Hsa-miR-1908-3p Mediates the Self-Renewal and Apoptosis of Human Spermatogonial Stem Cells via Targeting KLF2

Abstract

Spermatogenesis depends on precise epigenetic and genetic regulation of spermatogonial stem cells (SSCs). However, it remains largely unknown about the roles and mechanisms of small noncoding RNA in regulating the self-renewal and apoptosis of human SSCs. Notably, we have found that Homo sapiens-microRNA (hsa-miR)-1908-3p is expressed at a higher level in human spermatogonia than pachytene spermatocytes. MiR-1908-3p stimulated cell proliferation and DNA synthesis of the human SSC line. Allophycocyanin (APC) Annexin V and propidium iodide staining, determined by flow cytometric analysis and TUNEL assays, showed that miR-1908-3p inhibited early and late apoptosis of the human SSC line. Furthermore, Kruppel-like factor 2 (KLF2) was predicted and verified as the target of miR-1908-3p, and, significantly, KLF2 silencing resulted in the increase of proliferation and DNA synthesis, as well as reduction of apoptosis of the human SSC line. Moreover, KLF2 silencing ameliorated the decrease in the proliferation and DNA synthesis and the enhancement in the apoptosis of the human SSC line caused by miR-1908-3p inhibition. Collectively, these results implicate that miR-1908-3p stimulates the self-renewal and suppresses the apoptosis of human SSCs by targeting KLF2. This study thus provides a novel epigenetic regulatory mechanism underlying the fate determinations of human SSCs, and it offers new endogenous targets for treating male infertility.

Keywords: KLF2; apoptosis; hsa-miR-1908-3p; human spermatogonial stem cells; self-renewal.

Graphical abstract

Figure 1

Identification of the Human SSC Line (A) RT-PCR revealed gene expression of GFRA1, GPR125, THY1, UCHL1, PLZF, RET, and VASA in the human SSC line. RNA, without RT but with PCR, was used as a negative control, and ACTB served as the loading control of total RNA. (B–J) Immunocytochemistry showed the expression of GFRA1 (B), GPR125 (C), THY1 (D), VASA (E), UCHL1 (F), PLZF (G), RET (H), SV40 (I), and isotype IgGs (J) in the human SSC line. (B–J) Scale bars, 10 μm.

Figure 2

Effect of miR-1903-3p on the Proliferation of the Human SSC Line (A) Fluorescence microscope and phase-contrast microscope displayed transfection efficiency of miR-1908-3p mimics and inhibitor using the FAM-labeled siRNA oligonucleotides. Scale bars, 40 μm. (B) Real-time PCR demonstrated the relative levels of miR-1908-3p in the human SSC line after transfection of miR-1908-3p mimics, miRNA mimics control, miR-1908-3p inhibitor, and miRNA inhibitor control for 24 h. The level of miR-1908-3p was quantified with U6 as a loading control. (C and D) CCK-8 assays displayed the growth curve of the human SSC line treated with miR-1908-3p mimics and miRNA mimics control (C), as well as the miR-1908-3p inhibitor and miRNA inhibitor control (D) for 5 days. ∗p < 0.05, statistically significant differences compared to miRNA mimics control; #p < 0.05, statistically significant differences compared to miRNA inhibitor control.

Figure 3

Influence of miR-1903-3p on the DNA Synthesis of the Human SSC Line (A) Western blots demonstrated PCNA expression in the human SSC line at 48 h after transfection of miRNA mimic control, miR-1908-3p mimics, miRNA inhibitor control, and miR-1908-3p inhibitor. ACTB served as the loading control of protein. (B) The relative expression of PCNA in the human SSC line at 48 h after transfection of miR-1908-3p mimics to miRNA mimics control and miR-1908-3p inhibitor to miRNA inhibitor control through normalization to the signals of their loading control.(C–F) EDU incorporation assays showed the percentages of EDU-positive cells in the human SSC line treated with miRNA mimics control compared to the miR-1908-3p mimics for 48 h (C and D), as well as miR-1908-3p inhibitor compared to the miRNA inhibitor for 48 h (E and F). (C and E) Scale bars, 40 μm. ∗p < 0.05, statistically significant differences compared to miRNA mimics control; #p < 0.05, statistically significant differences compared to miRNA inhibitor control.

Figure 4

Impact of miR-1908-3p on the Apoptosis of the Human SSC Line (A–D) APC Annexin V and flow cytometric analysis showed the percentages of early and late apoptosis in the human SSC line affected by miRNA mimics control and miR-1908-3p mimics (A and B) and miRNA inhibitor control and miR-1908-3p inhibitor (C and D) for 48 h. (E–H) TUNEL assays displayed the percentages of TUNEL-positive cells in the human SSC line treated with miRNA mimics control and miR-1908-3p mimics (E and F) and miR-1908-3p inhibitor and miRNA inhibitor (G and H) for 48 h. (E and G) Scale bars, 40 μm. ∗p < 0.05, statistically significant differences compared to miRNA mimics control; #p < 0.05, statistically significant differences compared to miRNA inhibitor control.

