LncRNA PCBP1-AS1 induces osteoporosis by sponging miR-126-5p/PAK2 axis

Abstract

Aims: Long non-coding RNAs (lncRNAs) act as crucial regulators in osteoporosis (OP). Nonetheless, the effects and potential molecular mechanism of lncRNA PCBP1 Antisense RNA 1 (PCBP1-AS1) on OP remain largely unclear. The aim of this study was to explore the role of lncRNA PCBP1-AS1 in the pathogenesis of OP.

Methods: Using quantitative real-time polymerase chain reaction (qRT-PCR), osteogenesis-related genes (alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), and Runt-related transcription factor 2 (RUNX2)), PCBP1-AS1, microRNA (miR)-126-5p, group I Pak family member p21-activated kinase 2 (PAK2), and their relative expression levels were determined. Western blotting was used to examine the expression of PAK2 protein. Cell Counting Kit-8 (CCK-8) assay was used to measure cell proliferation. To examine the osteogenic differentiation, Alizarin red along with ALP staining was used. RNA immunoprecipitation assay and bioinformatics analysis, as well as a dual-luciferase reporter, were used to study the association between PCBP1-AS1, PAK2, and miR-126-5p.

Results: The expression of PCBP1-AS1 was pre-eminent in OP tissues and decreased throughout the development of human bone marrow-derived mesenchymal stem cells (hBMSCs) into osteoblasts. PCBP1-AS1 knockdown and overexpression respectively promoted and suppressed hBMSC proliferation and osteogenic differentiation capacity. Mechanistically, PCBP1-AS1 sponged miR-126-5p and consequently targeted PAK2. Inhibiting miR-126-5p significantly counteracted the beneficial effects of PCBP1-AS1 or PAK2 knockdown on hBMSCs' ability to differentiate into osteoblasts.

Conclusion: PCBP1-AS1 is responsible for the development of OP and promotes its progression by inducing PAK2 expression via competitively binding to miR-126-5p. PCBP1-AS1 may therefore be a new therapeutic target for OP patients.

Fig. 1

The expression of PCBP1 Antisense RNA 1 (PCBP1-AS1) was elevated in osteoporosis (OP) tissues and reduced with human bone marrow-derived mesenchymal stem cell (hBMSC) osteoblast development. a) and b) Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the amount of PCBP1-AS1 expression in OP tissues and hBMSCs. c) to f) The expression levels of osteogenesis-related genes (Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), osteocalcin (OCN), and alkaline phosphatase (ALP)) were detected by qRT-PCR. g) Calcium deposits in hBMSCs were detected by Alizarin red staining. h) By subcellular fractionation and qRT-PCR analysis, the amount of PCBP1-AS1 expression in the nuclear and cytoplasmic fractions of hBMSCs was determined. The data represent the mean (standard deviation). *p < 0.05, **p < 0.001 compared with normal or zero days, calculated with one-way analysis of variance. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mRNA, messenger RNA.

Fig. 2

PCBP1 Antisense RNA 1 (PCBP1-AS1) inhibited human bone marrow-derived mesenchymal stem cell (hBMSC) osteoblast differentiation. a) Transfection efficiency of PCBP1-AS1 overexpression vectors (OE-lnc) or si-PCBP1-AS1 (si-lnc) was recognized by quantitative real-time polymerase chain reaction (qRT-PCR). b) Cell proliferation of hBMSCs was measured by Cell Counting Kit-8 (CCK-8) assay. c) to f) qRT-PCR was used to determine the degree of osteogenesis-related gene expression for the genes Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), osteocalcin (OCN), and alkaline phosphatase (ALP). g) ALP quantification identified ALP activity. h) Calcium deposits in hBMSCs were detected by Alizarin red staining. The data represent the mean (standard deviation). *p < 0.05, **p < 0.001 compared with empty vector (OE-NC), ^^p < 0.001 compared with negative control (si-NC); calculated using one-way analysis of variance. mRNA, messenger RNA; OD, optical density.

Fig. 3

PCBP1 Antisense RNA 1 (PCBP1-AS1) acted as a sponge for microRNA (miR)-126-5p in human bone marrow-derived mesenchymal stem cells (hBMSCs). a) Three miRNAs were overlapped from Starbase and GSE91033. b) Quantitative real-time polymerase chain reaction (qRT-PCR) detected the levels of miR-378g, miR-126-5p, and miR-424-5p in our osteoporosis (OP) samples. c) The correlation between miR-378g/miR-126-5p/miR-424-5p expression and PCBP1-AS1 expression in OP samples. d) The predicted binding sites of miR-126-5p with PCBP1-AS1 through Starbase. e) In hBMSCs co-transfected with mimic-NC or miR-126-5p mimic and PCBP1-AS1-wild type (WT) or PCBP1-AS1-mutant (MUT), the relative luciferase activity was assessed. f) Binding of miR-126-5p to PCBP1-AS1 was determined by RNA immunoprecipitation (RIP) assay. g) The expression level of miR-126-5p in transfected hBMSCs was detected by qRT-PCR. h) qRT-PCR was applied to determine the miR-126-5p expression level in hBMSCs with osteoblast differentiation. The data signify the mean (standard deviation). **p < 0.001 compared with mimic-NC, immunoglobulin G (IgG), or zero days. ^^p < 0.001 compared with negative control (si-NC). All p-values calculated using one-way analysis of variance. Ago2, Argonaute 2; hsa, Homo sapiens; IgG, immunoglobulin G; lnc, long non-coding; ns, not significant.

