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. 2016 Feb 5;11(2):e0148532.
doi: 10.1371/journal.pone.0148532. eCollection 2016.

MicroRNA-27b Regulates Mitochondria Biogenesis in Myocytes

Linyuan Shen  1 , Lei Chen  2 , Shunhua Zhang  1 , Jingjing Du  1 , Lin Bai  1 , Yi Zhang  3 , Yanzhi Jiang  4 , Xuewei Li  1 , Jinyong Wang  2 , Li Zhu  1
Affiliations

MicroRNA-27b Regulates Mitochondria Biogenesis in Myocytes

Linyuan Shen et al. PLoS One. .

Abstract

MicroRNAs (miRNAs) are small, non-coding RNAs that affect the post-transcriptional regulation of various biological pathways. To date, it is not fully understood how miRNAs regulate mitochondrial biogenesis. This study aimed at the identification of the role of miRNA-27b in mitochondria biogenesis. The mitochondria content in C2C12 cells was significantly increased during myogenic differentiation and accompanied by a marked decrease of miRNA-27b expression. Furthermore, the expression of the predicted _target gene of miRNA-27b, forkhead box j3 (Foxj3), was also increased during myogenic differentiation. Luciferase activity assays confirmed that miRNA-27b directly _targets the 3'-untranslated region (3'-UTR) of Foxj3. Overexpression of miRNA-27b provoked a decrease of mitochondria content and diminished expression of related mitochondrial genes and Foxj3 both at mRNA and protein levels. The expression levels of downstream genes of Foxj3, such as Mef2c, PGC1α, NRF1 and mtTFA, were also decreased in C2C12 cells upon overexpression of miRNA-27b. These results suggested that miRNA-27b may affect mitochondria biogenesis by down-regulation of Foxj3 during myocyte differentiation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Mitochondria content and miRNA-27b expression during C2C12 cell differentiation.
(A) The change of mitochondrial content during myocyte differentiation was visualized by fluorescence staining (MitoTracker Green FM). 50% and 80% refer to the degree of cell confluence. Scale bar: 20 μm. (B) Quantification of green fluorescence performed with LSM imaging software. Fluorescence intensities are normalized to those measured at 50% cell confluence. Data represent means ± SD of three independent experiments and each one performed in duplicate (n = 6). (C) Relative mitochondrial DNA copy numbers were measured in a RT-PCR assay and calculated as the ratio of mitochondrial gene expression (ATP6, COX1 and ND2) to the expression of the nuclear DNA single copy gene (B2M). All data represent mean of three independent experiments performed in duplicate (n = 6). (D) Relative value of expression levels of miRNA-27b in C2C12 cells during myogenic differentiation (RT-PCR) (n = 6). (E) Relative value of expression levels of Foxj3 mRNA in C2C12 cells during myogenic differentiation (RT-PCR) (n = 6).GM: growth medium; DM: differentiation medium. Data are means ± SD; * P < 0.05, ** P < 0.01.
Fig 2
Fig 2. miRNA-27b _targets the 3’-UTR of Foxj3.
(A) Sequence alignment of miRNA-27b with 3’-UTR of human (Hsa), chimpanzee (Ptr), mouse (Mmu), rat (Rno), dog (Cfa) Foxj3 mRNA. Binding site and seed region of miRNA-27b are indicated in red. (B) Recombinant luciferase reporter plasmid containing the wild-type 3’-UTR of Foxj3, or one of the two mutant 3’-UTR of Foxj3, respectively. Fragment insertion was verified by Sanger Sequencing (bottom). (C) The repressive effect of miRNA-27b on the activity of Foxj3 as revealed in luciferase assays conducted with HeLa cells co-transfected with the wild-type or mutant Foxj3-3’UTR luciferase construct, and miR-27b mimic or mimic control. Data are means ± SD (n = 6), ** P < 0.01.
Fig 3
Fig 3. The role of miRNA-27b in mitochondrial biogenesis.
Expression of miRNA-27b (A) and its _target gene Foxj3 (B) in C2C12 cells transfected with miRNA-27b mimic or inhibitor. Results are normalized to negative control (NC). Data represent means ± SD of three independent experiments performed in duplicate (n = 6). (C) Levels of proteins related to mitochondrial biogenesis as detected in Western Blots (n = 6). (D) Mitochondrial content of myoblasts with upon increase or reduction of miRNA-27b levels, visualized after fluorescence staining (n = 6). (E) Protein bands were quantified by densitometry and normalized to β-Actin levels (n = 6). (F) Fluorescence intensities normalized to negative controls as measured with the LSM imaging software.(n = 6). (G) Mitochondrial DNA copy numbers normalized to negative controls as detected in a RT-PCR approach (n = 6). (H) Relative expression levels of mitochondrial genes ND2, ATP6 and COX II and nuclear encoded mitochondrial-resident gene VDAC were detected by RTPCR (n = 6). (J) Relative expression levels of downstream genes of Foxj3 (Mef2c, PGC1-α, NRF-1 and mtTFA) in myoblasts displaying an augmented or reduced content of miRNA-27b (n = 6). NC (negative control, no sequence similarities to any reported mouse gene sequence), Scale bar: 20 μm, All data are means ± SD, *P < 0.05, **P < 0.01.
Fig 4
Fig 4. miRNA-27b influences myogenic differentiation of C2C12 myoblasts.
(A) Effects of miRNA-27b on slow muscle fiber type formation in C2C12 cells as evaluated after immunofluorescence staining of MyHC I 5 days after differentiation. Scale bar: 20 μm. (B) Green fluorescence intensities normalized to negative controls as measured with the LSM imaging software. Data represent means ± SD of three independent experiments performed in duplicate (n = 6). (C) Relative expression levels of myogenic regulatory factors Myf5, MyoD, MRF4 and MyoG in cells with an augmented or reduced content of miRNA-27b as detected by RT-PCR (n = 6). (D) Relative expression levels of myosin heavy chain isoforms MyHC I, MyHC IIa, MyHC IIb and MyHC IIx upon increase or reduction of miRNA-27b expression as detected by RT-PCR (n = 6). (E) Composition of myosin heavy chains (MyHC I, MyHC IIa, MyHC IIb and MyHC IIx) in cells with an augmented or reduced expression of miRNA-27b as detected by RT-PCR (n = 6). NC (negative control, no sequence similarities to any reported mouse gene sequence), All data are means ± SD, *P < 0.05, **P < 0.01.
Fig 5
Fig 5. Possible pathway used by miRNA-27b to suppress mitochondria biogenesis.
Foxj3 and Mef2c are _target genes of miRNA-27b and are suppressed by it. The expression of downstream gene of Foxj3 (Mef2c, PGC1c, NRF1 and mtTFA), which play important roles in promoting mitochondrial biogenesis, is reduced upon suppression of Foxj3 by miRNA-27b. Mef2c and PGC1 also have other functions that may alter the slow fiber type composition and thus diminish their mitochondria content.
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Grants and funding

The study was supported by the Sichuan Sci & Tech Support Program (No. 2013NZ0041 and 2013NZ0056), the earmarked fund for China Agriculture Research System (No. CARS-36-05B), and Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13083). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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