Figure 5

KLF2 Is a Direct Downstream Target of miR-1908-3p in the Human SSC Line (A) Target genes of miR-1908-3p were predicted with three bioinformatics tools (TargetScan, miRTarBase, and miRDB). (B) Real-time PCR demonstrated the relative level of KLF2 mRNA in the human SSC line after transfection of miR-1908-3p mimics, miRNA mimics control, miR-1908-3p inhibitor, and miRNA inhibitor control for 24 h. The level of KLF2 mRNA was quantified with ACTB. (C) Western blots demonstrated KLF2 expression in the human SSC line at 48 h after transfection of miRNA mimic control, miR-1908-3p mimics, miRNA inhibitor control, and miR-1908-3p inhibitor. ACTB served as the loading control of protein. (D) The relative expression of KLF2 in the human SSC line at 48 h after transfection of miR-1908-3p mimics to miRNA mimics control and miR-1908-3p inhibitor to miRNA inhibitor control through normalization to the signals of their loading control.(E) The binding site of miR-1908-3p to the wild type and a mutated type of KLF2 mRNA. (F and G) Dual luciferase reporter assays validated the targeting of miR-1908-3p to wild-type KLF2 (F) or mutated KLF2 (G) after transfection of miR-1908-3p mimics compared to the miRNA mimics control. ∗p < 0.05, statistically significant differences compared to miRNA mimics control; #p < 0.05, statistically significant differences compared to miRNA inhibitor control.

Figure 6

Influence of KLF2 Silencing on the Proliferation, DNA Synthesis, and Apoptosis of the Human SSC Line (A) Real-time PCR demonstrated the relative level of KLF2 mRNA in the human SSC line after transfection of KLF2 siRNA 1, KLF2 siRNA 2, and KLF2 siRNA 3 compared to the control siRNA for 24 h. The level of KLF2 mRNA was quantified with ACTB as a loading control. (B) Western blots showed the relative protein level of KLF2 in the human SSC line affected by KLF2 siRNA 1, KLF2 siRNA 2, and KLF2 siRNA 3 compared to the control siRNA for 48 h. The band densities of KLF2 protein were quantified with ACTB as a loading control. (C) CCK-8 assays showed the proliferation of the human SSC line treated with KLF2 siRNA 1 compared with the control siRNA for 5 days. (D) Western blots revealed the relative protein level of PCNA in the human SSC line affected by KLF2 siRNA 1, KLF2 siRNA 2, and KLF2 siRNA 3 compared to the control siRNA for 48 h. The band densities of PCNA protein were quantified with ACTB as a loading control. (E) EDU assays showed the DNA synthesis of human SSC line treated with KLF2 siRNA 1 compared to the control siRNA for 48 h. Scale bars, 40 μm. (F and G) The percentages of apoptosis in the human SSC line affected by KLF2 siRNA 1 or control siRNA for 48 h, as determined by flow cytometric analysis (F) and TUNEL assays (G). (G) Scale bars, 40 μm. ∗p < 0.05, statistically significant differences compared to control siRNA.

Figure 7

The Function of the miR-1908-3p Inhibitor in the Effect of KLF2 Silencing on the Proliferation and DNA Synthesis of the Human SSC Line (A) Western blots demonstrated KLF2 expression in the human SSC line at 48 h after transfection of miRNA inhibitor control, miR-1908-3p inhibitor, and miR-1908-3p inhibitor and KLF2 siRNA 1. ACTB served as the loading control of protein. (B) The relative expression of KLF2 in the human SSC line at 48h after transfection of miR-1908-3p inhibitor to miRNA inhibitor control, and miR-1908-3p inhibitor and KLF2 siRNA 1 to miR-1908-3p inhibitor through normalization to the signals of their loading control. (C) CCK-8 assays demonstrated the proliferation of the human SSC line treated with miRNA inhibitor control, miR-1908-3p inhibitor, and miR-1908-3p inhibitor and KLF2 siRNA 1 of 5 days. (D) Western blots demonstrated PCNA expression in the human SSC line at 48 h after transfection of miRNA inhibitor control, miR-1908-3p inhibitor, and miR-1908-3p inhibitor and KLF2 siRNA 1. ACTB served as the loading control of protein. (E) The relative expression of PCNA in the human SSC line at 48 h after transfection of miR-1908-3p inhibitor to miRNA inhibitor control, and miR-1908-3p inhibitor and KLF2 siRNA 1 to miR-1908-3p inhibitor through normalization to the signals of their loading control. (F) EDU assays showed the DNA synthesis of human SSC line treated with miRNA inhibitor control, miR-1908-3p inhibitor, and miR-1908-3p inhibitor and KLF2 siRNA 1 for 48 h. (G) Qualification of EDU-positive cells in the human SSC line affected by miRNA inhibitor control, miR-1908-3p inhibitors, and miR-1908-3p inhibitors and KLF2 siRNA 1 in the human SSC line. (F) Scale bars, 40 μm. ∗p < 0.05, statistically significant differences compared to miRNA inhibitor control; #p < 0.05, statistically significant differences compared to miR-1908-3p inhibitor.

Figure 8

The Role of the miR-1908-3p Inhibitor in the Influence of KLF2 Silencing on the Apoptosis of the Human SSC Line (A–D) The percentage of apoptosis in the human SSC line affected by miRNA inhibitor control, miR-1908-3p inhibitor, and miR-1908-3p inhibitor and KLF2 siRNA 1 for 48 h, as determined by flow cytometric analysis (A and B) and TUNEL assays (C and D). (C) Scale bars, 40 μm. ∗p < 0.05, statistically significant differences compared to miRNA inhibitor control; #p < 0.05, statistically significant differences compared to miR-1908-3p inhibitor.