miR-21-5p Regulates the Proliferation and Differentiation of Skeletal Muscle Satellite Cells by Targeting KLF3 in Chicken

Abstract

The proliferation and differentiation of skeletal muscle satellite cells (SMSCs) play an important role in the development of skeletal muscle. Our previous sequencing data showed that miR-21-5p is one of the most abundant miRNAs in chicken skeletal muscle. Therefore, in this study, the spatiotemporal expression of miR-21-5p and its effects on skeletal muscle development of chickens were explored using in vitro cultured SMSCs as a model. The results in this study showed that miR-21-5p was highly expressed in the skeletal muscle of chickens. The overexpression of miR-21-5p promoted the proliferation of SMSCs as evidenced by increased cell viability, increased cell number in the proliferative phase, and increased mRNA and protein expression of proliferation markers including PCNA, CDK2, and CCND1. Moreover, it was revealed that miR-21-5p promotes the formation of myotubes by modulating the expression of myogenic markers including MyoG, MyoD, and MyHC, whereas knockdown of miR-21-5p showed the opposite result. Gene prediction and dual fluorescence analysis confirmed that KLF3 was one of the direct target genes of miR-21-5p. We confirmed that, contrary to the function of miR-21-5p, KLF3 plays a negative role in the proliferation and differentiation of SMSCs. Si-KLF3 promotes cell number and proliferation activity, as well as the cell differentiation processes. Our results demonstrated that miR-21-5p promotes the proliferation and differentiation of SMSCs by targeting KLF3. Collectively, the results obtained in this study laid a foundation for exploring the mechanism through which miR-21-5p regulates SMSCs.

Keywords: miR-21-5p; muscle development; myogenic markers; myotubes.

Figure 1

Expression of miR-99a-5p in chickens. (A) miR-21-5p sequence of different species: gga, chicken; bta, cattle; rno, rat; hsa, homo sapiens; mml, macaca mulatta; mdo, monodelphis domestica. (B) The miRNAs with the highest expression in embryoni (E7, E11, E17 and D1) chicken skeletal muscle. (C) Expression of miR-21-5p in different tissues of 3-day-old chickens. (D) Expression of miR-21-5p in skeletal muscle of chicken at four embryonic stages. The results were expressed as mean ± SEM. (n = 3). Different lowercase letters above bars indicate significant differences (p < 0.05). miR, microRNA.

Figure 2

miR-21-5p promotes the proliferation of chicken SMSCs. (A,B) After transfection with miR-21-5p inhibitor and mimic, the expression of miR-21-5p in SMSCs was determined by using qRT-PCR. (C,D) The mRNA expression of PCNA, CDK2, and CCND1 after 24 h transfection of miR-21-5p mimic or inhibitor in SMSCs cells. (E–G) The protein expression of PCNA and CDK2 after 48 h transfection of miR-21-5p inhibitors and miR-21-5p mimics in SMSCs determined by Western blot analysis. β-Tubulin was used as a reference gene. (H,I) Cell viability was measured using the cell counting kit-8 (CCK-8). (J–M) 5-ethynyl-20-deoxyuridine (EdU) staining of transfected SMSCs and the calculation of the proliferation rate. The results were expressed as mean ± SEM. (n = 3). * p < 0.05 and ** p < 0.01.

Figure 3

miR-21-5p promotes the differentiation of chicken SMSCs. (A) The expression level of miR-21-5p during proliferation (GM) and differentiation of SMSCs. DM24, DM36, DM48, and DM72 represent SMSCs which were induced to differentiate for 24, 36, 48, and 72 h, respectively. (B,C) The mRNA expression levels of MyoG, MyoD, MyHC, and MyF5 after 24 h of overexpression and inhibition of miR-21-5p in SMSCs. (D–F) The protein expression of MyoG and MyHC after 48 h of transfection of miR-21-5p mimic and inhibitor in SMSCs using Western Blot. (G,H) Anti-Myosin heavy chain (MyHC) immunofluorescence staining after the transfection of miR-21-5p mimic and inhibitor in SMSCs. DAPI (blue), cell nuclei; Merge: the fusion of SMSCs into primary myotubes. (I,L) Relative myotube area of chicken SMSCs following miR-21-5p overexpression and inhibition. (J,M) Immunofluorescent staining for MyHC in SMSCs myotubes showed that the average number of nuclei per myotubes and (K,N) Myotube diameter. The results were expressed as mean ± SEM. (n = 3). * p < 0.05; ** p < 0.01.

Figure 4

miR-21-5p directly targets KLF3 Gene. (A) Prediction of target genes of gga-miR-21-5p using DIANA, TargetScan, and miRDB. (B) The complementary pairing of gga-miR-21-5p with the targeted gene KLF3 3′UTR or mutated UTR. (C) Diagram of the construction of dual-luciferase reporter vectors containing the wild or mutant KLF3 3′-UTR sequences. hRluc means renilla luciferase; hluc2 means firefly luciferase. (D) Validation of wild-type plasmid and mutant plasmid. WT means wild-type vector; Mut represents mutant vector. (E) Chicken DF-1 cells were co-transfected with KLF3-3′-UTR wild or mutant dual-luciferase vector and the miR-21-5p mimic or mimic NC. The relative luciferase activity was assayed 48 h later. (F,G) After transfection with miR-21-5p mimics, miR-21-5p inhibitors or NC, the expression of KLF3 was determined by q-PCR and Western blot. The results were expressed as mean ± SEM. (n = 3). * p < 0.05; ** p < 0.01.

Figure 5

Knockdown of KLF3 facilitates chicken SMSCs proliferation. (A) Relative expression of KLF3 in different chicken tissues. (B) The knockdown efficiency of KLF3 gene in SMSCs by three small siRNAs were detected by qRT-PCR. (C) The protein expression level of KLF3 after interference by si-KLF3-77 was detected by Western blotting. (D) The mRNA expression of CDK2, PCNA, and CCND1 after 24 h of KLF3 knockdown. (E) CCK-8 assays for SMSCs after KLF3 knockdown. (F,G) The protein expressions of MyHC and MyoG after 48h of inhibition of the KLF3 gene in SMSCs using Western Blot. (H) Results of EdU assay for SMSCs after inhibition of KLF3 for 48 h, where EdU (red) fluorescence is used as an indicator of proliferation and nuclei are indicated by Hochest (blue) fluorescence. (I) The quantitative data of proliferating PSCs number in panel G. The results were expressed as mean ± SEM. (n = 3). ** p < 0.01.

Figure 6

Knockdown of KLF3 facilitates chicken SMSCs differentiation. (A) The expression level of KLF3 during the proliferation (GM) and differentiation of SMSCs. DM24, DM36, DM48, and DM72 represent SMSCs which were induced to differentiate for 24, 36, 48, and 72 h, respectively. (B) The mRNA expression levels of MyoD, MyHC, MyoG, and MyF5 after 24h knockdown of KLF3 in SMSCs cells. (C,D) The protein expression levels of MyHC and MyoG after 48 h inhibition of KLF3. (E) Immunofluorescence analysis of SMSCs after KLF3 inhibition. (F) Relative myotube area of chicken SMSCs following KLF3 knockdown. (G,H) Immunofluorescent staining for MyHC in SMSCs myotubes showed that the average number of nuclei per myotubes and Myotube diameter. The results were expressed as mean ± SEM. (n = 3). * p < 0.05; ** p < 0.01.

Figure 7

Schematic diagram of miR-21-5p mediated proliferation and differentiation of chicken skeletal muscle satellite cells.