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First published online August 22, 2007
doi: 10.1242/10.1242/jcs.010728

Journal of Cell Science 120, 3045-3052 (2007)
Published by The Company of Biologists 2007
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The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis by targeting HSP60, HSP70 and caspase-9 in cardiomyocytes

Chaoqian Xu1, Yanjie Lu1,2, Zhenwei Pan1,2, Wenfeng Chu1,2, Xiaobin Luo2,3,4, Huixian Lin2,3,4, Jiening Xiao2,3,4, Hongli Shan1, Zhiguo Wang2,3,4,* and Baofeng Yang1,2,*

1 Department of Pharmacology (the State-Province Key Laboratories of Biomedicine-Pharmaceutics of China), Harbin Medical University, Harbin, Heilongjiang 150086, People's Republic of China
2 Institute of Cardiovascular Research, Harbin Medical University, Harbin, Heilongjiang 150086, People's Republic of China
3 Research Center, Montreal Heart Institute, Montreal, PQ H1T 1C8, Canada
4 Department of Medicine, University of Montreal, Montreal, PQ H3C 3J7, Canada


Figure 1
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  		Fig. 1. MiR-1 and miR-133 produce opposing effects on cardiomyocyte apoptosis. (A) Examples of TUNEL staining showing the effects of various constructs, as labeled, on chromosomal condensation. (B) Pro-apoptotic effect of miR-1 and anti-apoptotic effect of miR-133, determined by ELISA quantification of DNA fragmentation expressed as optical density (OD) values. Ctl, control; WT, wild-type; MT, mutant. (C) Promoting effect of miR-1 and antagonizing effect of miR-133 on H2O2-induced apoptosis determined by ELISA. The lines represent fits to the Hill equation to define the concentration of H2O2 ([H2O2]o) for half-maximum cell death (IC50). AMO-1 and AMO-133 are anti-miRNA oligonucleotides specific for miR-1 and miR-133, respectively (same below). (D) Antagonizing effect of miR-1 and promoting effect of miR-133 on cell survival, as determined by MTT, in the presence of various concentrations of H2O2. The symbols are experimental data points and the lines represent fits to the Hill equation. Data in A-D were obtained from the rat ventricular cell line H9c2 cells. (E) Promoting effect of miR-1 and antagonizing effect of miR-133 on apoptosis, as determined by ELISA, induced by various concentrations of H2O2 in isolated neonatal rat ventricular myocytes (same below). Note that co-transfection of wild-type (WT) miR-1 and WT miR-133 abrogated the effects seen with WT miR-1 alone, whereas co-transfection of miR-1 and MT miR-133 produced effects similar to transfection of WT miR-1 alone. (F) Antagonizing effect of miR-1 and promoting effect of miR-133 on cell survival, as determined by MTT, in the presence of various concentrations of H2O2. Data are presented as means ± s.e.m. (n=8, 6, 6, 8 and 8, respectively). *P<0.05 versus (vs) Ctl; +P<0.05 vs WT miR-1.

 

Figure 2
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  		Fig. 2. The sequences showing the unique sites of miRNA::mRNA complementarity between miR-1 and HSP60 or HSP70, and between miR-133 and caspase-9 (Casp9) for both human (H) and rat (R) genes. The matched base pairs are bold and connected by a vertical line and the G:U/U:G wobble is indicated by bold letters connected by dots. The GenBank accession numbers of the genes are indicated in the brackets and the positions of the target sites are numbered. Note that there is only a single target site for miR-1 in HSP60 or HSP70 and the complementarity is limited to the 3'UTRs of these genes, whereas there are multiple target sites in Casp9 and the sites are distributed throughout the whole mRNA sequence.

 

Figure 3
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  		Fig. 3. Post-transcriptional repression of HSP60 and HSD70 by miR-1 and caspase-9 (Casp9) by miR-133. (A-C) Western blot analysis of HSP60, HSP70 and Casp9 (total) under various conditions with protein samples from H9c2 cells. The bar charts in the lower panels represent the densitometric measurements of western blots of HSP60, HSP70 and Casp9 expression. (D) Effects of miR-133 on Casp9 activity. (E) Effects of AMO-1 and AMO-133, respectively, on protein levels of HSP60, HSP70 and Casp9 in H9c2 cells. (F) Effects of miR-1 and miR-133 on mRNA levels of HSP60, HSP70 and Casp9 in H9c2, as determined by real-time RT-PCR. Data presented as means ± s.e.m. (n=8, 6, 6, 5, 6, and 6, for A-F, respectively). *P<0.05 vs Ctl; +P<0.05 vs WT miR-1 or WT miR-133.

 

Figure 4
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  		Fig. 4. Effects of miR-1 and miR-133 on caspase 3 (Casp3) protein levels and activities in H9c2 cells. (A) Immunoblotting analysis of Casp3 protein levels with and without miR-1 or miR-133 treatment. The antibody against the total Casp3 recognized the 35 kDa band representing Casp3. AP+: antibody pretreated with its antigenic peptide; Ctl: cells treated with Lipofectamine 2000 only; miR-1 and miR-133: cells transfected with miR-1 and miR-133, respectively and with Lipofectamine 2000. n=8 experiments for each group. (B) Regulation of Casp3 activity by miR-1 and miR-133. Transfection was performed with Lipofectamine 2000 and measurements were made 24 hours after transfection. *P<0.05 vs Ctl; +P<0.05 vs miR-1 alone or miR-133 alone or H2O2 alone; n=5 for each group.

 

Figure 5
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  		Fig. 5. Verification of HSP60 (A), HSP70 (B) and Casp9 (C) as cognate targets of miR-1 and miR-133, respectively, for post-transcriptional repression. Data on luciferase reporter activities show the interaction between miR-1 and HSP60 and HSP70 3'UTRs and between miR-133 and Casp9 mRNA. WT, wild type; MT, mutant, AMO-1 and AMO-133, miR-1- and miR-133-specific antisense inhibitors, respectively (see Fig. 2). Shown are means ± s.e.m. (n=5 batches of cells for each bar in A-C). *P<0.05 vs Ctl; +P<0.05 vs WT miR-1 or WT miR-133.

 

Figure 6
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  		Fig. 6. Comparison of miR-1 and miR-133 expression levels under various conditions, measured by real-time RT-PCR. (A) miR-1 and miR-133 levels without and with transfection of exogenous miR-1 and miR-133. The data are averaged from three batches of H9c2 cells. *P<0.05 vs non-transfected cells. (B) Enhanced expression of miR-1 and miR-133 induced by H2O2 (150 µM) in H9c2 cells (n=5 batches of cells for each group). *P<0.05 vs Ctl; +P<0.05 vs miR-1 or miR-133. (C) Comparison of miR-1 and miR-133 expression levels in various cell lines indicated. H9c2, rat ventricular cell line (n=6 batches of cells); A549, human lung cancer cell line (n=7 batches of cells); HEK293, human embryonic kidney cell line (n=7 batches of cells). MiR-1 and miR-133 levels are expressed as relative levels by normalizing to the values obtained from H9c2 cells. *P<0.05 vs H9c2.

 

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© The Company of Biologists Ltd 2007