Endocrinol Metab.  2023 Feb;38(1):156-173. 10.3803/EnM.2022.1516.

MicroRNA-181a-5p Curbs Osteogenic Differentiation and Bone Formation Partially Through Impairing Runx1-Dependent Inhibition of AIF-1 Transcription

• Liu J
• Chang X
• Dong D

• KMID: 2539829
• DOI: http://doi.org/10.3803/EnM.2022.1516

Abstract

Background
Evidence has revealed the involvement of microRNAs (miRNAs) in modulating osteogenic differentiation, implying the promise of miRNA-based therapies for treating osteoporosis. This study investigated whether miR-181a-5p influences osteogenic differentiation and bone formation and aimed to establish the mechanisms in depth.
Methods
Clinical serum samples were obtained from osteoporosis patients, and MC3T3-E1 cells were treated with osteogenic induction medium (OIM) to induce osteogenic differentiation. miR-181a-5p-, Runt-related transcription factor 1 (Runx1)-, and/or allograft inflammatory factor-1 (AIF-1)-associated oligonucleotides or vectors were transfected into MC3T3-E1 cells to explore their function in relation to the number of calcified nodules, alkaline phosphatase (ALP) staining and activity, expression levels of osteogenesis-related proteins, and apoptosis. Luciferase activity, RNA immunoprecipitation, and chromatin immunoprecipitation assays were employed to validate the binding relationship between miR-181a-5p and Runx1, and the transcriptional regulatory relationship between Runx1 and AIF-1. Ovariectomy (OVX)-induced mice were injected with a miR-181a-5p antagonist for in vivo verification.
Results
miR-181a-5p was highly expressed in the serum of osteoporosis patients. OIM treatment decreased miR-181a-5p and AIF-1 expression, but promoted Runx1 expression in MC3T-E1 cells. Meanwhile, upregulated miR-181a-5p suppressed OIM-induced increases in calcified nodules, ALP content, and osteogenesis-related protein expression. Mechanically, miR-181a-5p targeted Runx1, which acted as a transcription factor to negatively modulate AIF-1 expression. Downregulated Runx1 suppressed the miR-181a-5p inhibitor-mediated promotion of osteogenic differentiation, and downregulated AIF-1 reversed the miR-181a-5p mimic-induced inhibition of osteogenic differentiation. Tail vein injection of a miR-181a-5p antagonist induced bone formation in OVX-induced osteoporotic mice.
Conclusion
In conclusion, miR-181a-5p affects osteogenic differentiation and bone formation partially via the modulation of the Runx1/AIF-1 axis.

Keyword

MicroRNAs; Transcription factors; Ovariectomy; Osteoporosis; Osteogenesis

Figure

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  Fig. 1. miR-181a-5p was upregulated in the serum of patients with osteoporosis and downregulated during osteogenic differentiation. (A) Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to detect miR-181a-5p expression in the serum of osteoporosis patients and normal controls (normal group, n=48; osteoporosis group, n=56). (B) qRT-PCR was used to detect miR-181a-5p expression in MC3T3-E cells during osteogenic differentiation. aP<0.05 vs. day 0 of induction; bP<0.01 vs. the normal group or day 0 of induction.

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  Fig. 2. miR-181a-5p inhibited osteogenic differentiation in vitro. (A) Alizarin red S staining was used to detect mineralization in MC3T3-E1 cells transfected with miR-181a-5p-mimic and miR-181a-5p-inhibitor. (B, C) Alkaline phosphatase (ALP) staining and an ALP enzyme assay were conducted to detect osteogenic differentiation in MC3T3-E1 cells transfected with the miR-181a-5p-mimic and miR-181a-5p-inhibitor. (D, E) The expression levels of osteogenesis-related proteins (osteocalcin [OCN], osteopontin [OPN], collagen type I [Col1A1], and ALP) in MC3T3-E1 cells transfected with the miR-181a-5p-mimic and miR-181a-5p-inhibitor were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assays. (F) Flow cytometry analysis of cell apoptosis in MC3T3-E1 cells transfected with the miR-181a-5p-mimic and miR-181a-5p-inhibitor. Comparisons among multiple groups were conducted using one-way analysis of variance, followed by the Tukey multiple-comparison test. The experiment was repeated three times. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. aP<0.05 vs. the mimic-negative control (NC) group or inhibitor-NC group; bP<0.01 vs. the mimic-NC group or inhibitor-NC group.

