lncRNA-Six1 Is a Target of miR-1611 That Functions as a ceRNA to Regulate Six1 Protein Expression and Fiber Type Switching in Chicken Myogenesis

Emerging studies indicate important roles for non-coding RNAs (ncRNAs) as essential regulators in myogenesis, but relatively less is known about their function. In our previous study, we found that lncRNA-Six1 can regulate Six1 in cis to participate in myogenesis. Here, we studied a microRNA (miRNA) that is specifically expressed in chickens (miR-1611). Interestingly, miR-1611 was found to contain potential binding sites for both lncRNA-Six1 and Six1, and it can interact with lncRNA-Six1 to regulate Six1 expression. Overexpression of miR-1611 represses the proliferation and differentiation of myoblasts. Moreover, miR-1611 is highly expressed in slow-twitch fibers, and it drives the transformation of fast-twitch muscle fibers to slow-twitch muscle fibers. Together, these data demonstrate that miR-1611 can mediate the regulation of Six1 by lncRNA-Six1, thereby affecting proliferation and differentiation of myoblasts and transformation of muscle fiber types.


Introduction
Since the implementation of the Human Genome Project, scientists have been trying to explore the biological significances of genomic nucleic acid sequences. Protein coding genes only account for a small portion (1.5%) of the genome, while more than 85% of the genome is transcribed to non-coding RNA (ncRNA) [1,2]. Myogenesis is a process including myoblast proliferation, differentiation and myotube formation and is controlled by a series of myogenic regulatory factors. These factors can regulate myoblasts to withdraw from the cell cycle, express muscle-specific genes, and prevent the expression of other cell-or tissue-specific genes. The muscle growth of chickens is a complex quantitative characteristic which is directly regulated by major genes. However, recent studies have found that ncRNA also plays an important role [3][4][5][6].
All cells were incubated at 37 • C with an atmosphere of 5% CO 2 .
2.5. RNA Extraction, cDNA Synthesis and Quantitative Real-Time PCR (qRT-PCR) Total RNA was extracted from tissues or cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) as recommended by the supplier. The integrity and quantity of RNA were assessed using 1.5% agarose gel electrophoresis and a spectrophotometer (ND-2000, NanoDrop Inc., Wilmington, DE, USA).
A real-time qPCR with iTaq Universal SYBR Green Supermix Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used, and analysis was performed with the 2 −∆∆Ct method, as described previously [29]. Chicken β-actin and U6 were used as internal controls. Primers for RT-qPCR are shown in Table S1.

RNA Oligonucleotide and Plasmid Construction
Gga-miR-1611 mimics and mimic negative control (NC), gga-miR-1611 inhibitor, inhibitor NC, and small interfering RNAs (siRNAs) used for the knockdown of Six1 were designed and synthesized by RiboBio (Guangzhou, China). The siRNA and antisense oligonucleotide (ASO) that were used for the specific knockdown of lncRNA-Six1 in the cytoplasm and nucleus, respectively, and non-specific siRNA NC duplex, were also designed and synthesized. Oligonucleotide sequences in this study are shown in Table S2. lncRNA-Six1 and Six1 overexpression plasmids were constructed as in our previous study [28]. For the pmirGLO dual-luciferase miRNA target reporter vector, the segment sequence of lncRNA-Six1 and Six1 3 untranslated region (UTR) that contained the putative miR-1611 binding sequence was amplified by PCR, and then subcloned into XhoI and SalI restriction sites in the pmirGLO dual luciferase reporter vector (Promega, Madison, WI, USA). Mutant plasmids were generated by changing the binding site of miR-1611 from AGCCCTC to GATTTCT. The major primers used in this study are listed in Table S3.

Dual-Luciferase Reporter Assay
Cells were seeded in a 96-well plate. After being co-transfected, (a) wild-type or mutant-type lncRNA-Six1 position 1 dual-luciferase reporter, (b) wild-type or mutant-type lncRNA-Six1 position 2 dual-luciferase reporter, and (c) wild-type or mutant-type Six1-3 UTR dual-luciferase reporter, with miR-1611 mimic or mimic NC, the firefly and Renilla luciferase activities were measured at 48 h post-transfection using a Dual-GLO Luciferase Assay System Kit (Promega, USA), following the manufacturer's instructions. The luminescent signal was quantified using a Fluorescence/Multi-Detection Microplate Reader (BioTek, Winooski, VT, USA), and firefly luciferase activities were normalized to Renilla luminescence in each well.

