A multifunctional protein EWS regulates the expression of Drosha and microRNAs

Abstract

EWS (Ewing's Sarcoma) gene encodes an RNA/DNA-binding protein that is ubiquitously expressed and involved in various cellular processes. EWS deficiency leads to impaired development and early senescence through unknown mechanisms. We found that EWS regulates the expression of Drosha and microRNAs (miRNAs). EWS deficiency resulted in increased expression of Drosha, a well-known microprocessor, and increased levels of miR-29b and miR-18b. Importantly, miR-29b and miR-18b were directly involved in the post-transcriptional regulation of collagen IV alpha 1 (Col4a1) and connective tissue growth factor (CTGF) in EWS knock-out (KO) mouse embryonic fibroblast cells. The upregulation of Drosha, miR-29b and miR-18b and the sequential downregulation of Col4a1 and CTGF contributed to the deregulation of dermal development in EWS KO mice. Otherwise, knockdown of Drosha rescued miRNA-dependent downregulation of Col4a1 and CTGF proteins. Taken together, our data indicate that EWS is involved in post-transcriptional regulation of Col4a1 and CTGF via a Drosha-miRNA-dependent pathway. This finding suggests that EWS has a novel role in dermal morphogenesis through the modulation of miRNA biogenesis.

Figure 1

EWS deficiency alters the level of miRNAs. (a) The heat map of miRNA array showed that relative expressions of miR-29b and miR-18b are highly increased in EWS KO MEF. The relative expression of miRNAs is displayed as colors: higher (red) or lower (green). The heat map represents the average of two samples. (b) RT-PCR analysis verified that the levels of pri-miR-29b and pri-miR-18b are decreased in subcellular fractions of EWS KO MEF compared with EWS WT MEF. The intron and exon of mouse GAPDH were amplified as markers of nuclear and cytoplasmic RNA, respectively. The graphs (right panel) represent the average±S.E.M. of three separate experiments. (c) qRT-PCR analysis verified that miR-29b and miR-18b are significantly increased in EWS KO MEF. Let-7f-1 was decreased in EWS KO MEF. The data represent the average±S.E.M. of three separate experiments. (d) qRT-PCR analysis showed that overexpression of EWS significantly reduces miR-29b and miR-18b levels in EWS KO MEFs. The data represent the average±S.E.M. of three separate experiments. Significantly different at *, P<0.05; **, P<0.005

Figure 2

Col4a1 and CTGF are targeted by miR-29b and miR-18b under EWS deficiency condition. (a) A biological network delineating cellular processes associated with cell adhesion, cell proliferation and cytoskeleton organization, development and morphogenesis is represented by functionally enriched 46 genes. Node colors denote the downregulation (green with red circle) or no change (gray) of mRNA expression under EWS deficiency. Genes in the network were grouped according to their interaction and function. The lines represent activation information obtained from KEGG pathways. (b) qRT-PCR analysis verified that the mRNA levels of Col4a1 and CTGF were significantly reduced in EWS KO MEF. The data represent the average±S.E.M. of three separate experiments. (c) Western blot analysis confirmed that Col4a1 and CTGF were downregulated in EWS KO MEF. (d) RT-PCR analysis verified that mRNAs of Col4a1 (exon, intron1 and 2) and CTGF (exon and intron 1) the Col4a1 and CTGF are differently modulated in subcellular fractions of EWS WT and KO MEFs. The intron 1 and exon of mouse GAPDH were amplified as markers of nuclear and cytoplasmic RNA, respectively. The data represent the average±S.E.M. of three separate experiments. (e) Overexpression of miR-29b and miR-18b downregulated the mRNA levels of Col4a1 and CTGF in EWS WT MEFs. The mRNAs were detected by qRT-PCR. The data represent the average±S.E.M. of five separate experiments. Significantly different at *, P<0.05; **, P<0.005. (f) Overexpression of miR-29b and miR-18b reduced the protein level of Col4a1 and CTGF in EWS WT MEFs

