Identification of two miRNAs regulating cardiomyocyte proliferation in an Antarctic icefish

Summary The hemoglobinless Antarctic icefish develop large hearts to compensate for reduced oxygen-carrying capacity, which serves as a naturally occurred model to explore the factors regulating cardiogenesis. Through miRNAome and microRNAome comparisons between an icefish (Chionodraco hamatus) and two red-blooded notothenioids, we discovered significant upregulation of factors in the BMP signaling pathways and altered expression of many miRNAs, including downregulation of 14 miRNAs in the icefish heart. Through knocking down of these miRNAs, we identified two of them, miR-458-3p and miR-144-5p, involved in enlarged heart development. The two miRNAs were found to regulate cardiomyocyte proliferation by targeting bone morphogenetic protein-2 (bmp2). We further validated that activation of the miRNA-bmp2 signaling in the fish heart could be triggered by hypoxic exposure. Our study suggested that a few miRNAs play important roles in the hypoxia-induced cardiac remodeling of the icefish which shed new light on the mechanisms regulating cardiomyocyte proliferation in heart.

The miRNA mature sequences were downloaded using miRbase database, and the bmp2 3'UTR sequences were downloaded using NCBI database.target sequence prediction was performed using miRanda v3.3a software, and sequence comparison was performed and plotted using MEGA and GeneDoc software, with black shading representing a sequence conserved percentage of 100, gray representing a conserved percentage of 70, and light gray representing a conserved percentage of 40.Ventricle (μ m2) WT NC miR-458-3p miR-144-5p NC/ WT P value miR-4 5 8 -3 p / WT P value miR-4 5 8 -3 p / NC P value miR-144 -5 p/WT P value miR-144 -5 p Note: Small RNAs from total RNA preparations of miR-458-3p antagomir micro-injected, NC antagomir micro-injected, and WT zebrafish embryos heart tissues were isolated.The gene expression level was shown by fold changes.U6RNA was used as an internal control for qPCR analysis.The reactions were performed with three biological replicates, and each sample was assayed three times.The statistical significance was determined using a two-tailed unpaired Student t-test with *P < 0.05 (**P<0.01).
Supplementary Table 14.qRT-PCR results for miR-458-3p, miR-144-5p and bmp2 between the hypoxia acclimated zebrafish heart and the normoxia cultivated zebrafish heart, related to Figure 7. Note: Small RNAs from total RNA preparations of hypoxia acclimated or normoxia zebrafish heart tissues were isolated.The gene expression level was shown by fold changes.U6RNA was used as an internal control for qPCR analysis.The reactions were performed with three biological replicates, and each sample was assayed three times.
The statistical significance was determined using a two-tailed unpaired Student t-test with P < 0.05.
Gene Supplementary Table 15.qRT-PCR results for seven selected genes involved in cell cycle process between the hypoxia acclimated zebrafish heart and the normoxia cultivated zebrafish heartrelated to Figure 7. Note: The gene expression levels were shown by fold changes.β-actin was used as an internal control for qPCR analysis.All of the reactions were performed with three biological replicates, and each sample was assayed three times.The statistical significance was determined using a two-tailed unpaired Student t-test with P < 0.05.
the multiplicity of changes in gene expression in different samples, vertical coordinates represent the statistical significance of changes in gene expression, and scattered dots in the graph represent individual genes or miRNAs, gray dots represent genes or miRNAs with no significant differences, red dots represent significantly up-regulated differential genes or miRNAs, and blue dots represent significantly down-regulated differential genes or miRNAs.(A)Comparison of CH and GA differential genes under RNA-seq sequencing; (B) Comparison of CH and TB differential genes under RNA-seq sequencing; (C) Comparison of CH and GA differential mirrors under small RNA-seq sequencing; (D) Comparison of CH and TB differential mirrors under small RNA-seq sequencing; (E) Comparison of zebrafish small RNA-seq sequenced differential mirna under hypoxic and normoxic conditions Supplementary Fig.4 volcano plots for the RNA-seq and small RNA-seq data, related to Figure 2 and Figure 7.
The mapped reads of 12,774 unigenes were shown in scatterplot.Axis represents the biological replica.The Pearson's correlation coefficients (r) between the two replicates were calculated and the coefficients of determination (r 2 ) were shown in the Supplementary Fig.6Comparisons between the biological replication data sets of the C. hamatus (CH_1, CH_2 and CH_3) G. acuticeps (GA_1 and GA_2) and T. bernacchii (TB_1, TB_2 and TB_3),related to Figure 2 .plots.

Table 1 . Body mass, length, and heart-to-body mass ratio of T. bernacchii and
s heart shown on Figure1were from Number 1 fish samples of the two fish species.

Table 6 . Significantly upregulated genes invovled in cell cycle, cell division and cell proliferations, related to
Figure 2.

Table 7 .
qRT-PCR results for bmp2 and four selected genes involved in cell cycle process between the C. hamatus and T. bernacchii, related to Figure7.The gene expression levels were shown by fold changes.β-actin was used as an internal control for qPCR analysis.All of the reactions were performed with three biological replicates, and each sample was assayed three times.The statistical significance was determined using a two-tailed unpaired Student t-test with P < 0.05.
miRNA Supplementary

Table 9 .
The calculated expression levels of 6 selected miRNAs deduced from small RNA sequencing or quantitative real-time RT-PCR analysis, showing the consistency of differential expression of these miRNAs between C. hamatus and T. bernacchii detected by either method, related to Figure7.The gene expression levels were shown by fold changes.U6RNA was used as an internal control for qPCR analysis.All of the reactions were performed with three biological replicates, and each sample was assayed three times.The statistical significance was determined using a two-tailed unpaired Student t-test with P < 0.05.