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  Fig. 3. Runt-related transcription factor 1 (Runx1) was identified as a target gene of miR-181a-5p. (A) The binding site of miR-181a-5p to Runx1 was predicted by StarBase. (B, C) The binding relationship between miR-181a-5p and Runx1 was verified by a dual-luciferase reporter gene assay and an RNA immunoprecipitation assay. (D, E) Runx1 expression in MC3T3-E1 cells transfected with a miR-181a-5pmimic and miR-181a-5p-inhibitor was tested by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assays. (F, G) Runx1 expression in the serum of osteoporosis patients and normal controls was tested by qRT-PCR and Western blot assays (normal group, n=48; osteoporosis group, n=56). Comparisons between two groups were performed using the t test, and comparisons among multiple groups were conducted using one-way analysis of variance, followed by the Tukey multiple-comparison test. The experiment was repeated three times. WT, wild-type; MUT, mutant; RIP, RNA immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. aP<0.05 vs. the mimic-negative control (NC) group, inhibitor-NC group or normal group; bP<0.01 vs. the mimic-NC group or normal group; cP<0.001 vs. the immunoglobulin G (IgG) group.

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  Fig. 4. Downregulation of Runt-related transcription factor 1 (Runx1) inhibited osteogenic differentiation. (A, B) Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assays for the detection of Runx1 expression during osteogenic differentiation induction. (C, D) Runx1 expression in MC3T3-E1 cells transfected with sh-Runx1 and sh-negative control (NC) was determined by qRT-PCR and Western blot assays. (E) Alizarin red S (ARS) staining was utilized to detect mineralization in MC3T3-E1 cells transfected with sh- Runx1 and sh-NC. (F, G) Alkaline phosphatase (ALP) staining and an ALP enzyme assay were performed to detect osteogenic differentiation in MC3T3-E1 cells transfected with sh-Runx1 and sh-NC. (H, I) The expression levels of osteogenesis-related proteins (osteocalcin [OCN], osteopontin [OPN], collagen type I [Col1A1], and ALP) in MC3T3-E1 cells transfected with sh-Runx1 and sh-NC were tested by qRT-PCR and Western blot assays. (J) Flow cytometry was carried out to detect apoptosis in MC3T3-E1 cells transfected with sh-Runx1 and sh-NC. Comparisons between two groups were performed using the t test, and comparisons among multiple groups were conducted using one-way analysis of variance, followed by the Tukey multiple-comparison test. The experiment was repeated three times. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; APS, alkaline phosphatase staining; PI, propidium iodide; FITC, fluorescein isothiocyanate. aP<0.05 vs. day 0 of induction or sh-NC group; bP<0.01 vs. day 0 of induction or sh-NC group; cP<0.001 vs. the sh-NC group.

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  Fig. 5. miR-181a-5p suppressed osteogenic differentiation partially by downregulating Runt-related transcription factor 1 (Runx1). (A, B) Runx1 expression in MC3T3-E1 cells co-transfected with a miR-181a-5p-inhibitor and sh-Runx1 was tested by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assays. (C) Alizarin red S (ARS) staining was used to detect mineralization in MC3T3- E1 cells co-transfected with the miR-181a-5p-inhibitor and sh-Runx1 (×200). (D, E) Alkaline phosphatase (ALP) staining and an ALP enzyme assay were performed to measure osteogenic differentiation in MC3T3-E1 cells co-transfected with the miR-181a-5p-inhibitor and sh- Runx1. (F, G) The expression levels of osteogenesis-related proteins (osteocalcin [OCN], osteopontin [OPN], collagen type I [Col1A1], and ALP) in MC3T3-E1 cells co-transfected with the miR-181a-5p-inhibitor and sh-Runx1 were determined by qRT-PCR and Western blot assays. (H) Apoptosis in MC3T3-E1 cells co-transfected with miR-181a-5p-inhibitor and sh-Runx1 was measured by flow cytometry. Comparisons between two groups were performed using the t test. The experiment was repeated three times. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; APS, alkaline phosphatase staining; PI, propidium iodide. aP<0.05 vs. the miR-181a-5p-inhibitor+sh-negative control (NC) group; bP<0.01 vs. the miR-181a-5p-inhibitor+sh-NC group.

