CircRNA FUT10 regulates the regenerative potential of aged skeletal muscle stem cells by targeting HOXA9

Skeletal muscle is capable of repairing itself after injury to maintain the stability of its own tissue, but this ability declines with aging. Circular RNAs (circRNAs) are involved in cell aging. However, there is little research into their role and underlying mechanisms, especially in skeletal muscle stem cells (SkMSCs). In this study, we assessed circRNA FUT10 expression in aged and adult SkMSCs. We observed that circRNA FUT10 was upregulated in aged SkMSCs compared with that in adult SkMSCs. Furthermore, we identified putative miR-365-3p binding sites on circRNA FUT10, suggesting that this circRNA sponges miR-365a-3p. We also found that HOXA9 is a downstream target of miR-365a-3p and confirmed that miR-365a-3p can bind to circRNA FUT10 and the 3′-untranslated region of HOXA9 mRNA. This finding indicated that miR-365a-3p might serve as a “bridge” between circRNA FUT10 and HOXA9. Finally, we found that the circRNA FUT10/miR365a-3p/HOXA9 axis is involved in SkMSC aging. Collectively, our results show that the circRNA FUT10/miR365a-3p/HOXA9 axis is a promising therapeutic target and are expected to facilitate the development of therapeutic strategies to improve the prognosis of degenerative muscle disease.


INTRODUCTION
Age-related factors and progressive homeostatic decline are among the most common health issues for individuals at advanced age disease, such as amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy (DMD) and sarcopenia, et al [1,2]. Degenerative muscle disease, attributable to skeletal muscle aging, is a common agerelated disorder. Skeletal muscle can repair itself after injury. This process is largely dependent on skeletal muscle stem cells (SkMSCs), and their repairability declines with aging [3,4]. Therefore, exploring the mechanisms underlying skeletal muscle aging is essential for the clinical treatment of degenerative muscle disease. Circular RNAs (circRNAs) are a class of single-stranded noncoding RNA molecules, that are involved in age-related diseases [5][6][7][8]. Panda et al. reported that the senescence-associated circular RNA (SAC-RNA) circPVT1 is markedly reduced in senescent fibroblasts, promotes cell senescence and reverses cell proliferation by selectively targeting let-7 [9]. Moreover, Liang et al. reported that in lens epithelial cells, circRNA ZNF292 plays a role in resistance to oxidative damage by modulating miR-23b-3p in age-related cataracts (ARCs) [10]. Notably, recent studies suggest that circFUT10 promotes proliferation and inhibits differentiation via sponging let-7 [11]. In addition, circRNA FUT10 regulates myoblast cell survival and aging [12]. However, the role and mechanism underlying circRNA FUT10 activity in SkMSC aging is sparsely reported.
In this study, we examined circRNA FUT10, miR-365-3p, and HOXA9 expression in adult and aged SkMSCs. We also investigated the biological role of these molecules in aged SkMSCs. Finally, we determined whether the circRNA FUT10/miR-365a-3p/HOXA9 axis is involved in regulating SkMSCs aging. Our results will contribute to our understanding of degenerative muscular disease and identify therapeutic targets to improve disease prognosis.

Analysis of differentially expressed circRNAs between adult and aged SkMSCs
To further explore the potential functions of circRNA FUT10, we prepared a clustered heatmap. In total, 9,572 expressed circRNAs were detected using an Arraystar mouse circRNA microarray. We then identified circRNAs that were differentially expressed between adult and aged SkMSCs. Hierarchical clustering was performed to sort circRNAs according to their expression level ( Figure 1A). In the volcano plots ( Figure 1B), differentially expressed circRNAs were grouped by fold change (FC) ≥ 2.0, P < 0.05, and false discovery rate < Red and green indicate high and low relative expression, respectively. The red and green points in the plot represent the significantly differentially expressed circRNAs. (C) circRNA FUT10 mRNA expression in the above two groups. (D) MTT assay showing adult and aged SkMSC proliferation. (E) Immunofluorescence analysis of MyHC and MyoD expression in adult and aged SkMSCs. (F) Quantification of MyHC and MyoD. Each bar represents the mean ± SEM. *P < 0.05, **P < 0.01. All experiments were performed at least three times with duplication within each individual experiment. 0.05. This analysis 264 significantly different circRNAs in aged SkMSCs compared with adult SkMSCs. Of these, 152 circRNAs were upregulated, and 112 were downregulated. We selected circRNA FUT10, one of the most upregulated circRNAs and then verified it by qRT-PCR ( Figure 1D). MTT cell proliferation assays showed that adult SkMSCs have higher proliferative capacity than aged SkMSCs. In addition, MyoD and MyHC expression was measured in adult and aged SkMSCs. Immunofluorescence analysis indicated that adult SkMSCs show higher differentiation ability ( Figure 1E, 1F) than aged SkMSCs. Together, these data indicate that circRNA FUT10 is differentially expressed in adult and aged SkMSCs and plays a role in SkMSC proliferation and differentiation.

