LncRNA 2310043L19Rik inhibits differentiation and promotes proliferation of myoblast by sponging miR-125a-5p.

Although many long non-coding RNAs (lncRNAs) have been identified in muscle, some of their physiological functions and regulatory mechanisms remain elusive. Here we report the functional identification and characterization of a novel lncRNA 2310043L19Rik (lnc-231), which is highly expressed in muscle. The expression level of lnc-231 in skeletal muscle of young mice is higher than that in aged mice. Functional analysis showed that overexpression of lnc-231 restrained differentiation and promoted proliferation of myoblast, while inhibition of lnc-231 revealed completely opposite effects in vitro. RNA molecules of lnc-231 acted mechanistically as competing endogenous RNAs (ceRNA) to target miR-125a-5p, whereas miR-125a-5p binds to the 3’-UTR of E2F3 mRNA to inhibit its function. Collectively, lncRNA 2310043L19Rik promotes proliferation and inhibits differentiation of myoblast cells by attenuating the function of miR-125a-5p.


INTRODUCTION
Long non-coding RNAs are a class of RNAs longer than 200 nt with lower protein coding potential. They are located in the intergenic region that does not overlap with the annotated coding genes. LncRNA was originally proposed in the study of universal transcription of unknown coding regions by chip [1]. In the early 1990s, LincRNA Xist was found to be inactivated on the X chromosome (XCI) [2][3][4]. and it can inhibit the transcription of HOTAIR HOX family gene [5].
Skeletal muscle, the most plentiful tissue in domestic animals, is required for maintaining body movement and provides meat products to humans. The different regulatory mechanisms during the growth and development of skeletal muscle cause differences in muscle yield and meat quality. The growth and development of skeletal muscle is a complex process involving the proliferation, migration, differentiation, and the fusion of muscle stem cells, as well as the formation of muscle fibers [6]. Adult muscles have a strong adaptation capacity, enabling functional switches in response to altered conditions. For instance, adult skeletal muscles will change and adjust their functions during aging [7]. Deletion of small ankyrin 1 (sAnk1) isoforms results in structural and functional alterations in aging skeletal muscle fibers [8]. Deletion of pofut1 in mouse skeletal myofibers induces muscle aging-related phenotypes [9]. Moreover, there is a close correlationship between lncRNA expression and aging [10].
Studies of lncRNA functions have been carried out extensively in specific cell types, developmental stages, and diseases [11]. It has been demonstrated that lincRNAs play important roles in skeletal muscle AGING growth and development, muscle cell proliferation, migration, differentiation, and apoptosis [12].
Mammalian E2F, a family of transcription factors, regulates cell cycle progression through cooperating with other cell cycle regulatory genes [30]. E2F3 has been demonstrated to be essential for cardiac development and function [31]. The loss of E2F3 impaired the proliferation of vascular smooth muscle cells [32], suggesting that E2F3 plays a promoting role during the proliferation of myoblasts [33].
To study the role of lncRNA 2310043L19Rik in myogenesis, we induced differentiation of C2C12 in vitro to find differentially and upregulated lncRNA. The results showed that Lnc-231 was highly expressed in skeletal muscle. Lnc-231 inhibited myoblast differentiation and promote its proliferation via reducing the abundance of miR-125a-5p, which in turn restored the translation level of gene E2F3, and thereby promoting the proliferation of C2C12.

Expression pattern of LncRNA 2310043L19Rik
We found the differentially expressed lncRNA 2310043L19Rik (lnc-231) from previous sequencing results [17]. Interestingly, tissue expression profiles showed that lnc-231 was highly expressed in the skeletal muscle ( Figure 1A). This reminded us that lnc-231 may involve in the development of skeletal muscle. Inducing myoblast into myotube is an effective model in exploring myogenesis. To explore the role of lnc-231 in myogenesis, C2C12 differentiation was successfully induced in vitro, as suggested by a significant elevation of MyoG expression ( Figure 1B). Meanwhile, the expression of lnc-231 was significantly increased during differentiation ( Figure 1C). In addition, qRT-PCR results showed that Atrogin-1, a marker of muscle atrophy, expressed higher in skeletal muscle of aged mice than that in the adults' ( Figure 1D). In contrast, the expression of lnc-231 in aged skeletal muscle is lower than that in adult mice ( Figure 1E). Interestingly, these data suggested that the expression level of Atrogin-1 and lnc-231was negatively correlated during myogenesis.