Figure 3

Overexpression of EWS leads to increases of Col4a1 and CTGF expression. (a) Overexpression of EWS increased the protein levels of Col4a1 and CTGF were in EWS MEFs. (b) qRT-PCR analysis represented that overexpression of EWS increased the mRNA levels of Col4a1 and CTGF in EWS MEF cells. The data represent the average±S.E.M. of three separate experiments. Significantly different at *, P<0.05; **, P<0.005

Figure 4

EWS regulates Drosha expression. (a) The heat map of transcriptome analysis showed relative expressions of miRNA processing-related genes in between EWS WT and KO MEFs. Drosha among many microprocessors was increased in EWS KO cells, wherease other genes were barely changed. The relative expression level of genes was displayed as colors: higher (pale red) or lower (violet). (b) qRT-PCR verified that the mRNA level of Drosha was most significantly increased in EWS KO MEF. The data represent the average±S.E.M. of three separate experiments. (c) Western blot analysis confirmed that the protein level of Drosha elevated in EWS KO MEF. Two independent experiments were performed for western blot analysis. The graphs represent the average±S.E.M. of three separate experiments. (d) The confocal microscopy presented that the immunoreactivity of Drosha is highly increased in the nucleus of EWS KO MEF, whereas the immunoreactivity of DGCR8 is not changed. Scale bar, 10 μm. (e) Overexpression of EWS reduced the protein level of Drosha in a dose-dependent manner. The graph represents the average±S.E.M. of five separate experiments. (f) Overexpression of EWS decreased the mRNA level of Drosha both in EWS WT and KO MEFs. The data represent the average±S.E.M. of five separate experiments. Significantly different at *, P<0.05; **, P<0.005. (g) Overexpression of EWS reduced the protein levels of Drosha both in EWS WT and KO MEFs

Figure 5

Drosha regulates the processing of pri-miR-29b and pri-miR-18b and involves in Col4a1 and CTGF expression. (a) Northern blot analysis of miR-29b and miR-18b in EWS MEF cells. Mature forms of the miR-29b and miR-18b were highly expressed in EWS KO MEFs. U6 was used as a loading control. (b) RT-PCR analysis revealed that the levels of pri-miR-29b and pri-miR-18b are decreased by Drosha in a dose-dependent manner (c) qRT-PCR analysis verified that Drosha reduces the level of pri-miR-29b in a dose-dependent manner. The data represent the average±S.E.M. of three separate experiments. (d) Drosha increased the level of miR-29b in a dose-dependent manner. The data represent the average±S.E.M. of three separate experiments. (e) Knockdown of Drosha increased the level of pri-miR-29b. The data represent the average±S.E.M. of three separate experiments. Significantly different at *, P<0.05; **, P<0.005. (f) Knockdown of Drosha, DGCR8, and Dicer increased Col4a1 and CTGF protein levels both in EWS WT and KO MEFs

Figure 6

EWS KO mice show the alteration of Drosha, miRNAs, and Col4a1 and CTGF. (a) Drosha mRNA levels were increased in EWS KO mice. The data represent the average±S.E.M. of three separate experiments. (b) miR-29b and miR-18b levels were markedly increased in EWS KO mice. (c) Col4a1 and CTGF mRNA levels were reduced in EWS KO mice. The data represent the average±S.E.M. of three separate experiments. Significantly different at *, P<0.05; **, P<0.005. (d) The protein levels of Drosha were increased in EWS KO mice whereas the protein levels of Col4a1 and CTGF were downregulated in the skin tissues of EWS KO mice (n=2). (e) Nissl staining showed severely altered skin structure of EWS KO mice. (f) Col4a1 and CTGF immunoreactivity were decreased in EWS KO mice skin tissues. All scale bars: 100 μm. (g) A schematic diagram illustrates that the upregulation of Drosha under EWS deficiency accelerates the processing of pri-miR-29b and pri-miR-18b in the nucleus. Increased miR-29b and miR-18b negatively regulate the mRNAs of Col4a1 and CTGF in the cytoplasm. As a result, EWS deficiency leads to impaired dermal development