Muscle-specific microRNA miR-206 promotes muscle differentiation

Abstract

Three muscle-specific microRNAs, miR-206, -1, and -133, are induced during differentiation of C2C12 myoblasts in vitro. Transfection of miR-206 promotes differentiation despite the presence of serum, whereas inhibition of the microRNA by antisense oligonucleotide inhibits cell cycle withdrawal and differentiation, which are normally induced by serum deprivation. Among the many mRNAs that are down-regulated by miR-206, the p180 subunit of DNA polymerase alpha and three other genes are shown to be direct targets. Down-regulation of the polymerase inhibits DNA synthesis, an important component of the differentiation program. The direct targets are decreased by mRNA cleavage that is dependent on predicted microRNA target sites. Unlike small interfering RNA-directed cleavage, however, the 5' ends of the cleavage fragments are distributed and not confined to the target sites, suggesting involvement of exonucleases in the degradation process. In addition, inhibitors of myogenic transcription factors, Id1-3 and MyoR, are decreased upon miR-206 introduction, suggesting the presence of additional mechanisms by which microRNAs enforce the differentiation program.

Figure 1.

The muscle-specific expression of the miRNAs miR-1, -133, and -206. (A) RNase protection assays were performed with RNA from various tissues to detect miR-1, -206, and -133. 5S ribosomal RNA (rRNA) was used as a control. The different panels have different levels of exposure. The protected bands are indicated by arrows on the left. −, no RNA control; Co, colon; Kd, kidney; Ht, heart; Lv, liver; Bs, breast; Lu, lung; Si, small intestine; Sm, skeletal muscle; Ut, uterus; At, atrium; Sp, spleen; Te, testis; Ov, ovary; Bm, bone marrow; Br, brain; Cv, cervix; Pr, prostate tissue; Pt, prostate tumor; Pe, prostate epithelium. (B) During skeletal myogenesis of the C2C12 MB cell line, the miRNAs (bottom six panels) and several marker proteins for muscle differentiation (top four panels) were measured by RNase protection assays or immunoblots, respectively. The numbers at the bottom indicate the days after changing from GM to DM. In the immunoblot panels, molecular mass markers are indicated on the left and the myogenin band is specified by an arrow. The panels for miR-1, -133, and -206 are at comparable levels of exposure.

Figure 2.

The muscle-specific miRNAs induce the differentiation of MBs by regulating many genes. (A) Synthetic oligonucleotides complementary to miR-1, -133, and -206 were inserted downstream of Renilla luciferase ORF in pRL-CMV(MCS) to generate pRL1, pRL133, and pRL206, respectively. Luciferase assays were performed with pRL1 (white bar), pRL133 (black bar), and pRL206 (hatched bar) with the cotransfected RNA duplexes mimicking the miRNAs miR-1, -133, and -206, respectively (x axis). Sequential transfection of each RNA duplex (or miR-125b duplex as a control) and reporter plasmids in GM (serum+) was followed by luciferase assays 20 h after transfection. The y axis indicates relative Renilla luciferase activity, which is normalized as described in Materials and methods. (B) RNase protection assays were performed to measure the level of miR-133 (top) and -206 (middle), in MB transfected with the siRNA duplexes mimicking miR-133 or -206. 5S rRNA was used as a loading control (bottom). RNA from differentiated MTs is shown for comparison. (C) Immunoblots at the indicated days after the serum depletion of C2C12. C2C12 was transfected four times at 24-h intervals with miR-206 or GL2 negative control duplex before serum depletion. (D) C2C12 was transfected with miR-206 and GL2 as described in C, split at 5 × 10⁵ cells per well in a 6-well plate, and maintained in GM (serum+) for 3 d before immunostaining for MHC. The density of the cells can also be gauged in Fig. S1 B (available at http://www.jcb.org/cgi/content/full/jcb.200603008/DC1). (E) Quantification of MHC and myogenin expression in experiments as in D. MB, C2C12 MB in GM (serum+); MT, MT in DM (serum−); GL2 or 206, C2C12 in GM transfected with GL2 or miR-206 duplexes, respectively. Each value is a mean of triplicate. (F) Hierarchical cluster and heat map to show changes in mRNA relative to MBs in GM. Red and green represent increase and decrease of expression, respectively. Each row represents a single gene in the microarray. MT indicates C2C12 differentiated by serum deprivation, and other rows show C2C12 in GM transfected with the indicated duplexes. The 109 or 92 genes most up- or down-regulated after muscle differentiation are shown.