Flow Cytometric Analysis of the Cell Cycle
After transfection for 48 h, the cultured cells in growth media were collected and fixed overnight in 70% ethanol at −20 • C. Subsequently, the fixed cells were stained with propidium iodide (Sigma, Louis, MO, USA) (50 µg/mL), and then incubated at 37 • C in the dark for 30 min. Flow cytometry analysis was performed on a BD AccuriC6 flow cytometer (BD Biosciences, San Jose, CA, USA), and the data was processed using FlowJo software (7.6, Treestar Incorporated, Ashland, OR, USA).

5-Ethynyl-2'-Deoxyuridine (EdU) Assay
The primary myoblasts were exposed to 50 µM EdU (RiboBio, China) for 2 h at 37 • C after 48 h transfection. Subsequently, the cells were fixed in 4% paraformaldehyde for 30 min and neutralized using 2 mg/mL glycine solution, and then permeabilized by adding 0.5% Triton X-100. A solution containing EdU (Apollo Reaction Cocktail; RiboBio, China) was added, and the cells were incubated at room temperature for 30 min. The nuclear stain Hoechst 33,342 was then added, and incubation was continued for another 30 min. A fluorescence microscope (DMi8; Leica, German) was used to capture three randomly selected fields to visualize the number of EdU-stained cells.

CCK-8 Assay
Primary myoblasts were seeded in a 96-well plate and cultured in growth medium. After being transfected, cell proliferation was monitored using a TransDetect CCK (TransGen Biotech, Beijing, China), according to the manufacturer's protocol. Absorbance was measured using a Model 680 Microplate Reader (Bio-Rad, Hercules, CA, USA) by optical density at a wavelength of 450 nm.

Statistical Analysis
All results are presented as the mean ± S.E.M, based on at least three independent experiments for each treatment. Statistical significance was determined by one-sample or paired t-tests.

miR-1611 Regulates the Proliferation and Differentiation of Myoblasts, and Is Involved in the Formation of Skeletal Muscle Fibers
In order to unveil the functions of miR-1611, we performed overexpression and inhibition experiments to assess its role in myoblast proliferation and differentiation. After 48 h of transfection with a miR-1611 mimic, the relative expressions of miR-1611 were detected ( Figure 1A). Overexpression of miR-1611 led to a significant increase of the number of cells that progressed to G0/G1, and that reduced the number of S phase cells ( Figure 1B). Conversely, miR-1611 inhibition resulted in a smaller number of G0/G1, and increased the number S phase cells ( Figure 1B). Furthermore, using EdU and CCK-8 assays, we found that miR-1611 overexpression significantly repressed myoblast proliferation ( Figure 1C-E). On the contrary, its loss of function induces cell division ( Figure 1C,D,F). To further investigate the potential roles of miR-1611 in differentiation, we detected the relative expressions of miR-1611 after inducing the CPMs to differentiate in vitro ( Figure 1G). The expression level of miR-1611 showed a gradual decrease; hence, it is suggested that miR-1611 may play an important role in the process of myoblast differentiation. In the meantime, we also detected the expressions of myoblast differentiation marker genes, such as MYOD, MYOG, and MyHC, after overexpression or inhibition of miR-1611 ( Figure 1H). miR-1611 overexpression suppressed muscle differentiation marker gene expression, whereas the expressions of these genes were significantly induced after miR-1611 inhibition. Moreover, immunofluorescence staining also showed that the overexpression of miR-1611 could promote myoblast differentiation and induce myotube formation ( Figure 1I,J). On the contrary, the total area of myotube was significantly increased with the inhibition of miR-1611 ( Figure 1I,J).  In poultry, breast muscle is generally considered to be composed of fast-twitch muscle fibers with a larger muscle fiber diameter, while the leg muscles are mainly composed of slow-twitch muscle fibers with a smaller muscle fiber diameter [30][31][32]. Using H&E staining, we found that breast muscle has a larger area and diameter than leg muscle ( Figure S1A-C). Moreover, we also evaluated some of specific myosin heavy chain isoforms (for example, MYH1A for fast-twitch muscle fibers and MYH7B for slow-twitch muscle fibers) by immunohistofluoresence staining and Western blot ( Figure S1D-F). The result showed that MYH1A is highly expressed in breast muscle, while the expression of MYH7B is higher in leg muscle.
Tissue expression profiles for miR-1611 showed a differential expression between breast muscle and leg muscle, leading to speculation that miR-1611 may function in the formation of skeletal muscle fiber (Figure 2A). To further investigate the role of miR-1611 during the transformation of skeletal muscle fiber types, we detected the expression of a series of fast muscle genes (including Wnt4, Tnnc2, Tnnt3, and Srl) and a series of slow muscle genes (including Sox6, Tnnc1, Tnni1, and Tnnt1) ( Figure 2B,C). Overexpression of miR-1611 downregulated the expression of fast muscle genes and promoted slow muscle gene expression. Inhibition of its function drove the transformation of slow-twitch muscle fibers to fast-twitch muscle fibers. In all panels, the results are shown as mean ± SEM, and the data are representative of three independent assays. Statistical significances of differences between means were assessed using an unpaired Student's t-test. (* p < 0.05; ** p < 0.01) vs. NC, negative control.

