Article Figures & Data
Figures
- FIG 1
miRNAs regulated by CHD1L in mouse spermatogonial stem cells (mSSCs). Thy1+ mSSCs were infected with scrambled, nontargeted (sh-NT) or Chd1l gene-specific (sh-Chd1l) shRNA lentivirus. After 48 h, total RNAs, including small RNAs, were harvested and subjected to real-time quantitative PCR (RT-qPCR) and high-throughput miRNA sequencing (Sequencing) analysis, respectively. (A) RT-qPCR analysis of Chd1l gene expression. Data presented here are mean values ± standard errors of the means (SEM) from three independent experiments (n = 3). *, P < 0.05 (versus sh-NT). (B) Hierarchical cluster and heat map to show changes in miRNAs in CHD1L knockdown SSCs (sh-Chd1l) compared to expression levels in control SSCs (sh-NT). Red and green represent increase and decrease of expression, respectively. Each row represents the results for a single miRNA in small RNA sequencing analysis. The 20 miRNAs most up- or downregulated after CHD1L knockdown are shown. (C) KEGG pathways associated with the target genes of the miRNAs most regulated by CHD1L. (D) miRNA expression profile and validation. The miRNAs most up- or downregulated by CHD1L in mSSCs were selected and validated by standard RT-qPCR. Data shown here are mean values ± SEM from three independent experiments (n = 3), which are presented as log2 fold changes with the miRNA expression level in control mSSCs set as 0. *, P < 0.05 and FDR < 0.05 (for sequencing); #, P < 0.05 (for RT-qPCR).
- FIG 2
CHD1L regulates miR-486 in mouse spermatogonial stem cells (mSSCs) through a transcriptional mechanism. (A) C18-4 cells (mouse spermatogonial stem cell line) with Chd1l overexpression had downregulated miR-486. C18-4 cells were transfected with control (pcDNA3.1) or Chd1l overexpression (pcDNA3.1-Chd1l) plasmid. (B) Both mature and primary (pri-miR-486) miR-486 transcripts were upregulated in CHD1L knockdown SSCs. C18-4 cells were infected with scrambled, nontargeted (sh-NT) or Chd1l gene-specific (sh-Chd1l) shRNA lentivirus. (A and B) After 48 h, total RNAs, including small RNAs, were harvested and subjected to RT-qPCR analysis. (C) CHD1L regulates pri-miR-486 in mSSCs through a transcriptional mechanism. C18-4 cells infected with sh-NT or sh-Chd1l lentivirus were treated with an inhibitor of transcription (actinomycin D [ActD]; 1 µg/ml) for the indicated times. The result for pri-miR-486 at 0 h for both groups was set as 1.0 (arbitrary unit), and values at other time points calculated accordingly. (D and E) Neither the embryonic and adult skeletal muscle promoter nor the adult cardiac and skeletal muscle promoter for the Ank1.5 gene is required for CHD1L-mediated miR-486 expression in mSSCs. C18-4 cells infected with sh-NT or sh-Chd1l lentivirus were transfected with the indicated reporters. Luciferase activities were measured at 48 h posttransfection. The data presented here are representative mean values ± SEM from three independent experiments (n = 3). *, P < 0.05 (versus sh-NT).
- FIG 3
miR-486 controls SSC stemness gene expression and growth properties. (A to C) SSC stemness gene expression levels were downregulated by miR-486. C18-4 cells were transfected with miRNA negative control (Ctrl mimic) or miR-486 mimic, respectively. After 48 h, total RNAs, including small RNAs, and proteins were harvested and subjected to RT-qPCR (A and B) and Western blot analysis (C), respectively. (D) miR-486 inhibits SSC growth. C18-4 cells transfected with miRNA negative control (Ctrl mimic) or miR-486 mimic were subjected to cell proliferation analysis using an MTS assay kit. OD, optical density. (E) Immunofluorescence analysis of pH3-positive mouse SSCs. (F) miR-486 increases SSC apoptosis. C18-4 cells transfected with miRNA negative control (Ctrl mimic) or miR-486 mimic were harvested and subjected to apoptosis assay. Cells that stained positive for annexin V (x axis) but negative for propidium iodide (y axis) were considered apoptotic cells. (G) miR-486 promotes SSC cell cycle arrest at G0/G1 phase. C18-4 cells transfected with miRNA negative control (Ctrl mimic) or miR-486 mimic were harvested and subjected to cell cycle analysis. The data presented here are representative images or mean values ± SEM from three independent experiments (n = 3). *, P < 0.05 (versus Ctrl mimic).
