LncRNA MALAT1 mediates osteogenic differentiation in osteoporosis by regulating the miR-485-5p/WNT7B axis

Abstract

Introduction: Accumulating evidence demonstrates that long non-coding RNAs (lncRNAs) are associated with the development of osteoporosis.

Methods: This study aimed to investigate the effects of MALAT1 on osteogenic differentiation and cell apoptosis in osteoporosis. MALAT1 level, detected by RT-qPCR, was downregulated in hindlimb unloading (HU) mice and simulated microgravity (MG)-treated MC3T3-E1 cells. Moreover, osteogenic differentiation-related factor (Bmp4, Col1a1, and Spp1) levels were measured by RT-qPCR and Western blot. ALP activity was detected, and ALP staining was performed. Cell apoptosis was assessed by flow cytometry.

Results: The results revealed that MALAT1 upregulated the expression of Bmp4, Col1a1, and Spp1, and enhanced ALP activity. Knockdown of MALAT1 suppressed their expression and ALP activity, suggesting that MALAT1 promoted osteogenic differentiation. Additionally, MALAT1 inhibited apoptosis, increased Bax and caspase-3 levels, and decreased Bcl-2 level. However, knockdown of MALAT1 had opposite results. In MG cells, MALAT1 facilitated osteogenic differentiation and suppressed apoptosis. Furthermore, miR-485-5p was identified as a target of MALAT1, and WNT7B was verified as a target of miR-485-5p. Overexpression of miR-485-5p rescued the promotion of osteogenic differentiation and the inhibition of apoptosis induced by MALAT1. Knockdown of WNT7B abolished the facilitation of osteogenic differentiation and the suppression of apoptosis induced by downregulation of miR-485-5p.

Discussion: In conclusion, MALAT1 promoted osteogenic differentiation and inhibited cell apoptosis through the miR-485-5p/WNT7B axis, which suggested that MALAT1 is a potential target to alleviate osteoporosis.

Keywords: MALAT1; MiR-485-5p; WNT7B; osteogenesis differentiation; osteoporosis.

Figure 1

The expression of MALAT1 was downregulated MG-treated cells. (A) The expression of ALP in the MG-treated MC3T3-E1 cells was determined using RT-qPCR. (B) The mRNA expressions of Bglap, Runx2, and Col1a1 in the MG-treated MC3T3-E1 cells were measured by RT-qPCR. (C) The protein expressions of Bglap, Runx2, and Col1a1 in the MG-treated MC3T3-E1 cells were detected using Western blot. (D) ALP activity in the MG-treated MC3T3-E1 cells was detected using ALP staining. (E) Alizarin red staining was used to measure calcium deposit in the MG-treated MC3T3-E1 cells. (F, G) The differentially expressed lncRNAs in MG-treated MC3T3-E1 cells was expressed as heat maps and volcano maps. (H) The expression of MALAT1 in MG-treated MC3T3-E1 cells was detected using RT-qPCR. **p < 0.01, ***p < 0.001.

Figure 2

MALAT1 facilitated MC3T3-E1 cell osteogenic differentiation and suppressed apoptosis. (A) The transfection efficiency of MALAT1 was determined using RT-qPCR. Then, the MC3T3-E1 cells were treated with MG and transfected with MALAT1. (B) The expression of ALP was determined using RT-qPCR. (C) The mRNA expression of Bglap, Runx2, and Col1a1 was determined using RT-qPCR. (D) The protein expression of Bglap, Runx2, and Col1a1 was detected using Western blot. (E) The ALP activity of MC3T3-E1 cells. (F) Alizarin red staining was used to measure calcium deposit. (G) Cell apoptosis was analyzed by flow cytometry, and cell apoptosis rate was quantified. (H) The protein expression of Bax, caspase-3, and Bcl-2 was detected using Western blot. **P<0.01, ***P<0.001.

Figure 3

MALAT1 improved bone phenotype in HU mice. (A) Micro-CT images of distal femurs of mice. The (B) BMD, BV/TV, Tb.N, Tb.Sp, Tb.Th, and TbPF were analyzed by Micro-CT. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

MALAT1 acted as a miRNA sponge of miR-485-5p. (A) The binding sites of wild-type (wt) MALAT1 and miR-485-5p were shown. Meanwhile, mutant (mut) MALAT1 sequences were also shown. (B) The targeting relationship between MALAT1 and miR-485-5p was confirmed by luciferase reporter assay. (C) The targeted relationship between MALAT1 and miR-485-5p was verified using RNA pull-down. (D) The expression of miR-485-5p was measured by RT-qPCR after overexpression or MALAT1 knockdown. (E) The Pearson method was applied for correlation analysis. (F, G) The expression of miR-485-5p in the HU mice and MG-treated MC3T3-E1 cell was detected using RT-qPCR. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

miR-485-5p attenuated osteogenic differentiation induced by MALAT1. (A) The MG-treated MC3T3-E1 cell was transfected with MALAT1 and miR-485-5p mimic. (A) The expression of ALP was detected by RT-qPCR. (B) The expression of Bglap, Runx2, and Col1a1 was detected by RT-qPCR. (C) The protein expression of Bglap, Runx2, and Col1a1 was detected by Western blot. (D) The ALP activity of MC3T3-E1 cells. (E) The calcium deposit detected using Alizarin red staining. (F) The apoptosis of MC3T3-E1 cells detected using flow cytometry. (G) The protein expression of Bax, Caspase-3, and Bcl-2 detected using Western blot. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

WNT7B is a direct target of miR-485-5p. (A) The potential target of miR-485-5p was predicted by Starbase2.0 and TargetScan7.2. (B) Potential binding sites for miR-485-5p to WNT7B were predicted by TargetScan. (C) The targeted relationship between miR-485-5p and WNT7B was confirmed by dual-luciferase reporter assay. (D) The targeted relationship between miR-485-5p and WNT7B was confirmed by RNA pull-down. (E) GO analysis of biological processes. (F) The expression of WNT7B was assessed by RT-qPCR after miR-485-5p overexpression and knockdown. (G) The expression of WNT7B in vivo. (H) The correlation between the expression of WNT7B and MALAT1 analyzed using Pearson analysis. (I) The expression of WNT7B detected using RT-qPCR. *p < 0.05. **p < 0.01. ***P<0.001.

Figure 7

WNT7B was a negative regulator of miR-485-5p on the osteogenic differentiation. The MG-treated MC3T3-E1 cell was transfected with miR-485-5p inhibitor and si-WNT7B. (A) The expression of ALP was detected by RT-qPCR. (B) The expression of Bglap, Runx2, and Col1a1 was detected by RT-qPCR. (C) The protein expression of Bglap, Runx2, and Col1a1 was detected by Western blot. (D) The ALP activity of MC3T3-E1 cells. (E) The calcium deposit detected using Alizarin red staining. (F) The apoptosis of MC3T3-E1 cells detected using flow cytometry. (G) The protein expression of Bax, Caspase-3, and Bcl-2 detected using Western blot. *p < 0.05, **p < 0.01, ***p < 0.001.