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  Fig. 6. The transcription factor Runt-related transcription factor 1 (Runx1) suppressed the transcription of allograft inflammatory factor-1 (AIF-1). (A) The JASPAR database was used to predict the binding site of AIF-1 and Runx1. (B, C) The relationship between Runx1 and AIF- 1 was verified by a dual-luciferase reporter gene assay and a chromatin immunoprecipitation assay. (D, E) The expression of AIF-1 in MC3T3-E1 cells transfected with sh-Runx1 was tested by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assays. (F, G) The expression of AIF-1 in the serum of osteoporosis patients and normal controls was tested by qRT-PCR and Western blot assays (normal group, n=48; osteoporosis group, n=56). Comparisons between two groups were performed using the t test. The experiment was repeated three times. MUT, mutant; WT, wild-type; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. aP<0.05 vs. the overexpression (oe)-negative control (NC) group or the sh-NC group; bP<0.01 vs. the normal group; cP<0.001 vs. the immunoglobulin G (IgG) group.

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  Fig. 7. miR-181a-5p may promote allograft inflammatory factor-1 (AIF-1) transcription by downregulating Runt-related transcription factor 1 (Runx1), thereby inhibiting osteogenic differentiation and promoting apoptosis in MC3T3-E cells. MC3T3-E cells were co-transfected with a miR-181a-5p-mimic and sh-AIF-1, a miR-181a-5p-mimic and sh-negative control (NC), mimic-NC, and sh-AIF-1, as well as mimic-NC and sh-NC: (A) miR-181a-5p expression was tested using quantitative real-time polymerase chain reaction (qRT-PCR). (B, C) Runx1 and AIF-1 expression was determined using qRT-PCR and Western blot assays. (D, E, F) Alizarin red S (ARS) staining, alkaline phosphatase (ALP) staining, and an ALP enzyme assay (D, ×200). (G, H) The expression levels of osteogenesis-related proteins (osteocalcin [OCN], osteopontin [OPN], collagen type I [Col1A1], and and ALP) were determined by qRT-PCR and Western blot assays. (I) Apoptosis was measured using flow cytometry. Comparisons among multiple groups were conducted using one-way analysis of variance, followed by the Tukey multiple-comparison test. The experiment was repeated three times. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; APS, alkaline phosphatase staining; PI, propidium iodide; FITC, fluorescein isothiocyanate. aP<0.05 vs. the mimic-NC+sh-NC group; bP<0.01 vs. the mimic-NC+sh-NC group; cP<0.05 vs. the miR-181a-5p-mimic+sh-NC group; dP<0.01 vs. the miR-181a-5p-mimic+sh-NC group.

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  Fig. 8. Downregulation of miR-181a-5p promoted bone formation in postmenopausal osteoporotic mice. (A, B, C) Expression of miR-181a- 5p, allograft inflammatory factor-1 (AIF-1), and Runt-related transcription factor 1 (Runx1) was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assays. (D) Micro-computed tomography for analyzing the changes in bone microstructure parameters of mouse femurs in each group. (E) Hematoxylin and eosin staining of mouse femurs (×200). Comparisons among multiple groups were conducted using one-way analysis of variance, followed by the Tukey multiple-comparison test (n=6). OVX, ovariectomy; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BMD, bone mineral density; Tb.Th, trabecular bone thickness; Tb.N, trabecular bone number; Tb.Sp., trabecular bone separation; BV/TV, bone volume fraction; BS/BV, bone surface/bone volume. aP<0.05 vs. the sham group; bP<0.01 vs. the sham group; cP<0.05 vs. the antagomir-NC group; dP<0.01 vs. the antagomir-negative control (NC) group.

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