- FIG 4
The stemness marker expression and growth properties of SSCs are regulated by miR-486. C18-4 cells were infected with control (sh-NT) or miR-486 (sh-miR-486) lentivirus. Cells were harvested and subjected to various analyses as indicated. (A to C) SSC stemness marker expression levels were upregulated by miR-486 inhibition. (D) miR-486 suppression increased SSC growth. (E) SSC proliferation (pH3-positive cells) was increased by miR-486 knockdown. (F) miR-486 knockdown decreased SSC apoptosis. Cells that stained positive for annexin V (x axis) but negative for propidium iodide (y axis) were considered apoptotic cells. The data presented here are representative images or mean values ± SEM from three independent experiments (n = 3). *, P < 0.05 (versus sh-NT).
- FIG 5
MMP2 is the novel target of miR-486 in SSCs. (A) The GO and KEGG pathway analyses of the mRNAs significantly downregulated by CHD1L knockdown in SSCs. Thy1+ mSSCs were infected with scrambled, nontargeted (sh-NT) or Chd1l gene-specific (sh-Chd1l) shRNA lentivirus. After 48 h, total RNAs were harvested and subjected to high-throughput mRNA sequencing analysis. The genes that displayed significant downregulation by Chd1l knockdown in SSCs were analyzed using DAVID. The top 10 KEGG pathways are presented here. (B) Venn diagram illustrating the downregulated genes observed in mRNA sequencing (RNA-seq) analysis and the miR-486 target genes predicted by miRWalk. (C) Immunohistochemistry analysis of MMP2 protein expression in mouse testis. dpp, days postpartum. (D) Western blot analysis of Chd1l and MMP2 protein expression in mouse testis. (E and F) MMP2 expression was suppressed in SSCs with Chd1l knockdown. (G to J) The MMP2 protein level, but not the mRNA level, was modulated by miR-486 in SSCs. (K) Schematic diagram illustrating the miR-486 sequence and the wild-type (WT) and mutated (MUT) miR-486 binding sites within the 3′UTR of Mmp2 gene reporters. (L and M) miR-486 binding site was required for miR-486-mediated Mmp2 gene repression. Control miRNA mimic (Ctrl mimic) or miR-486 mimic was cotransfected into C18-4 cells with the wild-type reporter (pmiR-mmp2-WT) or the reporter containing the mutated miR-486 binding site (pmiR-Mmp2-Mut). Transfected cells were cultured for 48 h before luciferase activity assay. The data presented here are representative images or mean values ± SEM from three independent experiments (n = 3). *, P < 0.05 (versus sh-NT or Ctrl mimic).
- FIG 6
Mmp2 inhibition reproduces the effects of miR-486 overexpression on SSC stemness gene expression and growth properties. (A to C) SSC stemness marker expression was suppressed by Mmp2 knockdown. C18-4 cells were transfected with control (si-NT) or one of two Mmp2-specific [si-mmp2 (#1 & #2)] siRNAs. After 48 h, total RNAs and proteins were harvested and subjected to RT-qPCR (A and B) and Western blot analysis (C), respectively. (D and E) Mmp2 knockdown decreased SSC growth (MTS assay) (D) and proliferation (pH3-positive cells) (E). (F) Mmp2 knockdown increased SSC apoptosis. Cells that stained positive for annexin V (x axis) but negative for propidium iodide (y axis) were considered apoptotic cells. (G) Mmp2 knockdown increased SSC G0/G1 cell cycle arrest. The data presented here are representative images or mean values ± SEM from three independent experiments (n = 3). *, P < 0.05 (versus si-NT).