LncRNA 2310043L19Rik regulates myoblast differentiation
To further explore the function of lnc-231 on myoblasts, we constructed an expressing vector encoding lnc 231 ( Figure 2A). Overexpression of lnc-231 inhibited the expression of MyHC and MyoG ( Figure 2B, 2C, 2E, 2F). In addition, the lnc-231 expressing vector significantly reduced MyHC mRNA ( Figure 2D) and protein levels ( Figure 2G). Moreover, lnc-231 overexpression inhibited the transition of myoblasts into the multi-nuclear myotubes and suppressed their differentiation. To further investigate the role of lnc-231, we designed three siRNAs against lnc-231. Knock down analysis showed that approximate 80% of lnc-231 was silenced upon siRNAs treatment ( Figure 3A), which was followed by an elevation of MyHC positive cell rates ( Figure 3B, 3C), MyHC mRNA/protein level, and MyoG protein level ( Figure 3D-3F).

LncRNA 2310043L19Rik regulates G0/G1 to S transition and proliferation of myoblasts
In the propidium iodide flow cytometry assays, overexpression of lnc-231 significantly increased the S phase cell ratio ( Figure 4A, 4B). On the contrary, the ratio of cells in the S phase decreased by knocking down lnc-231 ( Figure 4C, 4D). These data indicated that lnc-231 might play a positive role in regulating the G0/G1 to S transition of C2C12 cell cycle. Furthermore, overexpression of lnc-231 elevated the rate of EdU   AGING positive cells ( Figure 5A, 5B), while knockdown of lnc-231 caused a decrease in the rate of EdU-positive cells ( Figure 5C, 5D). These results suggested that lnc-231 might stimulate proliferation of myoblast. In addition, we detected the expression of the key proliferation marker gene Ki67 and cyclin-dependent kinases (CDK2, CDK4, and CDK6). qRT-PCR results showed increased mRNA levels of Ki67, CDK2, CDK4, and CDK6 in cells with lnc-231 overexpression ( Figure 5E).
In contrast, mRNA levels of Ki67, CDK2, CDK6 were reduced after silencing lnc-231 ( Figure 5F). Western blot results showed overexpression of lnc-231 raised protein levels of CDK2 and CDK6 ( Figure 5G), but suppressed expression of CDK2 and CDK6. In summary, these findings suggested that lnc-231 might promote cell proliferation by inducing cell cycle progression from G0/G1 to S through upregulating the expression of cell cycle-dependent kinases (CDK2/4/6). AGING LncRNA 2310043L19Rik regulates the expression of E2F3 by sponging miR-125a-5p To further explore the regulatory mechanism of lnc-231, we performed a nuclear separation experiment and found that cytoplasmic expression of lnc-231 was higher than that in the nucleus ( Figure 6A). This suggests that lnc-231 may act as a ceRNA by sponging miRNAs in the cytoplasm. Using bioinformatics analysis, we predicted miRNAs that bind to lnc-231, and identified miR-125a-5p as a candidate miRNA. To further verify whether lnc-231 is targeted by miR-125a-5p, we constructed a luciferase reporter vector ligating with lnc-231-wt and lnc-231-mut ( Figure 6B). The results showed that the luciferase activity was significantly decreased in the lnc-231-WT and miR-125a-5p co-transfected groups ( Figure 6C, showing that miR-125a-5p binds to lnc-231-WT. It has been reported that the E2F3 is a target gene of miR-125a-5p, which can inhibit the protein level of E2F3 and inhibit the proliferation of myoblasts [34]. Our results showed that the alterations of miR-125a-5p abundance affected either by overexpressing or knocking down lnc-231 ( Figure 7A, Figure 7E) had no effect on the expression of E2F3 mRNA ( Figure 7B, 7F). However, overexpression of lnc-231 improved the protein level of E2F3 ( Figure 7C, Figure 7D), while lnc-231 silencing suppressed the protein level of E2F3 ( Figure 7G, 7H). To assess whether lnc-231 might regulate the protein level of E2F3 via sponging miR-125a-5p, we designed a co-transfection assay. The results showed ( Figure 7I, 7J) that overexpression of miR-125a-5p inhibited E2F3 protein level and the decreased E2F3 protein level could be rescued by co-overexpression of lnc-231. AGING LncRNA 2310043L19Rik adsorbs miR-125a-5p to promote myoblast proliferation The results of EdU assay showed ( Figure 8A) that overexpression of miR-125a-5p significantly reduced the rate of EdU-positive cells ( Figure 8B). Compared with miR-125a-5p overexpression alone, co-overexpression of lnc-231 and miR-125a-5p elevated the rate of EdU positive cells. The rate of EdU-positive cells in lnc-231 overexpressed cells was higher than that cooverexpressed with lnc-231 plus miR-125a-5p. Flow cytometry results showed that overexpression of lnc-231 increased the percentage of S phase cells ( Figure 8C,  8D). Overexpression of miR-125a-5p arrested cells in the G0/G1 phase and reduced the population in the S phase, while Co-overexpression of lnc-231 and miR-125a-5p increased the fraction of S phase cells.