Figure 3.

Inhibition of the miRNAs by 2′-O-methyl antisense oligonucleotides inhibits differentiation. (A) C2C12 cells transfected with antisense oligonucleotide against GL2 or a mixture of antisense oligonucleotides against miR-1, -133, and -206 (anti-mix). Three transfections at 24-h intervals in GM (serum+) were followed by serum depletion (DM). 96 h after serum depletion, MHC expression (green) and BrdU incorporation (red) were detected by immunostaining. Blue indicates nuclei stained by DAPI. (B) Quantification of percentage of BrdU- and MHC-positive cells in samples prepared as described in panel A. Anti-(1+206) indicates a mixture of 2′-O-methyl antisense oligonucleotides against miR-1 and -206. Each value is a mean of triplicate. (C) Immunoblots at 48 and 96 h after serum depletion (DM). The transfection and induction of differentiation were performed as described in A. (D) Cell morphology visualized by MHC immunostaining as described in A (top). Results are quantitated at the bottom. Cells that were elongated were twofold longer than proliferating C2C12. Each value is a mean of triplicate experiments.

Figure 4.

miR-206 directly down-regulates DNA pol α during differentiation. (A) Northern hybridization (top) and immunoblots (bottom) for the largest subunit of DNA pol α (Pola1) at 24 and 96 h after transfection of the indicated duplexes into C2C12 in GM (serum+). MB and MT are shown for comparison. (B) Two predicted target sites of miR-206 in the 3′UTR of Pola1. The nucleotide coordinate of Pola1 is based on the mouse Refseq (available from GenBank/EMBL/DDBJ under accession no. NM_008892.1). M1 and M2, mutated (bold italic) from the seed matches (bold), are indicated. (C) Luciferase assays were performed to measure the effect of miR-206 transfection, as described in Fig. 2 A. pRL206 indicates insertion of a perfectly complementary target of miR-206 downstream of the luciferase gene in pRL-CMV. DNA pol α (3985–4657) and (4991–5345) indicates insertion of the indicated regions of Pola1 mRNA (available from GenBank/EMBL/DDBJ under accession no. NM_008892.1), respectively. M1 and M2 indicate 4991–5345 with point mutations in putative target sequences as described in B. (D) Northern blot of Pola1 (top) and its quantification (bottom) after transfection of antisense oligonucleotide against miR-206 (open squares) or GL2 (closed squares) during C2C12 differentiation as described in Fig. 3 A. The ethidium bromide staining of two rRNA bands is shown as loading control. Each band from Northern hybridization was quantified with ImageQuant 5.2 software and normalized to the total RNA amount from the intensity of 28S and 18S rRNA. The normalized value at day 0 was set at 1.

Figure 5.

