MicroRNA-449 and microRNA-34b/c function redundantly in murine testes by targeting E2F transcription factor-retinoblastoma protein (E2F-pRb) pathway

Abstract

MicroRNAs (miRNAs) mainly function as post-transcriptional regulators and are involved in a wide range of physiological and pathophysiological processes such as cell proliferation, differentiation, apoptosis, and tumorigenesis. Mouse testes express a large number of miRNAs. However, the physiological roles of these testicular miRNAs remain largely unknown. Using microarray and quantitative real time PCR assays, we identified that miRNAs of the microRNA-449 (miR-449) cluster were preferentially expressed in the mouse testis, and their levels were drastically up-regulated upon meiotic initiation during testicular development and in adult spermatogenesis. The expression pattern of the miR-449 cluster resembled that of microRNA-34b/c (miR-34b/c) during spermatogenesis. Further analyses identified that cAMP-responsive element modulator τ and SOX5, two transcription factors essential for regulating male germ cell gene expression, acted as the upstream transactivators to stimulate the expression of the miR-449 cluster in mouse testes. Despite its abundant expression in testicular germ cells, miR-449-null male mice developed normally and exhibited normal spermatogenesis and fertility. Our data further demonstrated that miR-449 shared a cohort of target genes that belong to the E2F transcription factor-retinoblastoma protein pathway with the miR-34 family, and levels of miR-34b/c were significantly up-regulated in miR-449-null testes. Taken together, our data suggest that the miR-449 cluster and miR-34b/c function redundantly in the regulation of male germ cell development in murine testes.

FIGURE 1.

Expression profiles of miR-449 cluster and miR-34 family in mice. A, qPCR analyses of expression levels of miR-449 in 11 tissues collected from adult male mice. B, germ cell depletion through busulfan treatment followed by qPCR detection of miR-449. miR-449 was abundant in control (untreated wild type) testes, whereas miR-449 was undetectable in busulfan-treated (germ cell-depleted) testes, suggesting that miR-449 is exclusively expressed in germ cells. Scale bar, 50 μm. C, qPCR analyses of expression levels of miR-449 and members of the miR-34 family during postnatal testicular development. D, localization of miR-449 in the mouse testis using in situ hybridization. Hybridization signals are in red (Texas Red), and the nuclei were counterstained with hematoxylin (blue). The inset is a control. Scale bar, 70 μm. E, an enlarged image of the field framed be the dashed box in D. Specific miR-449a hybridization signals were mainly detectable in the cytoplasm of spermatocytes and spermatids (arrowheads), whereas no signals were fund in somatic cell types and spermatogonia (arrows). Scale bar, 50 μm. Ctr, control. F, localization of miR-34a in the murine testis by in situ hybridization. Hybridization signals (red) were only detected in spermatogonia. Cell nuclei (blue) were counterstained with hematoxylin (blue). The inset is a control. Scale bar, 50 μm. Data are represented as mean ± S.E. (n = 3).

FIGURE 2.

CREMτ and SOX5 transactivate miR-449 expression by binding two conserved cis-elements upstream of Cdc20b. A, RT-PCR analyses of Cdc20b expression in multiple adult mouse tissues (upper panel) and developing mouse testes (lower panel). B, schematic representation of putative conserved CREMτ and SOX5 binding sites (highlighted with red boxes) in the promoter region of Cdc20b. C, mapping of the upstream transcriptional regulatory region of Cdc20b/miR-449 cluster by luciferase reporter assays. Luciferase plasmids containing a partial deletion or mutation in CREMτ and SOX5 binding sites were constructed as indicated. Normalized luciferase activity is represented as mean ± S.E. (n = 3). All groups were performed in triplicate. D, co-transfection luciferase reporter assays using the GC-2 cell line identified that both CREMτ (p < 0.05) and SOX5 (p < 0.01) proteins can efficiently activate the −1.2 kb-containing reporter construct. Various combinations of plasmids transfected into GC-2 cells are indicated below each group. Luc1.2k^C-MT, luciferase construct containing a mutation in the candidate CREMτ binding site; Luc1.2k^S-MT, luciferase reporter containing a mutation in the candidate SOX5 binding site. E, knockdown of CREMτ individually or knockdown of CREMτ and SOX5 simultaneously using siRNAs led to decreased levels of miR-449 in GC-2 cells (n = 3; p < 0.05). siCREMτ, siRNA designed against CREMτ mRNA; siSOX5, siRNA designed against SOX5. Small nucleolar RNU6B was concurrently amplified for data normalization. F, ChIP assays confirmed the binding of both CREMτ and SOX5 to the −1.2 kb promoter region upstream of Cdc20b gene. rep1, rep2, and distal represent three different PCR fragments designed against the genomic region as indicated. Luc, luciferase; mut, mutant; TSS, transcription start site; Ab, antibody; Con, control. Data are represented as mean ± S.E. (n = 3).