- FIG 7
miR-486 repression is required for CHD1L-mediated SSC stemness gene expression and growth properties. C18-4 cells infected with control (sh-NT) or Chd11 (sh-Chd1l) lentivirus were coinfected with control (sh-NT) or miR-486 (sh-miR-486) lentivirus as indicated. Infected cells were cultured for 48 h and subjected to RT-qPCR (A and C), Western blot analysis (B and D), MTS assay (E), pH3 immunofluorescence staining (F), apoptosis assay (G), or cell cycle assay (H). (A) RT-qPCR detection of Chd1l, miR-486, and Mmp2 gene expression levels. (B) Western blot detection of MMP2 protein expression in the infected cells. (C and D) miR-486 inhibition partially rescued the expression levels of the SSC stemness markers examined. (E and F) miR-486 repression partially released the inhibitory effects of Chd1l knockdown on SSC growth (E) and proliferation (F). (G) miR-486 knockdown almost blunted the apoptotic effects induced by Chd1l inhibition in SSCs. (H) The promotive effect of Chd1l knockdown on SSC cell cycle arrest at G0/G1 phase was almost abolished by miR-486 inhibition. The data presented here are representative images or mean values ± SEM from three independent experiments (n = 3). *, P < 0.05 (versus sh-NT); ǂ, P < 0.05 (sh-Chd1l/sh-miR-486 versus sh-Chd1l).
- FIG 8
miR-486 regulates SSC stemness gene expression and growth properties through modulating MMP2 protein expression. C18-4 cells infected with control (sh-NT) or miR-486 (sh-miR-486) lentivirus were cotransfected with control (si-NT) or Mmp2 (si-mmp2) siRNA as indicated. Transfected cells were cultured for 48 h and subjected to RT-qPCR (A and C), Western blot analysis (B and D), MTS assay (E), pH3 immunofluorescence staining (F), apoptosis assay (G), or cell cycle assay (H). (A) RT-qPCR detection of Chd1l, miR-486, and Mmp2 gene expression. (B) Western blot detection of MMP2 protein expression in the infected cells. (C and D) Stemness gene induction caused by miR-486 knockdown in SSCs was normalized by Mmp2 inhibition. (E and F) Mmp2 repression abrogated the promotive effects of miR-486 inhibition on SSC growth (E) and proliferation (F). (G and H) The inhibitory effects of miR-486 knockdown on SSC apoptosis and cell cycle arrest were completely reversed by Mmp2 gene inhibition. The data presented here are representative images or mean values ± SEM from three independent experiments (n = 3). *, P < 0.05 (versus sh-NT/si-NT); ǂ, P < 0.05 (sh-miR-486/si-mmp2 versus sh-miR-486).
- FIG 9
MMP2 regulates SSC stemness gene expression and growth through activating β-catenin signaling by cleaving N-cadherin. (A and B) MMP2 increased SSC stemness gene expression. C18-4 cells were treated with 10 ng/ml recombinant MMP2 for the indicated times. Total RNAs and proteins were harvested and subjected to RT-qPCR (A) and Western blot analysis (B), respectively. (C) MMP2 promoted SSC growth. C18-4 cells were incubated with vehicle control or 10 ng/ml recombinant MMP2 and subjected to cell proliferation analysis using an MTS assay kit. (D) Wnt/β-catenin/TCF/LEF signaling was activated by MMP2 in SSCs. C18-4 cells were transfected with either M50 Super 8x TOPFlash (plasmid 12456; Addgene) or M51 Super 8x FOPFlash (plasmid 12457p; Addgene) reporter plasmid for 44 h, followed by incubation with vehicle control or 10 ng/ml recombinant MMP2 for an additional 4 h. Cell lysates were harvested and subjected to a standard luciferase activity assay. (E) Immunofluorescence staining revealed the cellular locations of β-catenin and N-cadherin. (F) MMP2 cleaves N-cadherin. Recombinant N-cadherin (1,000 ng per reaction mixture volume) (ab112268; Abcam) was incubated with activated MMP2 (100 ng per reaction mixture volume) (ab174022; Abcam) in assay buffer for 4 h, followed by Western blot analysis with an antibody against N-cadherin (ab76057; Abcam). FL, full length recombinant N-cadherin protein. The data presented here are representative images or mean values ± SEM from three independent experiments (n = 3). *, P < 0.05 (versus 0 h or vehicle).