Inhibition of DNA synthesis by the down-regulation of DNA pol α promotes withdrawal from the cell cycle in the continued presence of serum. (A) DNA synthesis (top) and cell growth (bottom) were measured after the transfection of GL2 (white bar) or miR-206 (black bar) duplexes into C2C12 MB in GM (serum+). The same assays were performed at 4 d after serum depletion (MT; hatched bar) with a similar number of cells before serum depletion (MB; gray bar) for comparison. Each value is a mean of four values from two independent transfections. The x axis shows days after transfection, and the y axis shows ³H-thymidine incorporation or absorbance at 570 nm from the MTT assay. (B) Immunoblots as described in Fig. 4 A. Cdk2 kinase activity shows an autoradiogram of phosphorylated histone H1 (see Materials and methods). (C) Propidium iodide staining for DNA content and FACS to determine the number of cells transfected with GL2 or miR-206. The percentage of cells in G1, S, and G2 at 24 h are calculated and plotted. (D) For synchronization of cell cycle, cells were treated with 2 mM of thymidine for 8 h, released for 8 h, and treated with 2 μg/ml of aphidicolin for 8 h. During synchronization, miR-206 (bottom two right panels) or GL2 control duplex (left) was transfected at the onset of thymidine treatment and a second time at 4 h after release from thymidine. Propidium iodide–stained FACS profiles of arrested cells (0 h; middle) and cells at 12 h after release from aphidicolin (bottom) are shown, with that of asynchronous MBs (top) for comparison. (E) The percentage of cells in S phase (y axis) is quantified as described in C before (0 h) or at the indicated time points (x axis) after release from aphidicolin. Closed circles indicate GL2 transfected cells, and open circles indicate miR-206 transfected cells.

Figure 6.

Examination of down-regulated genes from microarray to determine whether they are direct targets of miR-206. (A and C) Northern blots for MyoD inhibitors or down-regulated genes containing putative miRNA targets, as described in Fig. 4 A. (B and D) The luciferase assays were performed with pRL-CMV derivatives containing the mRNA sequences of the genes in A and C, respectively, as described in Fig. 2 A. The difference from RL-CMV control is significant for B-ind1 (P = 0.0059), Cx43 (P = 0.06), and Mmd (P = 0.0006). (E) Northern blot and quantification of B-ind1 mRNA after transfection of antisense oligonucleotide during C2C12 differentiation, as described in Fig. 4 D. The level of miR-206 during differentiation (of anti-GL2 transfected cells) was measured by RNase protection assays (top) and quantitated (bottom, filled triangle with dotted line).

Figure 7.

The down-regulation of direct targets by miR-206 is posttranscriptional, dependent on target sites, and occurs through mRNA cleavage. (A) The schematic of B-ind1 mRNA (top) and 3′UTR of Pola1 mRNA (bottom). Vertical bars show potential binding sites of miR-206, predicted by miRanda program (Δg is less than −14 kcal/mol). M1 and M2 indicate point mutations at indicated sites (used in C and Fig. 4 C and described in B and Fig. 4 B). P, P1, and P2 indicate locations of each set of primers for the RACE-PCR (used in E and F and Figs. S3–S5, available at http://www.jcb.org/cgi/content/full/jcb.200603008/DC1). Cleavage sites for each treatment are indicated as described in the figure (see also Figs. S3–S5). The number of arrowheads corresponds to the frequency of cloning from RACE-PCR product. Although MB had very few cleavage products (E), we cloned a few to show that distribution of background cleavage sites is different from that seen in the presence of miRNAs (Fig. S4). (B) Among the several predicted target sites (A) of miR-206 in B-ind1 mRNA (available from GenBank/EMBL/DDBJ under accession no.NM_021345), two sites are shown as described in Fig. 4 B. (C) Luciferase assays were performed as described in Fig. 4 C, with pRL-CMV derivatives containing B-ind1 3′UTR without mutation (B-ind1 wild type [wt]) or with point mutations at both M1 and M2 (B-ind1 M1/M2; described in B). (D) Nuclear run-on assays to measure the transcription of B-ind1 and GAPDH upon differentiation (MB vs. MT) or after transfection of the indicated duplexes in GM (serum+). PBS, pBluescript vector negative control. (E) Agarose gel electrophoresis of RLM-RACE products from primer set P1 (A and Fig. S5 A), showing sites of cleavage of B-ind1 mRNA from C2C12 transfected with indicated duplexes in GM (serum+). Molecular size markers (in nucleotides) are indicated. (F) Quantitation of the amount of the RLM-RACE products from E as described in Materials and methods. The x axis indicates the relative intensity. Each value is a mean of triplicate measurements.