miR-106b-25 cluster [17], and the miR-17 family has been well documented to influence the survival, differentiation and functions of various kinds of mammalian cells [18,19]. However, the effects of miRs associated with the miR-17 family in myogenesis are controversial. MiR-17, miR-20a and miR-92a are reported to strongly repress myoblasts differentiation by targeting Enigma homolog 1 (ENH1)/Inhibitor of differentiation 1 (Id1) [20]. Over the same period, Luo reported that miR-20a/b promoted quail muscle clone 7 (QM-7) myoblasts differentiation by negatively regulating E2F1 [21]. Intriguingly, miR-106a-5p, a member of the miR-106a-363 cluster, was reported to down-regulate during myogenic differentiation [22,23], and its role in myogenic differentiation deserves to be analyzed. PI3K (p85α), encoded by PIK3R1 gene, is a key protein involved in the PI3K/AKT signaling pathway [24], which is essential for myogenic differentiation [25,26]. It has been recently shown that PI3K/AKT signaling controls muscle-abundant miRs (myomiR) maturation during C2C12 myoblasts differentiation [27]. Insulin and insulin-like growth factor 1 (IGF1) are known as physiological activators of PI3K/AKT signaling in different cell types, including C2C12 myoblasts [28]. Administration of IGF1 promotes myoblast proliferation, differentiation [25,29], and induces myotube hypertrophy [30] by activating the PI3K/AKT signaling pathway. However, regulation of PIK3R1 by miR-106a-5p and how miR-106a-5p responds to IGF1 stimuli to regulate myogenesis are still poorly understood.
In this study, we analyzed the expression profiles of miR-106a-5p and determined its role and mechanism on myogenesis. Our study identified miR-106a-5p as a novel negative regulator for myogenesis, and miR-106a-5p could repress differentiation and promote atrophy by blocking the PI3K/AKT signaling pathway through targeting PIK3R1.

Ethics Statement
All experimental mice were operated on in accordance with approved guidelines of the Animal Care and Use Committee of the Northwest A and F University, Yangling, China (NWAFU-314020038) and the guidelines of the Animal Use Committee of the Chinese Ministry of Agriculture (Beijing, China). Furthermore, C2C12 and HEK29T cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Cell Culture
The C2C12 myoblasts (ATCC, USA) were used to determine the function of miR-106a-5p during myogenesis. HEK293T (ATCC, USA) was employed for the luciferase reporter analysis. Cells were cultured in Dulbecco's Modified Eagle Media (DMEM, Gibco, ThermoFisher, Waltham, MA, USA) supplemented with 10% FBS (Gibco) and 100 IU/mL penicillin-streptomycin at 37 • C with humidified 5% CO 2 atmosphere. Upon shifting to a 2% horse serum containing medium, C2C12 myoblasts were induced to fusion and differentiation. The medium was changed every day.

Animals
The C57BL/6 male mice were purchased from the Fourth Military Medical University Animal Center (Xi'an, China) and raised in a controlled temperature (25 ± 1 • C) with a 12 h light/12 h dark cycle. Tissues were collected from two-month and six-month old mice. All procedures with mice were in accordance with approved guidelines of the Animal Care and Use Committee of the Northwest A and F University.

Transfections and Treatment of Myoblasts and Myotubes
To test the effects of miR-106a-5p on differentiating cells, myoblasts were seeded in 6-well or 12-well plates and transfected with 50 nM FAM-labeled miR-106a-5p agomir or negative control (GenePharm, Shanghai, China) using Lipo Plus (Sagecreation, Beijing, China) in Opti-EME (Gibco) according to the manufacturer's instructions. Furthermore, 75 nM IGF1 recombinant protein (Sino Biological, Beijing, China), a PI3K-AKT signaling activator, was used to recover the effects of miR-106a-5p.
To test the effects of miR-106a-5p on well-differentiated myotubes (5 days post-differentiation), myotubes were incubated with 50 µM Dexamethasone (DEX) for 36 h, then cells were harvested for further analysis. Furthermore, myotube transfection (50 nM miR-106a-5p agomir or negative control) was performed with Lipo Plus (Sagecreation) according to the manufacturer's instructions, and cells were harvested 36 h after transfection.