Fig. 4

PCBP1 Antisense RNA 1 (PCBP1-AS1) affected human bone marrow-derived mesenchymal stem cell (hBMSC) osteoblast differentiation by sponging microRNA (miR)-126-5p. hBMSCs were co-transfected hBMSCs with inhibitor-NC, miR-126-5p inhibitor (inhibitor), negative control (si-NC), si-PCBP1-AS1 (si-lnc), or si-PCBP1-AS1 + miR-126-5p inhibitor (si-lnc + inhibitor). a) The expression level of miR-126-5p in hBMSCs was detected by quantitative real-time polymerase chain reaction (qRT-PCR). b) Cell proliferation of hBMSCs was measured by Cell Counting Kit-8 (CCK-8) assay. c) to f) The expression level of osteogenesis-related genes (Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), osteocalcin (OCN), and alkaline phosphatase (ALP)) were determined with the help of qRT-PCR. g) ALP activity was determined by ALP quantification. h) Calcium deposits in hBMSCs were detected by Alizarin red staining. The data represent the mean (standard deviation). *p < 0.05, **p < 0.001 compared with inhibitor-NC, ^^p < 0.001 compared with si-NC, #p < 0.05, ##p < 0.001 compared with si-lnc+ inhibitor; calculated using one-way analysis of variance. mRNA, messenger RNA; OD, optical density; si-circ, silence circle RNA.

Fig. 5

Group I Pak family member p21-activated kinase 2 (PAK2) was a direct target of microRNA (miR)-126-5p in human bone marrow-derived mesenchymal stem cells (hBMSCs). a) A total of 25 genes were overlapped from TargetScan, GSE35958, and Starbase. b) The enrichments of PAK2, mitogen-activated protein 3 kinase 2 (MAP3K2), glycogen synthase kinase 3 beta (GSK3B), Runt-related transcription factor 2 (RUNX2), collagen, type I, alpha 1 (COL1A1), and lysyl oxidase (LOX) were detected in hBMSCs by RNA pull-down assay. c) Targetscan showed the predicted binding sequences of PAK2 for miR-126-5p. d) The relative luciferase activity was determined in hBMSCs co-transfected with PAK2-wild type (WT) or PAK2-mutant (MUT) and mimic-negative control (NC) or miR-126-5p mimic. e) PAK2 expression in osteoporosis (OP) tissues was determined with quantitative real-time polymerase chain reaction (qRT-PCR). f) A negative association between miR-126-5p and PAK2. g) and h) PAK expression in transfected hBMSCs was measured by g) qRT-PCR and h) western blot. The data represent the mean (standard deviation). *p < 0.05, **p < 0.001 compared with Bio-NC or mimic-NC. ^^p < 0.001 compared with inhibitor-NC. All p-values calculated using one-way analysis of variance. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; hsa, Homo sapiens; mRNA, messenger RNA; UTR, untranslated region.

Fig. 6

MicroRNA (miR)-126-5p regulated osteoblast differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) by targeting group I Pak family member p21-activated kinase 2 (PAK2). hBMSCs were co-transfected hBMSCs with inhibitor-NC, negative control (si-NC), miR-126-5p inhibitor (inhibitor), and si-PAK2 or si-PAK2 + miR-126-5p inhibitor (si-PAK2 + inhibitor). a) and b) PAK expression was measured by a) quantitative real-time polymerase chain reaction (qRT-PCR) and b) western blot. c) Cell proliferation of hBMSCs was measured by Cell Counting Kit-8 (CCK-8) assay. d) to g) The expression level of osteogenesis-related genes (Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), osteocalcin (OCN), and alkaline phosphatase (ALP)) detected by qRT-PCR. h) ALP activity was detected by ALP staining and quantification. i) Calcium deposits in hBMSCs were detected by Alizarin red staining. The data represent the mean (standard deviation). *p < 0.05, **p < 0.001 compared with inhibitor-NC, ^^p < 0.001 compared with si-NC, #p < 0.05, ##p < 0.001 compared with si-PAK2+ inhibitor; calculated using one-way analysis of variance. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mRNA, messenger RNA; OD, optical density.

Fig. 7

The mechanism of PCBP1 Antisense RNA 1 (PCBP1-AS1)/microRNA (miR)-126-5p/group I Pak family member p21-activated kinase 2 (PAK2) axis in osteoporosis. The upregulation of PCBP1-AS1 could induce osteoporosis by downregulating miR-126-5p to enhance PAK2 expression.