Tables
- TABLE 1
Primers used in this study
Gene Primer sequence (5′–3′) Application Gapdh F GCCTCAAGATCAGCAAT RT-qPCR Gapdh R AGGTCCACCACTGACACGTT RT-qPCR Chd1l F GACCTGAGTTTGGGTGATG RT-qPCR Chd1l R CGGATAAGTAGTTCGGTA RT-qPCR Oct4 F GGCGTTCTCTTTGGAAAGGTGTTC RT-qPCR Oct4 R CTCGAACCACATCCTTCTCT RT-qPCR Nanog F GCTGAGATGCCTCACACGGAG RT-qPCR Nanog R TCTGTTTCTTGACTGGGACCTTGTC RT-qPCR Mmp2 F CCCCATGTGTCTTCCCCTTC RT-qPCR Mmp2 R GTCAGTATCAGCATCGGGGG RT-qPCR Plzf F GTGCCTCGCCATACCAGTGTAC RT-qPCR Plzf R CCCCTTTTCTTTTCTGTTATTCTTTT RT-qPCR c-Myc F GGTGGGTCGTCGAGTGCTAG RT-qPCR c-Myc R AGTGGTTACCGCCTTGTTGTTA RT-qPCR Mmp2 3′UTR (WT) F CGAGCTCGGCCCTATCATCTTCATCGC pmiR-Mmp2 Mmp2 3′UTR (WT) R GCTCTAGATATTTATACTTGTTTGCCATTT pmiR-Mmp2 Mmp2 3′UTR (MUT) F CCTGGCATGGGGCAGTGGGCTGACGAT
GTGGCCAAGGAAATCpmiR-Mmp2 Mmp2 3′UTR (MUT) R GATTTCCTTGGCCACATCGTCAGCCCA
CTGCCCCATGCCAGGpmiR-Mmp2 U6 F CTCGCTTCGGCAGCACA RT-qPCR U6 R AACGCTTCACGAATTTGCGT RT-qPCR miR-486 F ACACTCCAGCTGGGCGGGGCAGCTCAGTA RT-qPCR miR-486 R TGGTGTCGTGGAGTCG RT-qPCR miR-486 shRNA TCCTGTACTGAGCTGCCCCGAG miR-486 sh 1,080-bp upstream region F GGGGTACCCCCTTGTAACTCCCAGTGGCTG pGL3-miR-486 1,080-bp upstream region R GAAGATCTGATGAAGGTCCACATCCTCCT pGL3-miR-486 Intron F GGGGTACCGTAACACCAGGCAAGCGGCAG pGL3-miR-486 Intron R GAAGATCTCTGGAGTTAGAAGAAGGGAAA pGL3-miR-486 Distal region F GGGGTACCCCCTTGTAACTCCCAGTGGCTG pGL3-miR-486 Distal region R GAAGATCTGAGAGGCTCACAGCACAGGA pGL3-miR-486 Middle region F GGGGTACCCAACCCAGCCTGAGGAGAGC pGL3-miR-486 Middle region R GAAGATCTCTTGGTCTCAGGAAACACAT pGL3-miR-486 Proximal region F GGGGTACCATACAGCACCTGCTGTCTCC pGL3-miR-486 Proximal region R GAAGATCTGATGAAGGTCCACATCCTCCT pGL3-miR-486