Real-Time Quantitative PCR
The total RNA was extracted with Trizol reagent (TakaRa, Ostu, Japan) as recommended by the manufacturer, and the concentration and quality were analyzed by the NanoDrop 2000 (ThermoFisher). Then complementary DNA (cDNA) was synthesized by reverse transcription kit (TakaRa). Real-Time quantitative PCR (RT-qPCR) was performed using Applied Biosystems qPCR instrument (ThermoFisher) and SYBR green PCR Master Mix (Vazyme, Nanjing, China). The expressions of all coding genes were normalized to β-actin, and U6 small RNA was the internal reference when testing the level of miR-106a-5p. miRs quantification was determined by using Bulge-loop TM miRNA qRT-PCR Primer Set (one RT primer and a pair of qPCR primers in each set) specific for miR-106a-5p, designed and synthesized by RiboBio (Guangzhou, China) and other primers which were synthesized by Invitrogen (Shanghai, China). The sequences were shown in Table 1.

Western Blotting Analysis
Cells were harvested in radioimmunoprecipitation assay (RIPA) lysis buffer (Applygen Technologies Inc., Beijing, China) supplemented with protease and phosphatase inhibitor cocktail (Cwbiotech, Jiangsu, China). Protein concentration was determined by BCA Protein Assay Kit (Cwbiotech) and 25 µg proteins per sample were loaded and separated using a 5% stacking gel and a 10% separating gel. Separated proteins were transferred to PVDF membrane (CST, Boston, MA, USA), and then the membrane was blocked in 5% BSA buffer for 2 h at room temperature, and incubated with primary antibodies against MyoD Imaging and quantification of the bands were carried out by Gel Doc XR system (Bio-Rad, Hercules, CA, USA) and Image Lab software (Bio-Rad).

Immunofluorescence Analysis
Differentiated C2C12 myotubes were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and then blocked in 5% BSA for 30 min. Later, myotubes were sequentially incubated with anti-myosin heavy chain monoclonal antibody (1:200, #MAB4470, R and D Systems) overnight and an Alexa Fluor 594-conjugated anti-mouse IgG (1:1000, #SA00006-3, Proteintech, Chicago, IL, USA) for 1 h at 4 • C. Finally, nuclei were stained with DAPI. Images were captured with a fluorescence microscope (Nikon, Tokyo, Japan). The myotubes with 1-3, and > 4 nuclei were counted, respectively. The differentiation index was determined as the percentage of MyHC-positive nuclei among total nuclei, and the myotube fusion index was determined as the distribution of the nucleus number in total myotubes according to a previous report [31].

Statistical Analyses
Values are presented as the mean ± standard error of the mean (SEM), and statistical significance of differences was determined by Student's t-test or one-way analysis of variance (ANOVA) by IBM SPSS Statistics 22.0 (Armonk, NY, USA). A p-value of < 0.05 was considered statistically significant.

The Expression Profiles of miR-106a-5p in Mice
MiR-106a-5p was highly conserved among detected species based on the nucleotide sequences ( Figure 1A). MiR-106a-5p was widely expressed in various tissues in two-month mice, especially in heart and kidney, but a small amount of expression was detected in skeletal muscle ( Figure 1B). Further, a significantly higher expression was found in Extensor Digitorum Longus (EDL) and Soleus muscle (SOL) of six-month old mice than that of two-month ( Figure 1C). Furthermore, the expression of miR-106a-5p gradually decreased during myogenic differentiation in C2C12 cell line ( Figure 1D), while the expression levels of myogenic markers MyoD, MyoG, and MyHC were significantly up-regulated ( Figure 1E-G).

MiR-106a-5p Contributes to C2C12 Myotubes Atrophy by Suppressing PI3K-AKT Signaling Pathway
Expressions of miR-106a-5p, as well as MAFbx and MuRF1 in C2C12 myotubes, were significantly elevated with the treatment of 50 µM DEX ( Figure 4A). DEX significantly reduced the diameter of C2C12 myotubes ( Figure 4B,C), indicating muscle atrophy. In addition, expressions of miR-106a-5p together with MAFbx, not MuRF1 were much higher in tibialis anterior (TA) muscles of six-month mice than that of two-month mice ( Figure 4D).

Discussion
Previous studies reveal a critical requirement for miR-106a at the early mammalian development. MiR-106a is differentially expressed in developing mice embryos and functions to control differentiation of stem cells [32].
In addition, miR-106a is down-regulated in myoblasts differentiation [22,23]. In our study, miR-106a-5p was observed to decrease in a time-dependent manner during myogenic differentiation in C2C12 cells and is relatively lower expressed in adult skeletal muscle. These data indicate that miR-106a-5p might be a negative regulator for myogenesis. However, there was no significant difference in the expression of miR-106a-5p in fast EDL and slow SOL muscle, suggesting miR-106a-5p might have limited effects on muscle fiber type transition.
Given that miR-106a-5p was down-regulated in C2C12 myogenic differentiation, miR-106a-5p agomir was used to explore the effects of miR-106a-5p on myogenesis. Here, transfection of miR-106a-5p agomir significantly reduced the differentiation index, fusion index as well as the expression levels of myogenic markers, suggesting miR-106a-5p could dramatically repress myogenic differentiation. Meanwhile, enforced expression of miR-106a-5p significantly reduced the level of p-AKT (ser473) in C2C12 myoblasts. It has been well documented that PI3K/AKT is a crucial signaling pathway to promote myogenesis and induce muscle hypertrophy [33,34]. IGF1 is a stimulator of PI3K/AKT signaling during muscle differentiation [35]. In our study, administration of 75 nM IGF1 recombinant protein totally reversed the inhibitory effects of miR-106a-5p. Taken together, miR-106a-5p might interrupt C2C12 myoblasts differentiation by disrupting AKT activity.
Furthermore, miR-106a-5p is previously reported to be upregulated in limb-girdle muscular dystrophies types 2A and 2B and is involved in muscular disorders [36]. In the present study, miR-106a-5p accompanied with atrophic factors were up-regulated in aged muscles and DEX-treated myotubes, and overexpression of miR-106a-5p was sufficient to reduce the diameters of well-differentiated myotubes in vitro, indicating miR-106a-5p might be involved in muscle atrophy. During miR-106a-5p-induced muscle atrophy, decreased phosphorylated AKT (ser473) was observed. Similarly, miR-18a, a member of miR-17-92 cluster, is demonstrated to decrease during myogenic differentiation and promotes muscle atrophy by targeting IGF1 [37]. Collectively, our data indicate that miR-106a-5p might induce myotube atrophy through blocking the AKT signaling pathway.
In our study, pan-PI3K (p85α) protein levels were repressed by enforced miR-106a-5p agomir in both differentiating and well-differentiated myotubes. PI3K (p85α), encoded by PIK3R1 gene, is a regulatory subunit of PI3Ks and essential for myoblasts proliferation and differentiation [24,38]. Germline deletion of the PIK3R1 gene leads to impaired muscle growth, and a significant reduction in muscle weight and fiber size [39]. Moreover, PI3KR1 was identified as the target of miR-128a [40] and miR-29b in muscle cells [41]. Here, the luciferase reporter assay showed that miR-106a-5p directly bound to the PIK3R1 3 UTR, and PI3KR1 is a novel target of miR-106a-5p in C2C12 cells.
In conclusion, miR-106a-5p is identified as a novel repressor of myogenesis, and miR-106a-5p represses differentiation and promotes atrophy by blocking the PI3K-AKT signaling pathway through targeting PIK3R1.