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

Affiliations
  • 1Department of Orthopedic, The First Affiliated Hospital of Harbin Medical University, Harbin, China
  • 2Department of Nephrology, The First Affiliated Hospital of Harbin Medical University, Harbin, China

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

  • 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.

  • 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.

  • 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.

  • 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.

  • 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.

  • 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.

  • 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.

  • 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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