Effect of circRNA FUT10 on SkMSCs proliferation and differentiation
To further explore the potential functions of circRNA FUT10 in adult and aged SkMSCs, we designed circRNA FUT10 overexpression vectors and short hairpin RNA (shRNA) to knock down circRNA FUT10 ( Figure 2A). These constructs were transfected into AGING SkMSCs. EdU assays showed that circRNA FUT10overexpression inhibited adult SkMSC proliferation, while suppressing circRNA FUT10 with shRNA promoted aged SkMSC proliferation ( Figure 2B, 2C). Next, the effect of circRNA FUT10 on differentiation was evaluated by western blot of the differentiation markers MyoD and MyHC. circRNA FUT10 overexpression reduced MyoD and MyHC expression and inhibited the differentiation of adult SkMSCs, while circRNA FUT10 suppression promoted the differentiation of aged SkMSCs ( Figure 2D, 2E). These data indicate that circRNA FUT10 regulates proliferation and differentiation in adult and aged SkMSC.

Effect of HOXA9 on SkMSCs proliferation and differentiation
To further explore the potential function of HOXA9 in SkMSCs, HOXA9 mRNA ( Figure 6A) and protein ( Figure 6B, 6C) expression was evaluated in aged SkMSCs. Next, HOXA9 overexpression and knock down, respectively, caused the upregulation and downregulation of HOXA9 mRNA ( Figure 6D) and protein ( Figure 6E, 6F). MTT assays showed that HOXA9 overexpression inhibited adult SkMSC proliferation, while the suppression of HOXA9 promoted aged SkMSC proliferation ( Figure 6G). In addition, the effect of HOXA9 on SkMSC differentiation was evaluated by western blot of MyoD and MyHC ( Figure  6H). HOXA9 overexpression inhibited adult SkMSC differentiation, while the suppression of HOXA9 promoted aged SkMSC differentiation ( Figure 6I). These data indicate that HOXA9 is highly expressed in aged SkMSCs, and that HOXA9 inhibits SkMSC differentiation.

DISCUSSION
Accumulating evidence has demonstrated that circRNAs are involved in cell aging. A better understanding of the molecular mechanism involved in the development and progression of degenerative muscle disease may force us to explore the underlying AGING mechanisms of SkMSCs aging. In this study, we investigated the expression of circRNA FUT10 in aged SkMSCs. Furthermore, we identified its roles in SkMSC aging and showed that circRNA FUT10 binds miR365a-3p and HOXA9. We also validated that circRNA FUT10 positively regulates HOXA9 by sponging miR-365-3p in SkMSC aging.
Recently, the regulatory potential of circRNAs and their role in the development and progression of muscle diseases have been reported [15,25,26]. Studies have also suggested that circRNA is involved in cellular aging [27][28][29]. One recent study reported that circLMO7 regulates myoblast differentiation and survival via miR-378a-3p [30]. circBBS9 is suggested to play active roles in muscle aging by mediating the benefits of aerobic training intervention [31]. Interestingly, circRNA FUT10 has been reported to promote proliferation and inhibit cell differentiation [11], as well as to reduce proliferation and facilitate myoblast differentiation [12].
Furthermore, we used TargetScan analysis to predict targets with miR-365a-3p binding sites. Among the candidates, HOXA9 was verified as a downstream target of miR-365a-3p by luciferase reporter assays. Several studies report that HOXA9 is involved in cell proliferation [38][39][40]. One study demonstrated that HOXA9 may promote denervated muscle atrophy by strongly upregulating MLL1 and WDR5 expression and inhibiting myogenic differentiation [23]. Furthermore, HOXA9 deletion improves satellite cell function and muscle regeneration in aged mice, whereas HOXA9 overexpression mimics age-associated defects in satellite cells from young mice, which can be rescued by inhibiting HOXA9-targeted developmental pathways. Clearly, HOXA9 is related to satellite cell aging. Our results demonstrate that HOXA9 is overexpressed in aged SkMSCs compared with control samples and that miR-365a-3p reverses the suppressive effect of HOXA9 on SkMSC proliferation and differentiation. In accordance with these observations, some studies reported that HOXA9 is the direct target of miR-365a-3p [41][42][43]. Furthermore, circRNA FUT10 downregulation suppressed SkMSC proliferation and viability. These effects were reversed by miR-365a-3p overexpression and HOXA9 silencing. Finally, we examined the percentage of Pax7+ cells derived from primary cultures of different muscle tissues. Our results indicate that the circRNA FUT10/miR-365a-3p/HOXA9 axis regulates SkMSC aging.
In summary, we observed that circRNA FUT10 is highly expressed in aged SkMSCs and suppresses cell proliferation and differentiation. circRNA FUT10 competitively binds miR-365a-3p to attenuate the suppressive effect of miR-365a-3p on HOXA9. These findings provide insights into SkMSC aging and provide a potential target for the treatment of degenerative muscle disease.