FIGURE 3.

Lack of testicular phenotype in miR-449^−/− mice. A, gross morphology of testes from adult male WT, miR-449^+/−, and miR-449^−/− mice. B, average testicular weight of adult WT, miR-449^+/−, and miR-449^−/− mice. C–E, hematoxylin-eosin-stained testicular sections of WT (C), miR-449^+/− (D), and miR-449^−/− (E) mice. Scale bar, 70 μm. F–H, hematoxylin-eosin-stained cauda epididymal sections of WT (C), miR-449^+/− (D), and miR-449^−/− (E) mice. Scale bar, 100 μm. I, cauda epididymal sperm counts in WT, miR-449^+/−, and miR-449^−/− male mice. Data are represented as mean ± S.E. (n = 3). J, volcano plot showing differentially expressed mRNAs between miR-449 KO and wild type testes determined by Illumina microarray analyses. The x axis represents -fold changes (KO versus WT) in log₂ scale (red vertical line marks the 2-fold change). The y axis shows p value significance for each gene-specific analysis of variance in the −log₁₀ scale (red horizontal line means p = 0.05). Only six genes (Cyp2c55, Gbf1, Itgad, Pbx4, Plk1, and Vav2) can be considered as significantly deregulated between KO and WT (fold change >2; p < 0.05). Data are represented as mean ± S.E. (n = 3).

FIGURE 4.

Shared mRNA targets between miR-449 and miR-34b/c and potential compensatory effects of miR-34b/c in miR-449 knock-out mice. A, high similarity of mature sequences between members of the miR-449 cluster and the miR-34 family from humans, mice, and rats. The conserved motif “GGCAGUG” located in the “seed region” of each miRNA is highlighted in bold. B, qPCR analyses showing that levels of miR-34b and miR-34c, rather than miR-34a, were notably up-regulated in miR-449 KO mice as compared with those in wild type. *, p < 0.05. C, cell proliferation assays showing a time-dependent decrease in total cell number of GC-1 cells transfected with pre-miR-449a compared with the scrambled transfection control (Ctrol). D, qPCR analysis of levels of mRNAs for CCND1, BCL2, E2F2, E2F3, and MYC in HeLa cells transfected with pre-miR-449a. The values were normalized against GAPDH endogenous control. E, highly conserved miR-449a sequences among nine species. Sequences highlighted in bold indicate identical nucleotides. F, schematic illustration of the luciferase reporter assay. The 3′-UTR sequence for each target gene was inserted downstream of the luciferase coding sequence in pGL3.0 control vector (Promega) as described previously (19). G, luciferase reporter assays demonstrated that miR-449 was capable of repressing expression of genes encoding factors belonging to the E2F-pRb pathway, including CCND1, BCL2, E2F2, E2F3, and MYC (n = 3; *, p < 0.05). SCRAMBLE, a non-sense double strand siRNA showing no homology to any mRNA in the murine genome. Data are represented as mean ± S.E. (n = 3).

FIGURE 5.

Exhaustive correlation analyses between miR-449a/b- and miR-34b/c-induced mRNA transcriptomic changes in human airway mucociliary epithelial cells. A, significant down-regulation (p < 1.27e−09; two-tailed Wilcoxon rank sum test) of mRNAs (red line) possessing predicted binding motifs for the miR-449a/b seed region as compared with mRNAs lacking miR-449a/b binding motifs (black line). B, significant down-regulation (p < 6.720e−29; two-tailed Wilcoxon rank sum test) of mRNAs (red line) possessing predicted binding motifs for the miR-34b/c seed sequence compared with mRNAs lacking miR-34b/c binding motifs (black line). C, mRNA transcriptomic changes induced by miR-449a/b are highly correlated with those by miR-34b/c. -Fold changes are represented as log₂ scale. Pearson's correlation coefficient r = 0.86.

FIGURE 6.

Proposed model for miR-449/miR-34 to act through E2F-pRb pathway in control of male germ cell development during murine testicular development and spermatogenesis. In this model, CREMτ and SOX5 activate the expression of miR-449 upon meiotic initiation. miR-449 in collaboration with miR-34b/c then suppresses the expression of factors that belong to the E2F-pRb pathway, including CDK4/6, CCND1, CDK25A, MYC, and E2F1/2/3/5. These changes further cause reduced levels of E2F proteins and an increase in hypophosphorylated pRb protein level, which will further reduce E2F activities and thus affect male germ cell development during both the meiotic and haploid phases of spermatogenesis.