LINC01133 promotes the osteogenic differentiation of bone marrow mesenchymal stem cells by upregulating CTNNB1 by acting as a sponge for miR-214-3p

Abstract

Background: Osteoporosis results from decreased bone mass and disturbed bone structure. Human bone marrow mesenchymal stem cells (hBMSCs) demonstrate robust osteogenic differentiation, a critical process for bone formation. This research was designed to examine the functions of LINC01133 in osteogenic differentiation.

Methods: Differentially expressed lncRNAs affecting osteogenic differentiation in hBMSCs were identified from the GEO database. A total of 74 osteoporosis patients and 70 controls were enrolled. hBMSCs were stimulated to undergo osteogenic differentiation using an osteogenic differentiation medium (OM). RT-qPCR was performed to evaluate LINC01133 levels and osteogenesis-related genes such as osteocalcin, osteopontin, and RUNX2. An alkaline phosphates (ALP) activity assay was conducted to assess osteogenic differentiation. Cell apoptosis was detected using flow cytometry. Dual luciferase reporter assay and RIP assay were employed to investigate the association between miR-214-3p and LINC01133 or CTNNB1. Loss or gain of function assays were conducted to elucidate the impact of LINC01133 and miR-214-3p on osteogenic differentiation of hBMSCs.

Results: LINC01133 and CTNNB1 expression decreased in osteoporotic patients but increased in OM-cultured hBMSCs, whereas miR-214-3p showed an opposite trend. Depletion of LINC01133 suppressed the expression of genes associated with bone formation and ALP activity triggered by OM in hBMSCs, leading to increased cell apoptosis. Nevertheless, this suppression was partially counteracted by the reduced miR-214-3p levels. Mechanistically, LINC01133 and CTNNB1 were identified as direct targets of miR-214-3p.

Conclusions: Our study highlights the role of LINC01133 in positively regulating CTNNB1 expression by inhibiting miR-214-3p, thereby promoting osteogenic differentiation of BMSCs. These findings may provide valuable insights into bone regeneration in osteoporosis.

Keywords: Apoptosis; Bone marrow mesenchymal stem cells; LINC01133; Osteogenic differentiation.

Fig. 1

The levels of LINC01133 in patients with OP. The volcano plot (A) and heatmap (B) from the GEO database (GESE113359 dataset) were used to detect differentially expressed LncRNAs in human bone marrow mesenchymal stem cells (hBMSCs) on days 0 and 10 of osteogenic differentiation. C-D. Patients with OP had significantly lower levels of LINC01133 in serum and bone tissue compared to controls. E. Diagnostic value of ROC curves for analyzing serum LINC01133 in patients with OP. *** P < 0.001

Fig. 2

The expression of LINC01133 and osteogenesis-relative genes in hBMSCs cultured in PM and OM. A-C, the mRNA levels of OCN, OPN, and RUNX2 in hBMSCs were analyzed in both OM and PM at the same time points (0, 3, 7, 14, and 21 days). D. Flow cytometry was conducted to examine the apoptosis of hBMSCs during the OM-induced osteogenic differentiation. E. RT-qPCR was employed to analyze the mRNA levels of LINC01133 in hBMSCs cultured for the same time (0, 3, 7, 14, and 21 days) in OM and PM. PM, proliferation medium; OM, osteogenic differentiation medium. * P < 0.05, *** P < 0.001

Fig. 3

Effect of silencing LINC01133 on cell viability, apoptosis, and osteogenic differentiation of hBMSCs cells. A. LINC01133 levels were detected by the RT-qPCR after siRNA transfection. B. The expression of LINC01133 was detected after transfection of si-LINC01133 in OM-cultured hBMSCs. C. si-LINC01133 regulated ALP activity in OM-induced hBMSCs. D. RT-qPCR analysis of the effect of si-LINC01133 on the levels of osteogenic differentiation markers OCN, OPN, and RUNX2. E. Flow cytometry was used to detect the effects of si-LINC01133 on OM-induced cell apoptosis of hBMSCs. ** P < 0.01, *** P < 0.001

Fig. 4

LINC01133 directly targets miR-214-3p. A. Subcellular fractionation assay detects the distribution of LINC01133 in hBMSCs. B. The binding sequences between miR-214-3p and LINC01133. Dual luciferase reporter assay (C) and RIP assay (D) were conducted to verify the correlation between miR-214-3p and LINC01133. E. The serum miR-214-3p levels in the subjects. F. The Pearson coefficient correlation was employed to examine the relationship between miR-214-3p and LINC01133 in patients with OP. G. RT-qPCR was employed to analyze the mRNA levels of miR-214-3p in hBMSCs cultured for the same time (0, 3, 7, 14, and 21 days) in OM and PM. H. Effect of silencing LINC01133 in OM-induced hBMSCs on miR-214-3p levels. *** P < 0.001

Fig. 5

Effects of miR-214-3p and LINC01133 on proliferation, apoptosis, and osteogenic differentiation of hBMSCs. A. Inhibition of miR-214-3p levels in hBMSCs by transfection with miR-214-3p inhibitor. B. miR-214-3p levels in the hBMSCs under OM induced downregulation of LINC01133. C. Co-regulation of miR-214-3p and LINC01133 on ALP activity in hBMSCs. D. Effects of co-regulation of miR-214-3p and LINC01133 on mRNA levels of osteogenic differentiation markers were analyzed by RT-qPCR. E. Flow cytometry used to detect the effects of miR-214-3p inhibitors on OM-induced cell apoptosis of hBMSCs. ** P < 0.01, *** P < 0.001

Fig. 6

CTNNB1 is the target of miR-214-3p. A. The overlapping targets of miR-214-3p were predicted by the online software Targetscan, miRDB, ENCORI, microT, miRWalk, and EVmiRNA. B. The PPI network was constructed through the overlapping targets and the top 10 hub genes in the PPI network were also displayed according to node degree. C. The binding sequences between miR-214-3p and CTNNB1. Dual luciferase reporter assay (D) and RIP assay (E) were conducted to verify the correlation between miR-214-3p and CTNNB1. F. The serum CTNNB1 levels in the subjects. The Pearson coefficient correlation was employed to examine the relationship between CTNNB1 with LINC01133 (G) and miR-214-3p (H). I. RT-qPCR was employed to analyze the mRNA levels of CTNNB1 in hBMSCs cultured for the same time (0, 3, 7, 14, and 21 days) in OM and PM. J. LINC01133 and miR-214-3p regulated CTNNB1 levels in hBMSCs. *** P < 0.001