Cell culture and transfection
Isolated single myofiber-associated cells were prepared using limb muscles obtained from 2-week-old and 6month-old female BALB/c mice (30-200 g, maintained in a 12:12 h light/dark cycle at 23° C and 50-70% humidity). Animal experiments were approved by The Institutional Animal Care and Use Committee at The Third Affiliated Hospital of Southern Medical University (Guangzhou, China). All animals were purchased from The Guangdong Medical Laboratory Animal Center (Guangzhou, China). SkMSCs were dissected from mice tibialis muscles using enzymatic dissociation (0.2% collagenase, Gibco;CA, USA) at 37° C for 60 min. Following be filtered by a 80-µm filter (Bioss, Beijing, China), cells were stained for the isolation of particular cell populations by flow cytometry and fluorescence-activated cell sorting (FACS). Then, cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 20% FBS (HyClone; GE Healthcare life Sciences) and 1% chick embryo extract (Gemini Bio Products). Cells differentiation was induced by DMEM with 2% heatinactivated horse serum (Gibco; Thermo Fisher Scientific, Inc.). The overexpression vectors (Roche, Switzerland) for circRNA FUT10 (circ-OE) and HOXA9 (HOXA9-OE), miR-365a-3p mimic and inhibitor (Sigma-Aldrich; Merck KGaA) and the short harpin RNA (shRNA) (Bioss, Beijing, China) for circRNA FUT10 (circ-SI) and small interfering RNA (siRNA) (Invitrogen, CA, USA) for HOXA9 (HOXA9-SI) were pretransfected into SkMSCs, respectively. After cells were plated for six hours, the vectors were added. The above vectors were delivered into SkMSCs by using a Lipofectamine 2000 reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. RNA can be extracted after 24 hours, and the protein can be extracted after 48 hours.

RNA pull-down assay
RNA pull-down assays were performed to identify the binding sites of miR-365a-3p with circRNA FUT10 and the 3′ UTR regions of HOXA9 mRNA. The biotin-labeled circRNA FUT10 and HOXA9 probes (Invitrogen, CA, USA) were designed and constructed. Then, the above probe-streptavidin Dynabeads were incubated with SkMSCs at 37° C for 12 hours. Finally, real time qPCR was performed to assess miR-365a-3p enrichment according to the manufacturer's instructions.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
An MTT assay was conducted to evaluate the role of circRNA FUT10 in SkMSCs. SkMSCs stably transfected with NC or HOXA9-SI or HOXA9-OE were seeded into 96-well plates at a density of 4×10 4 cells/well and incubated for 24 hours. Then, 20 μL of MTT solution (5 mg/ml) was added into each well and incubated for an additional 4 hours. After adding 160 μL/well of DMSO to each well, the optical density (OD) value of each well was measured at 450 nm by a spectrophotometer (Philips, China).

5-Ethynyl-2′deoxyuridine (EdU) assay
Cells were seeded at a density of 8×10 4 cells/well in 6well plates and cultured for 24 h at 37° C. then, cells were treated with 10 µM EdU working solution growth medium for 2 h in the dark. Then, the cells were treated with 4% paraformaldehyde for 20 min, followed by 2 mg/ml glycine and 0.5% Triton X-100 for 15-20 min at room temperature. After incubated with Hoechst 33342 for about half an hour, cells were subjected to an AxioVision 4.8 camera attached to an Axio Observer Z1 inverted microscope (Carl Zeiss, Inc.).

Flow cytometry assay
To assess stem phenotype conversion, cells were resuspended in 200 μL PBS and incubated with anti-Pax7-BV510 (4 μl/ ml) at 4° C for 1 hour. After the cells were washed three times with PBS and suspended in 100 μL PBS, the analysis was performed on a Flow CytoFLEX (Beckman Coulter, USA).

Statistical analysis
Statistical significance was determined by performing Student's t-test for comparisons between two groups and one-way analysis of variance followed by NDTukey's post hoc test for comparisons between more than two groups using the GraphPad 5.0 (GraphPad Software, La Jolla, CA). Unless otherwise stated all data are expressed as the mean ± SEM. A difference was considered statistically significant at a level of P < 0.05.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The study was approved by the Ethics Review Committee of The Third Affiliated Hospital of Southern Medical University (Guangzhou, China). The animal study followed the Guidelines for the Animal Care and Use approved by The Third Affiliated Hospital of Southern Medical University.

AUTHOR CONTRIBUTIONS
Benggang Qin and Gang Chen conceived and designed the study. Menghai Zhu, Peng Zou, Chong Lian conducted the experiments. Menghai Zhu and Benggang Qin analysed the data and wrote the paper.