Transcription factor GTF2I regulates osteoclast differentiation through mediating miR-134-5p and MAT2A expressions

Abstract

This study explored the possible effect of transcription factor GTF2I on the differentiation of osteoclasts and its regulation on the miR-134-5p/MAT2A axis. RANKL-induced osteoclasts were measured for expressions of GTF2I, miR-134-5p, and MAT2A. The number and size of osteoclasts were assessed after TRAP staining. The expressions of osteoclast differentiation biomarkers, NFATC1, TRAP, and CTSK, were detected as well. The relationships of the GTF2I/miR-134-5p/MAT2A axis were verified by ChIP, dual luciferase, and RNA pull-down assay. In vivo experiments were conducted on ovariectomized (OVX)-treated mice to determine the effect of GTF2I overexpression on osteoclast differentiation and bone loss. RANKL-induced osteoclasts had suppressed expressions of GTF2I and miR-134-5p and increased expression of MAT2A. GTF2I overexpression or miR-134-5p overexpression contributed to decreased cell number and size and suppressed cell differentiation, whereas such an effect can be abolished by overexpression of MAT2A. GTF2I can bind the miR-134-5p promoter to regulate its expression, whereas miR-134-5p can negatively regulate MAT2A expression. The protective effect of GTF2I overexpression against bone loss and cell differentiation was verified by in vivo experiments. Collectively, these results indicate that GTF2I can mediate miR-134-5p expression to increase MAT2A expression, contributing to the suppression of osteoclast differentiation.

Keywords: GTF2I; MAT2A; OVX; miR‐134‐5p; osteoclast differentiation.

FIGURE 1

GTF2I overexpression restricts osteoclastogenesis. (A) BMMs biomarker F4/80 was detected by immunofluorescence (N = 3); (B‐C) RT‐qPCR and western blot detected the mRNA and protein expressions of GTF2I (N = 3); (D) osteoclastogenesis was observed using TRAP staining (N = 3); (E‐F) RT‐qPCR and western blot detected the mRNA and protein expressions of NFATC1, TRAP, and CTSK (N = 3). Data were expressed as mean ± standard deviation. Comparisons among multiple groups were conducted using one‐way ANOVA followed by Tukey's multiple comparisons test for post‐hoc analysis. Cellular experiments were repeated 3 times. *p < 0.05, **p < 0.01, ***p < 0.001 when compared with the control group; ^# p < 0.05, ^## p < 0.01 when compared with the RANKL + oe‐NC group. BMMs, bone marrow macrophages.

FIGURE 2

GTF2I promotes GTF2I expression by binding its promoter to mediate osteoclastogenesis. (A) RT‐qPCR detected the expression of miR‐134‐5p (N = 3); (B‐C) ChIP and dual‐luciferase reporter gene assay confirmed the binding of GTF2I with miR‐134‐5p (N = 3); (D) after cell transfection, RT‐qPCR detected the expression of miR‐134‐5p (N = 3); (E) osteoclastogenesis was assessed using TRAP staining (N = 3); (F‐G) after cell transfection, the expressions of NFATC1, TRAP, and CTSK were detected by RT‐qPCR and western blot (N = 3). Data were expressed as mean ± standard deviation. Comparisons between two groups were conducted using the t‐test, whereas comparisons among multiple groups were conducted using one‐way ANOVA followed by Tukey's multiple comparisons test for post‐hoc analysis. Cellular experiments were repeated 3 times. *p < 0.05, **p < 0.01 when compared with the control, oe‐NC, IgG, or oe‐NC + in‐NC group; ^# p < 0.05, ^## p < 0.01 when compared with the oe‐GTF2I + in‐NC group.

FIGURE 3

miR‐134‐5p negatively regulates MAT2A expression. (A) the mRNAs that can bind miR‐134‐5p were predicted; (B) binding sites of miR‐134‐5p with MAT2A; (C‐D) MAT2A mRNA and protein expressions were detected by RT‐qPCR or western blot (N = 3); (E‐F) RNA pull‐down and dual‐luciferase reporter gene assay confirmed the binding of miR‐134‐5p with MAT2A (N = 3). Data were expressed as mean ± standard deviation. Comparisons between two groups were conducted using the t‐test. Cellular experiments were repeated 3 times. *p < 0.05, **p < 0.01 when compared with the control, mimic NC, or NC probe group.