miR-1611 Is a Direct Target of the ceRNA Network between lncRNA-Six1 and Six1
To further investigate the molecular mechanism of how miR-1611 exerts its biological effect, we predicted its target lncRNAs and genes on an RNAhybrid (https://bibiserv.cebitec.uni-bielefeld. de/rnahybrid) [33]. Interestingly, miR-1611 was found to contain potential binding sites for both lncRNA-Six1 and Six1, implying a new mechanism by which lncRNA-Six1 regulates Six1 ( Figure 3A). Dual-luciferase reporter assays were carried out to confirm whether miR-1611 can directly interact with lncRNA-Six1 and Six1 ( Figure 3B-D). The results showed that miR-1611 could perfectly bind with and target to interact with both lncRNA-Six1 position 1 and the 3 UTR of Six1. More importantly, overexpression of miR-1611 significantly decreased the expression level of lncRNA-Six1 and Six1, whereas the expression of lncRNA-Six1 and Six1 were upregulated with the inhibition of miR-1611 ( Figure 3E,F). In addition, the effects of miR-1611 on myoblast proliferation and differentiation, and the formation of skeletal muscle fibers, were negated with the co-overexpression with lncRNA-Six1 or Six1 (Figure 3G-J). Taken together, these results demonstrated that lncRNA-Six1 can function as a ceRNA for miR-1611 to regulate Six1.   Figure 4B). Meanwhile, lncRNA-Six1 overexpression resulted in a decrease in G0/G1 phase cells, and an augmented number of cells that progressed to the S phase ( Figure 4C), as well as significantly promoting myoblast proliferation ( Figure 4D-F). However, G0/G1 cells increased, while S phase cells decreased significantly ( Figure 4C), and myoblast proliferation was significantly suppressed with lncRNA-Six1 interference (Figure 4D,E,G). In addition, the expression of lncRNA-Six1 was upregulated after the induction of CPM differentiation in vitro ( Figure 4H). The transfection of pSDS-lncRNA-Six1 induced the expression of the myoblast differentiation marker gene, while the interference of lncRNA-Six1 downregulated its expression ( Figure 4I). Immunocytofluorescence staining showed that lncRNA-Six1 overexpression facilitated myoblast differentiation, which was associated with increased total areas of myotubes ( Figure 4J). In contrast, the differentiation of myoblasts was significantly restrained with lncRNA-Six1 knockdown ( Figure 4K).
To evaluate the role of lncRNA-Six1 in the muscle fiber phenotype, we assayed the expressions of fast and slow muscle genes after lncRNA-Six1 overexpression or knockdown ( Figure 5A,B). The expressions of multiple fast-type muscle genes were significantly promoted by lncRNA-Six1 overexpression, while the interference of lncRNA-Six1 activated the slow muscle program.  In all panels, data are presented as means ± S.E.M. of three independent assays. Statistical significance of differences between the means was assessed using an unpaired Student's t-test. (* p < 0.05; ** p < 0.01) vs. NC, negative control.