DISCUSSION
In this study, we demonstrated that a long non-coding RNA 2310043L19Rik(lnc-231) is highly expressed in skeletal muscle, promotes proliferation and inhibits myoblast differentiation. Lnc-231 acts as a ceRNA to sponge miR-125a-5p and negatively regulates the abundance of miR-125a-5p in myoblasts, resulting in the restoration of protein expression of its target gene E2F3, and thereby promoting the cell cycle progression of muscle cells by inducing G0/G1 to S transition ( Figure 9).
It has been reported that lnc-231 is significantly elevated during muscle cell differentiation. [17]. This is consistent with our tissue expression profiling results, which showed that lnc-231 had higher expression in AGING skeletal muscle than in other tissues, suggesting that lnc-231 may be involved in the regulation of skeletal muscle development. Interestingly, the muscular expression of lnc-231 was significantly higher in young mice than that in aged mice; however the expression of Atrogin-1 (a marker gene of muscle atrophy) in aged mice was significantly higher than that in young mice. Since apoptosis of skeletal muscle cells is believed to cause muscle atrophy [35][36][37], we speculated that lnc-231 may inhibit the apoptosis of skeletal muscle cells, although the specific mechanism remains to be uncovered.
We also found that overexpression of lnc-231 inhibited myoblast differentiation, while knockdown of lnc-231 promoted myoblast differentiation. In agreement with this, lncRNA SYISL has been reported to promote myoblast proliferation and fusion but inhibit myogenic differentiation through interacting with polycomb AGING repressive complex 2 [19]. The inhibitory effects of lncRNA on differentiation might be achieved via absorbing several types of differentiation-related miRNAs [38]. Therefore, the specific mechanism of lnc-231-induced inhibition of differentiation still needs further investigations.
In addition, our results showed that lnc-231 promoted the G0/G1-to-S phase progression in myoblasts, and hence promoting cell proliferation. Using bioinformatics analysis and luciferase reporter vector assays, we demonstrated that lnc-231 can serve as a direct target for miR-125a-5p. As a ceRNA, it can absorb miR-125a-5p to reduce the cytoplasmic abundance of miR-125a-5p, and increase the expression of E2F3 protein. It has been reported that miR-125a-5p targets E2F3 to inhibit the proliferation of myoblasts. Our results showed that miR-125a-5p overexpression inhibited the expression of E2F3 protein levels, which is consistent with previous studies [34].
In conclusion, lnc-231 inhibits myoblast differentiation, and serves as a ceRNA that adsorbs miR-125a-5p to change its enrichment, thereby promoting the translation of the target gene E2F3, which in turn promotes cell cycle progression by activating the G0/G1 to S transition. Our results suggested the role of lnc-231/miR-125a-5p/E2F3 pathway in regulating myoblast proliferation and differentiation. Indicatively, further investigations of this pathway might contribute to a better understanding of the mechanisms underlying muscle atrophy and skeletal muscle regeneration in the future.

Ethics statement
All experimental procedures on the mice were conducted according to the Guidelines for Experimental Animals of the Ministry of Science and Technology (Beijing, China). The study protocol was reviewed and approved by the ethics committee of Nanjing Agricultural University. Animals were humanely euthanized as necessary, to reduce suffering.

Animals and cells
BALB/c mice were obtained from Qinglongshan Laboratory Animal Company (Nanjing, China). Mice were housed in a room of Nanjing Agricultural University under conventional conditions with appropriate temperature and humidity and fed a standard diet. Four mice at 8 weeks of age were used to collect different tissues to measure the expression of 2310043L19Rik by quantitative PCR (qPCR). Mouse C2C12 cells were cultured in growth medium (GM) (26) consisting of 89% high-glucose DMEM, 10% (v/v) fetal bovine serum, and 1% penicillin-streptomycin (both from Thermo Fisher Scientific) at a constant temperature of 37°C in 5% CO2. Similar to induction of differentiation of C2C12 cells, mouse C2C12 myoblasts were induced in differentiation medium (DM) consisting of 97% DMEM, 2% horse serum (Thermo Fisher Scientific), and 1% penicillin-streptomycin.

