Functional analysis of miRNA-146b during myotube differentiation in chicken myoblasts

In poultry industry as well as livestock, the precise genetic information is being required for improving the economic traits. Thus, functional genomic studies are widely conducted to build the genetic and genomic information for faster, healthier and more-efficient animal production. Especially, chicken myoblast cells which are required to muscle development and regeneration are important because chicken growth performance is closely related to muscle mass. In this study, we induced expression of microRNA-146b (miR-146b) mediated by piggyBac transposon system in chicken myoblast (pCM cells). Subsequently, we analyzed and compared the proliferation and differentiation capacity, and also examined the expression patterns of related genes between regular pCM (rpCM) cells and miR-146b overexpressing pCM (pCM-146b OE) cells. The overexpression of miR-146b showed that the increased proliferation and up-regulated gene expression related to cell proliferation. In addition, the next generation sequencing (NGS) analysis were performed to compare the global gene expression patterns between rpCM cells and pCM-146b OE cells. We found that the higher proliferation rate of pCM-146b OE cells resulted from up-regulation of the cell cycle related gene sets. Moreover, miR-146b overexpression indicated that inhibitory effect of myotube differentiation in chicken myoblast cells. Collectively, these results demonstrated that miR-146b is closely related to proliferation and differentiation of chicken myogenic cells as a modulator of post-transcription.


Introduction
Since the whole-genome sequencing information of avian species were revealed, the numerous suggestions such as increasing muscle mass, enhancing muscle regeneration capability and reducing fatty acid accumulation have been proposed to improve growth performance. Especially, exploring the useful genes or genetic markers is important to understand the biological function(s) and regulatory pathway(s) to determine the economically important traits in poultry industry  Mineno et al. 2006). In addition, some miRNAs were reported to control myogenesis process in mammals (Kim et al. 2006, Chen et al. 2010). miRNA-146b (miR-146b) is well-conserved in most vertebrates and has many biological functions such as innate immunity, inflammation and cell senescence (Taganov et al. 2006;Bhaumik et al. 2009;Perry et al. 2009

Materials and Methods
Primary chicken myoblast (pCM) cell culture and induction of myotube differentiation Primary chicken myoblast (pCM) cells were isolated from pectoralis major of 10-dayold male chick embryos and maintained in Medium 199 (Invitrogen), supplemented with 10% fetal bovine serum (FBS; HyClone), 2% chicken serum (Sigma-Aldrich) and 1 × antibiotic-antimycotic (Invitrogen) ). These cells were cultured in an incubator 37℃ in an atmosphere of 5% CO 2 and 60-70% relative humidity. To induce myotube differentiation at 80% confluency of cells, after washed one time by using PBS, the differentiation medium containing 0.5% FBS and 1 × antibioticantimycotic was changed. The differentiation medium was replaced with fresh differentiation medium daily.
The cytomegalovirus (CMV) and elongation factor-1 (EF-1) promoter controlled the expression of GFP-miRNA-146b and puromycin resistance gene, respectively ( Fig. 1A). The miRNA-146b were synthesized as 5'-gct ggt gac gtc ccc tat gga att gag ttc tcc gct gtg aca ctt caa act gag aac tga att cca tag gcg atg tgg tca gca − 3' Quantitative RT-PCR analysis Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Total RNA was quantified using a NanoDrop 2000 (Thermo Scientific), and 2ug RNA were used for cDNA synthesis using random primers (Invitrogen) under standard conditions. Quantitative RT-PCR for miRNA was conducted by using High-specificity miRNA QPCR Core Reagent Kit (Agilent Technology, Santa Clara, CA, USA). Each 20 ul RT-PCR reaction mix contained 2 ul cDNA, 2.5 ul PCR buffer, 1 ul dNTP mixture (2.5 mM), 1unit Taq DNA polymerase, and 10 pmol forward and reverse primer (Table 1). Quantitative RT-PCR analysis was performed using the iCycler iQ Real-time PCR detection system (Bio-Rad) and EvaGreen (Biotium, Fremont, CA, USA). The PCR parameters were as follows: an initial incubation at 94℃ for 5 min, followed by 40 cycles at each condition ( Table 1). The reaction was terminated by a final incubation at 72℃ for 10 min, and melting curve profiles were analyzed for the amplicons.