Six1 Is a Critical Factor in Myogenesis, Participating in the Transformation of Skeletal Muscle Fiber Types
To determine the function of Six1 in myogenesis, we constructed a Six1 overexpression vector and synthesized the specific siRNA ( Figure 6A). Six1 overexpression led to a decrease in the number of G0/G1 cells, and increased the number of cells in the S phase ( Figure 6B), as well as significantly facilitated myoblast proliferation as judged by EdU incorporation and CCK-8 assay ( Figure 6C-E). Inversely, the opposite results were shown with Six1 knockdown (Figure 6B-D,F). During myoblast differentiation, the expression of Six1 was significantly upregulated ( Figure 6G). At the same time, the expression of myoblast differentiation marker genes was found to be significantly promoted after the overexpression of Six1, whereas the interference of Six1 restrained its expression ( Figure 6H). Furthermore, Six1 overexpression significantly promoted the formation of myotubes and increased the size of the myotube area ( Figure 6I,J). In contrast, Six1 interference suppressed myoblast differentiation ( Figure 6I,J).  In a previous study, we identified the tissue expression profile of Six1 by qRT-PCR [28]. Compared with leg muscles, we also found by immunohistofluorescence that Six1 is expressed more in breast muscle ( Figure 7A,B). Overexpression of Six1 significantly upregulated the fast muscle genes, and downregulated the slow muscle genes, while the inverse results were observed with the knockdown of Six1 ( Figure 7C,D). Taken together, we argue that Six1 plays a critical role, not only in myoblast development, but also in the transformation of muscle fibers from one type to the other in skeletal muscle development.

Discussion
The present study reveals a role of miR-1611 in the proliferation and differentiation of myoblasts, as well as the transformation of skeletal muscle fiber types. Our findings are partly based on the function of miR-1611 in directly interacting with lncRNA-Six1 and Six1 (Figure 8). miRNAs are well-known to function as important regulators and mediators in myogenesis. In chickens, many miRNAs were found to widely participate in muscle developmental processes in several different ways [5,[10][11][12]34]. In this study, we first analyzed the function of miR-1611 in myogenesis, which is specifically expressed in chickens. Overexpression of miR-1611 repressed the proliferation and differentiation of myoblasts. In addition, miR-1611 is highly expressed in slow-twitch fibers, and it could active the slow muscle program.
Recently, a new pattern of gene expression has been put forward regarding the interaction of RNA transcripts, referred to as ceRNAs [23]. There is evidence that lncRNAs can function as ceRNAs to protect mRNAs by acting as molecular sponges for miRNAs, thereby modulating the de-repression of miRNA targets and imposing an additional level of posttranscriptional regulation [35][36][37].
In the current study, using in silico analysis, we identified that miR-1611 contains binding sites for lncRNA-Six1 and Six1. A dual-luciferase reporter assay validated the direct binding of the miR-1611 response elements on lncRNA-Six1 transcript and Six1 3 UTR. Moreover, our rescue experiment showed that the inhibition effect of miR-1611 in regulation of Six1 could be neutralized when lncRNA-Six1 is overexpressed. These data indicated that miR-1611 is a crucial mediator for lncRNA-Six1 to function as a ceRNA, thus regulating Six1.
It is worth noting that lncRNAs have also been demonstrated to function during muscle growth [38,39]. For instance, the noncoding RNA steroid receptor RNA activator (SRA) was reported to be an enhancer of the myogenic differentiation and myogenic conversion of non-muscle cells, through the co-activation of MyoD activity [40,41]. Moreover, lnc-MD1 [24], Yam-1 [42], H19 [25,35,43], and Malat1 [26] are also believed to be important regulators of myogenesis. In our study, we identified that lncRNA-Six1 could regulate the expression of Six1 by acting as a ceRNA. lncRNA-Six1 overexpression significantly promoted the proliferation of myoblasts, and this induced myoblast differentiation. At the same time, lncRNA-Six1 is involved in the formation of skeletal muscle fibers, and it can promote the conversion of the muscle fiber phenotype.
Six1 is an evolutionarily, highly conserved transcription factor that is widely involved in skeletal muscle development [44][45][46]. Recent research has shown that Six1 determined proliferation and differentiation of the muscle lineage by targeting the myogenic regulatory factor family [44]. Moreover, Six1 was also reported to be able to drive the transformation of slow-twitch fibers to the fast-twitch phenotype in skeletal muscle [44,47]. In the current study, we found Six1 facilitated myoblast proliferation and differentiation. Furthermore, overexpression of Six1 could upregulate fast muscle genes, downregulate slow muscle genes, and induce the transformation of slow-twitch muscle fibers to fast-twitch muscle fibers.
Altogether, the results of our study demonstrate that miR-1611 can act as a regulator to mediate the regulation of Six1 by lncRNA-Six1, thus affecting myoblast proliferation and differentiation, as well as being involved in the transformation of skeletal muscle fiber types.