FIGURE 4

miR‐134‐5p inhibits osteoclastogenesis through mediating MAT2A expression. (A‐B) RT‐qPCR and western blot detected MAT2A expression (N = 3); (C) osteoclastogenesis was assessed using TRAP staining (N = 3); (D‐E) NFATC1, TRAP, and CTSK expressions were detected using RT‐qPCR and western blot (N = 3). Data were expressed as mean ± standard deviation. Comparisons among multiple groups were conducted using one‐way ANOVA followed by Tukey's multiple comparisons test for post‐hoc analysis. Cellular experiments were repeated 3 times. *p < 0.05, **p < 0.01 when compared with the mimic NC + oe‐NC group. ^# p < 0.05, ^## p < 0.01 when compared with the miR‐134‐5p mimic + oe‐NC group.

FIGURE 5

MAT2A can abolish the suppressive effect of GTF2I overexpression on osteoclast differentiation. (A‐B) RT‐qPCR and western blot detected MAT2A expression (N = 3); (C) TRAP staining observed osteoclastogenesis (N = 3); (D‐E) RT‐qPCR and western blot detected the expressions of NFATC1, TRAP, and CTSK (N = 3); data were expressed as mean ± standard deviation. Comparisons among multiple groups were conducted using one‐way ANOVA followed by Tukey's multiple comparisons test for post‐hoc analysis. Cellular experiments were repeated 3 times. *p < 0.05, **p < 0.01 when compared with the NC group. ^# p < 0.05, ^## p < 0.01 when compared with the oe‐GTF2I + oe‐NC group. The NC group was set for both the oe‐GTF2I + oe‐NC group and oe‐GTF2I + oe‐MAT2A group.

FIGURE 6

GTF2I mediates osteoporosis in mice through regulating the miR‐134‐5p/MAT2A axis. (A‐B) RTq‐PCR and western blot detected GTF2I, miR‐134‐5p, and MAT2A expressions (N = 10); (C) H&E staining for morphological observation (N = 10); (D‐K) micro‐CT analyze the bone microenvironment, BV/TV, BMD, Tb.Th, Tb.N, Tb.Sp, Ct.Ar, and Ct.Th (N = 10); (L‐M) RT‐qPCR and western blot detected expressions of NFATC1, TRAP, and CTSK (N = 10); data were expressed as mean ± standard deviation. Comparisons among multiple groups were conducted using one‐way ANOVA followed by Tukey's multiple comparisons test for post‐hoc analysis. *p < 0.05, **p < 0.01, when compared with the sham group. ^# p < 0.05 when compared with the OVX + oe‐NC group. Tb.Th, trabecular thickness; BMD, bone mass density; BV/TV, bone volume/tissue volume; Tb.N, trabecular number; Tb.Sp, trabecular segregation; Ct.Ar, cortical area; Ct.Th, cortical thickness. Abbreviation: BMM, bone marrow macrophages; RTq‐PCR, reverse transcription‐quantitative polymerase chain reaction.

FIGURE 6

GTF2I mediates osteoporosis in mice through regulating the miR‐134‐5p/MAT2A axis. (A‐B) RTq‐PCR and western blot detected GTF2I, miR‐134‐5p, and MAT2A expressions (N = 10); (C) H&E staining for morphological observation (N = 10); (D‐K) micro‐CT analyze the bone microenvironment, BV/TV, BMD, Tb.Th, Tb.N, Tb.Sp, Ct.Ar, and Ct.Th (N = 10); (L‐M) RT‐qPCR and western blot detected expressions of NFATC1, TRAP, and CTSK (N = 10); data were expressed as mean ± standard deviation. Comparisons among multiple groups were conducted using one‐way ANOVA followed by Tukey's multiple comparisons test for post‐hoc analysis. *p < 0.05, **p < 0.01, when compared with the sham group. ^# p < 0.05 when compared with the OVX + oe‐NC group. Tb.Th, trabecular thickness; BMD, bone mass density; BV/TV, bone volume/tissue volume; Tb.N, trabecular number; Tb.Sp, trabecular segregation; Ct.Ar, cortical area; Ct.Th, cortical thickness. Abbreviation: BMM, bone marrow macrophages; RTq‐PCR, reverse transcription‐quantitative polymerase chain reaction.