Statistical analysis
Statistical analysis was conducted using the SAS version 9.4 software (SAS Institute, Cary, USA). The significance of differences was analyzed using a general linear model procedure and the differences among groups were deemed to be significant when p < 0.05.

miR-146b overexpression in chicken myoblast cells
Based on our previous report of miRNA expression system (Lee et al. 2019), we designed and constructed piggyBac transposon-mediated miR-146b overexpression vector (piggyBac CMV-GFP-miRNA-146b, Fig. 1A). Two copies of miR-146b were simultaneously transcribed with GFP transgene under CMV promoter (Fig. 1A). Thus, this miRNA expression cassette system could be efficiently utilized not only for overexpression of the targeted miRNA but also for GFP visualization in the transfected cells. Both of the stable transgene-expressing cells, regular pCM (rpCM) and miR-146b overexpression cells (pCM-146b OE cells) showed no difference of morphological features (Fig. 1B). Quantitative RT-PCR (qRT-PCR) was conducted to determine the overexpression of miR-146b in pCM-146b OE cells. The expression level of miR-146b was significantly up-regulated in pCM-146b OE cells compared to that of regular pCM (Fig. 1C).

Characterization of pCM-146b OE cells in undifferentiated state
To examine gene expression patterns of the myogenic markers and targets of miR- Overexpression of miR-146b improved the proliferation of chicken myoblast cells Intriguingly, pCM-146b OE cells showed the higher proliferative growth rate. To exclude influence of GFP which was inserted in miR-146b overexpression vectors, the proliferative analysis was compared between pCM-GFP cells and pCM-146b OE cells. The result showed that pCM-146b OE cells also have higher growth rate than that of pCM-GFP cells 3 days after in vitro culture (Fig. 3A). Furthermore, we analysis cell proliferation-related genes (CCND3, IRF2, WNT5A and PDGFRB) by qRT-PCR. All of transcripts which were reported as positive regular of proliferation were up-regulated in pCM-146b OE cells (Fig. 3B). These results suggested that miR-146b has an effect on the skeletal muscle proliferation in chicken myoblast cells.  Table 2). Subsequently, these gene sets were compared by Heatmap visualization to examine the different expression patterns of gene sets between in between regular pCM and pCM-146b OE cells (Fig. 4C). To validate the DEGs from mRNA sequencing analysis, we selected and analyzed the gene expression patterns of six up-regulated genes (CCNB2, CDC20, KIF23, KPNA2, PLK1 and TOP2A) particularly related to cell cycle regulation.
All of six cell cycle regulation related transcripts were highly up-regulated in pCM-146b OE cells (Fig. 5A). To understand the functional interactions of the upregulated genes and their neighbor genes, we applied STRING analysis to the interactions between these genes (Fig. 5B). These results supported that the effect of miR-146b on the skeletal muscle proliferation and also it influenced the regulatory pathways of cell cycling in chicken myoblast cells. Overexpression of miR-146b influences myotube differentiation in chicken myoblast cells Subsequently, we compared and analyzed the myotube differentiation capacity between regular pCM and pCM-146b OE cells. Overexpression of miR-146b dramatically reduced the myotube differentiation and formation during myogenic process in pCM-146b OE cells (Fig. 6). pCM-146b OE cells showed lower number of the differentiated myotubes and less myotube differentiation formation compared to regular pCM cells (Fig. 6A). The area of differentiated myotubes was significantly decreased in pCM-146b OE cells after 4 days of myogenic induction (Fig. 6B).
Western blotting results after the myogenic differentiation similarly showed the expression patterns in the undifferentiated stage (Fig. 7A). The expression of Pax7 was still down-regulated while the expression of MyoD was up-regulated in pCM-146b OE cells. Furthermore, Desmin, a myogenic differentiation terminal marker, was also down-regulated in pCM-146b OE cells. Additionally, we investigated expression of ID1 which is closely associated with muscle differentiation by binding E proteins (Fig. 7B).  . 6). Similarly, the expression of desmin which is a myotube terminal differentiation marker was down-regulated in pCM-146b OE cells. Moreover, the expression of ID1 protein which is an inhibitor of myogenic differentiation in muscle was up-regulated in pCM-146b OE cells (Fig. 7). ID1 protein competitively suppresses E protein/MyoD complex because it has more high affinity for the E-

Conclusion
In this study, we produced the miR-146b overexpression chick myoblast cells and conducted a functional assay during myogenic proliferation and differentiation.
Comparing with the regular pCM cells, pCM-146b OE cells show that higher proliferation rates and lower differentiation rates. pCM-146b OE cells have higher expression of cell proliferation related genes and cell cycle related genes.
Especially, increasing of myogenic proliferation suggested that miR-146b could enhance the cell proliferation and inhibit myogenic differentiation by regulating the expression of PDGFRB. Furthermore, pCM-146b OE cells demonstrated higher expression of the ID1, it assumed that miR-146b could be indirectly control the myogenic differentiation by regulating the expression of ID1. These results suggest that miR-146b acts as key regulator of myogenic proliferation and differentiation in chicken.
Declarations Figure 1 Design of chicken miRNA 146b expression vector and characterization of miR-146b overexpre Protein expression analysis during differentiation and ID1 expression analysis (A) Protein exp