qPCR
Total RNA was collected from mouse tissues and C2C12 cells by using Trizol reagent (Thermo Fisher Scientific). Next, the concentration and integrity of RNA were assessed with NanoDrop 2000 (Thermo Fisher Scientific) and denatured gel electrophoresis, respectively. cDNA synthesis for mRNA and miRNA detection was performed with PrimeScript Real Time Master Mix (Perfect Real Time; Takara Biotech, Dalian, China) and Mix-X miRNA First-Strand Synthesis Kit (TaKaRa, Dalian, China), respectively. qPCR for mRNA and miRNA detection was qRT-PCR was performed using AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China) for quantitative on a Step-One Plus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). The primers for quantitative analysis were shown in Table 1. The relative expression levels were calculated using the 2 ΔΔct method, and mouse GAPDH and U6 snRNA was used for normalization of mRNA and miRNAs expression levels as endogenous reference genes. Statistical analysis was performed using Prism 6 software (GraphPad Software, San Diego, CA), and data were expressed as mean ± SE unless otherwise noted. Differences were tested using two-tailed Student's t test and ANOVA test. P < 0.05 was considered significant difference. CTCGCTTCGGCAGCACA AACGCTTCACGAATTTGCGT

Cytoplasmic and nuclear lncRNA 2310043L19Rik
RNAs were extracted from C2C12 cells by using a Nuclear or Cytoplasmic RNA Purification Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. First, the cell pellet was resuspended in buffers from an RNA Purification Kit and centrifuged twice at 4°C. The supernatant was taken as the cytoplasmic fraction, and the pellet was saved as the nuclear fraction. RNAs of cytoplasmic and nuclear fractions were extracted with Trizol reagent. The relative levels of lncRNA 2310043L19Rik in both cytoplasmic and nuclear fractions were analyzed by Realtime qPCR.

Cell cycle flow cytometry
Cell cycle and cell viability analyses were performed in accordance with the manufacturer's instructions.

Immunofluorescence
After the transfection and differentiation processes, C2C12 cells cultured in a 12-well plate in DM for 4 d. After that, the cells were washed with PBS 3 times and fixed in 4% paraformaldehyde for 30 min, followed by 3 washes with PBS. The cells were subsequently incubated in ice-cold 0.5% Triton X-100 at room temperature for 15 min and further washed 3 times. Next, the cells were incubated in blocking solution (1% bovine serum albumin) at room temperature for 2 h. After 3 washes with PBS, the cells were incubated in MyHC and antibody (dilution 1:50; Developmental Studies Hybridoma Bank) and MyoG antibody (1:100, ABclone, Wuhan, China) at 4°C for 12 h. The cells were washed 3 times and incubated with Alexa Fluor 488-Conjugated Goat anti-mouse IgG (H+L) antibody (dilution 1:100; ZSGB-BIO, Beijing, China) and Rhodamine(TRITC)-Conjugated Goat anti-mouse IgG (H+L) antibody (dilution 1:100; ZSGB-BIO, Beijing, China) in the dark. After 1 h of incubation at room temperature, the cells were washed 3 times. The cell nuclei were stained with DAPI in the dark. After 3 washes, images were captured using confocal microscopy (LSM700META; Zeiss).

Cell viability analysis
Cell viability was determined by WST-8 assay using Cell Counting Kit-8 (CCK-8; Dojindo, CK04). This colorimetric strategy is based on the reduction of tetrazolium salt (WST-8) to a colored formazan product by active dehydrogenases in viable cells. The amount of formazan is therefore directly proportional to the percentage of living cells. Our experimental procedures were performed according to the manufacturer's instructions. Briefly, C2C12 seeded in 24-well plates were grown to 90% confluency following 2 d of culture, and exposed to the indicated treatments as mentioned above. To measure cell viability, 50 ul of CCK-8 assay solution was added to each well containing 500 ul medium, and incubated at 37°C in a humid atmosphere containing 5% CO 2 for 2 h. Living cells promoted the formation of formazan, which was detectable at 450 nm under a microplate reader (Bio-Rad, Hercules, CA, USA).

Statistical analysis
Statistical analysis was performed using SPSS software (Chicago, IL, USA), and graphs were drawn with Prism v.6 (GraphPad Software, La Jolla, CA, USA). Student's t test was used for group comparisons. Multiple comparisons were performed by one-way ANOVA followed by Dunnett's post hoc test. Results are shown as means±SEM. Statistical significance was set at P < 0.05.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest.