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- Springer Journals
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- Copyright © The Author(s) 2023
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- 1471-2474
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- 10.1186/s12891-023-06136-z
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Abstract
Background Osteoporosis (OP) is a common bone disease marked by decreased bone strength. Increasing evidence suggests that long non-coding RNA (lncRNAs) play important roles in the occurrence and progression of OP. This study aimed to investigate the role and mechanism of LINC00205 in the osteogenic differentiation of human mesen- chymal stem cells (hMSCs) and OP. Methods Bone tissue samples were obtained from healthy controls and patients with osteoporosis with a spinal fracture (OP-Frx) or without a spinal fracture (OP-no-Frx). HMSCs were cultured and induced to undergo osteogenic differentiation. The expression of LINC00205, lysine (K)-specific methyltransferase 2C (KMT2C), and miR-26b-5p in bone tissues and cells was evaluated using western blotting and real-time quantitative reverse transcription polymer- ase chain reaction (qRT-PCR). The effects of LINC00205, miR-26b-5p, and KMT2C on calcium deposition, alkaline phos- phatase (ALP) activity, and mRNA levels of the osteogenic differentiation marker genes [ALP, osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2)] were investigated using alizarin red S staining, an ALP activity assay, and qRT-PCR, respectively. Dual-luciferase reporter assay was performed to ascertain the binding relationship between miR-26b-5p and LINC00205/KMT2C. Results LINC00205 and KMT2C were upregulated in patients with OP-Frx and OP-no-Frx, whereas miR-26b-5p was downregulated. Furthermore, LINC00205 and KMT2C expression decreased, whereas that of miR-26b-5p increased over time from day 7 to 21 of the osteogenic differentiation of hMSCs. The knockdown of LINC00205 and KMT2C significantly increased ALP activity, calcium deposition, and the expression of RUNX2, ALP, and OCN. In contrast, the inhibition of miR-26b-5p yielded the opposite result. These data suggest that LINC00205 inhibits the osteogenic dif- ferentiation of hMSCs by modulating the miR-26b-5p/KMT2C signaling axis. Conclusion LINC00205 promotes OP and is involved in spinal fractures. LINC00205 is also a potential negative regu- lator of the osteogenic differentiation of hMSCs. Keywords lncRNA LINC00205, Osteoporosis, Spinal fracture, miR-26b-5p, KMT2C, Human mesenchymal stem cells Hongtao Wang and Weilin Xu contributed equally to this work. *Correspondence: Juan Zhang juanzhang7@163.com Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wang et al. BMC Musculoskeletal Disorders (2023) 24:262 Page 2 of 14 The aim of the present study was to look into the role Introduction and mechanism of action of LINC00205 in the progres- Osteoporosis (OP) is one of the most prevalent bone dis- sion of OP, as well as its role in regulating the osteo- eases [1]. It is characterized by reduced bone strength genic differentiation of human mesenchymal stem cells and puts a person at a 40% lifetime risk of fractures [1]. (hMSCs). We performed a comprehensive bioinformatics Studies have shown that fractures in patients with OP analysis to identify the target miRNA of LINC00205 and most commonly occur in the wrist, hip, or spine [2]. then established a lncRNA–miRNA-mRNA regulatory Furthermore, osteoporotic spinal fractures significantly network. Based on the results of bioinformatics analy- increase the mortality rate for 12 months and increase sis, we hypothesized that LINC00205 might influence the risk of other medical complications [3, 4]. OP is the progression of OP by regulating the miR-26b-5p/ becoming a heavy burden on healthcare systems world- KMT2C axis. Therefore, we performed various rescue wide, and its medical and socioeconomic implications and functional experiments to investigate the role of the will further increase with the aging population [5]. LINC00205/miR-26b-5p/KMT2C axis in regulating oste- Long non-coding RNAs (lncRNAs) are non-protein ogenic differentiation of hMSCs and thus influencing OP coding RNAs transcribed by eukaryotic genomes [6]. progression. They are involved in mRNA processing, maturation, and stability by acting as microRNA (miRNA) sponges or competing for binding sites on mRNAs [6]. In addition, Materials and methods lncRNAs are involved in various cellular activities and Human samples pathogenesis, including OP pathogenesis [7]. For exam- Bone tissues from patients with OP (N = 24), divided into ple, plasma lncRNA NEF is downregulated in postmeno- two groups, namely those with spinal fracture (OP-Frx) pausal OP and is associated with long treatment cycles or without spinal fracture (OP-no-Frx), and from control and high relapse rates [8]. BCAR4, another lncRNA, is subjects admitted to People’s Hospital of Dongxihu Dis- downregulated during human mesenchymal stem cell trict were collected during hip arthroplasty. The patients (hMSC) differentiation and mitigates the progression of in control group suffered from bone injury caused by OP [9]. LINC00205, a newly studied lncRNA, has been external force such as sports, traffic accident, etc. This found to act as a carcinogen in lung cancer, liver can- study was approved by the Research Ethics Committee of cer, retinoblastoma, and other cancers by promoting the People’s Hospital of Dongxihu District. Written informed malignant proliferation of cancer cells [10–12]. However, consent was obtained from each patient enrolled in the the role of LINC00205 in OP remains to be elucidated. study. The inclusion criteria for patients with OP were The miRNAs are a class of non-coding RNAs (ncRNAs) a bone mineral density (BMD) of at least 2.5 standard that are known to degrade mRNA or inhibit its transla- deviations below the peak BMD (-2.5 T score) of healthy tion and consequently regulate gene expression [13–15]. young women in the lumbar spine, total hip, or femoral Accumulating evidence indicates that some miRNAs, neck after the age of 40 years. The exclusion criteria were including miR-205-5p, miR-151a-3p, and miR-16a-5p, a history of OP therapy, hormone replacement therapy, are involved in the development of OP [13–15]. They early menopause (< 40 years), abnormal menopause, can regulate osteoblast and osteoclast differentiations acute gastrointestinal inflammation, or chronic renal to participate in osteogenesis and osteoclastogenesis, failure. The clinical characteristics of the participants are thereby leading to OP [16]. For example, miR-214 and summarized in Supplementary Table 1. miR-135-5p have been reported to regulate the prolif- eration and osteogenic differentiation of hMSCs [17, 18]. Another miRNA, miR-26b-5p, was found to be upregu- Cell culture and osteoblast differentiation lated in hMSCs during osteogenesis and induce osteo- HMSCs were obtained from the Chinese Academy of genic differentiation [19]. Sciences (Shanghai, China). They were maintained in Lysine (K)-specific methyltransferase 2C (KMT2C) is α-modified Eagle’s medium (α-MEM) supplemented with a histone methyltransferase involved in transcriptional 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, co-activation. KMT2C is highly conserved and known USA) and 1% penicillin–streptomycin (Thermo Fisher to have evolved in unicellular eukaryotes [20]. Studies Scientific, Waltham, MA, USA) in an incubator at 37 have shown that KMT2C is mutated in various diseases ℃ with 5% C O To induce osteoblast mineralization, such as mental disabilities and cancer [21, 22]. A bioin- 100 nM dexamethasone, 10 mM β-glycerophosphate formatics tool was used in this study to identify the pos- disodium salt hydrate, and 50 μg/mL L-ascorbic acid sible binding sites between miR-26b-5p and KMT2C. The (Sigma-Aldrich, St. Louis, MO, USA) were added to the interaction of KMT2C and miR-26b-5p in the develop- hMSC culture for 3 weeks [23]. Alizarin red S (ARS) ment of OP has not yet been studied or reported. staining and alkaline phosphatase (ALP) activity assay W ang et al. BMC Musculoskeletal Disorders (2023) 24:262 Page 3 of 14 Table 1 Forward and reverse primers listed for real-time qPCR were collected 2 days post-transfection, and analyzed for transfection efficiency using qRT-PCR. Name Sequence (5’‑3’) miR-26b-5p forwardTTC AAG TAA TTC AGG ATA GGT qRT‑PCR analysis miR-26b-5p reverseGTG CGT GTC GTG GAGTC The RNeasy Mini Kit (QIAGEN, Hilden, Germany) LINC00205 forwardGGC TTT TGT GCC TGG AAG TG LINC00205 reverseGGG AAG TTC TGA GCT GGC AT was used to extract total RNA. The total RNA was then RUNX2 forwardCGG TAA AAT CTG CGT GCT CT reverse transcribed into cDNA using the PrimeScript RUNX2 reverseTTC CCT ACG AGG ATT TCA GC RT Kit (Takara, Tokyo, Japan). PCR was carried out in ALP forwardTGG ACC TCA TCA GCA TTT GG ALP reverseGAG GGA AGG GTC AGT CAG GTT an ABI 7500 Real-Time PCR System (Applied Biosys- OCN forwardCCA GCG ACT CTG AGT CTG ACAA tems, Foster City, CA, USA) using the Takara SYBR OCN reverseAAC GGT GGT GCC ATA GAT GC Green Master Mix. The expression levels of LINC00205, KMT2C forwardGCA GAA TCA AGG AGC TGT CTG KMT2C reverseTGC CTT GAG GTC AAC GTA CAA TTG KMT2C, alkaline phosphatase (ALP), runt-related tran- U6 forwardCTC GCT TCG GCA GCACA scription factor 2 (RUNX2), and osteocalcin (OCN) were U6 reverseAAC GCT TCA CGA ATT TGC GT normalized to that of GAPDH using the delta-delta Ct GAPDH forwardCGC TAA CAT CAA ATG GGG TG −∆∆Ct GAPDH reverseTTG CTG ACA ATC TTG AGG GAG (2 ) method [24]. The miRNA was isolated using miRNA Isolation Kit (Ambion, Austin, TX). Subsequently, miRNA was reverse transcribed into cDNA using the TaqMan miRNA was performed to confirm successful osteoblast differen - Reverse Transcription Kit (Applied Biosystems) accord- tiation of hMSCs. ing to the manufacturer’s protocol. PCR was performed according to the standard TaqMan microRNA assay Cell transfection protocol. U6 served as an endogenous control for miR- Small interfering RNAs (siRNAs) targeting LINC00205 26b-5p. All the primers used are listed in Table 1. (si-LINC00205), KMT2C (si-KMT2C), and negative con- trol siRNAs (si-NC) were acquired from Ribobio (Guang- Cell counting kit‑8 (CCK‑8) assay zhou, China). The miR-26b-5p inhibitor, NC inhibitor, In vitro cell proliferation was assessed using the CCK-8 miR-26b-5p mimics, and NC mimics were sourced from Kit (Beyotime, Shanghai, China) according to the manu- SwitchGear Genomics (Menlo Park, CA, USA). HMSCs facturer’s instructions. Briefly, hMSCs were plated in were transfected with 200 nM inhibitor, 100 nM mimic, 3 96-well plates at a density of 4 × 10 cells/well, allowed or 75 nM siRNA using the Lipofectamine 2000 Trans- to adhere to the dishes, and cultured for 24, 48, or 72 h. fection Reagent (Thermo Fisher Scientific). The cells CCK-8 solution (10 μL) was pipetted into each well, and Fig. 1 Confirmation of induction of osteoblastic differentiation of hMSCs. A Images of hMSCs stained with ARS on days 0, 7, 14, and 21 following treatment with the osteogenic medium. B The ALP enzymatic activity in hMSCs on days 0, 7, 14 and 21 following treatment with the osteogenic medium. **P < 0.001 when compared to day 0. All data were from three independent experiments Wang et al. BMC Musculoskeletal Disorders (2023) 24:262 Page 4 of 14 the cells were incubated further for 4 h. The absorbance (SDS-PAGE), followed by transfer to polyvinylidene flu - of each well was measured at a wavelength of 450 nm oride (PVDF) membranes (Millipore, Billerica, MA). using a microplate reader (Bio-Rad, Hercules, CA). Subsequently, the membranes were blocked with 5% skimmed milk at 37 °C for 1 h. Next, they were incu- bated overnight at 4 °C with KMT2C (Cat #MA5-38,554, Alkaline phosphatase (ALP) activity assay 1:2000, Invitrogen, Carlsbad, CA) or GAPDH (Cat HMSCs were collected, washed in phosphate-buffered #MA1-16,757, 1:2000, Invitrogen) antibodies. Thereaf - saline, and fixed with pre-cooled 95% ethanol for 1 h. ter, the membranes were incubated with a suitable HRP- Subsequently, the cells were lysed with radioimmu- conjugated secondary antibody (Cat # ab205719, 1:2000, noprecipitation assay (RIPA) buffer (Beyotime). The Abcam, Cambridge, UK) at 25 ℃ for 1 h. Finally, the pro- supernatant of the lysate was collected and the Alkaline tein bands were visualized using an ECL substrate (Mil- Phosphatase Assay Kit (Beyotime) was used to deter- lipore). The intensity of the protein bands was analyzed mine the ALP activity. Substrates and p-nitrophenol using the Image Lab Software (Bio-Rad). were added to the collected supernatant, and the solution was incubated for 10 min at 37 ℃. The absorbance was Statistical analysis quantified at a wavelength of 405 nm using a microplate Data from three independent experiments were analyzed reader (Bio-Rad). using GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA) and are expressed as mean ± standard devia- Alizarin red S (ARS) staining tion (SD). The Shapiro–Wilk test was performed to assess HMSCs were fixed in paraformaldehyde (4%) at 25 ℃ for whether the data had a normal distribution, and Fisher’s 30 min. Subsequently, the cells were stained with 0.1% F test/Brown-Forsythe test was done to check homogene- ARS (Beijing Chemical Factory, Beijing, China) at 37 ℃ ity of variance. Furthermore, Student’s t-test was used to for 5 min. The stained cells were visualized and images assess the differences between two independent groups. were captured using an optical microscope (Olympus, One-way analysis of variance (ANOVA) with Tukey’s Tokyo, Japan). post-hoc test was used to evaluate differences among mul - tiple groups. Statistical significance was set at P < 0.05. Luciferase assay LINC00205 and KMT2C wild-type sequences contain- Results ing the predicted miR-26b-5p binding site were inserted Knockdown of LINC00205 promotes osteogenic into the psiCHECK2.0 vector (Promega, Madison, differentiation of hMSCs WA) and labeled as LINC00205-WT and KMT2C-WT, Before commencing any experiments, we confirmed respectively. The corresponding mutant reporter vectors the osteoblastic differentiation of hMSCs using the ARS were labeled as LINC00205-MUT and KMT2C-MUT. staining and ALP activity assay. The ARS staining dem - HMSCs were transfected with 100 ng of reporter vector onstrated successful osteogenic differentiation of hMSCs and 50 nM of miR-NC or miR-26b-5p mimic. The Dual- after 21 days as evidenced by a greater number of calcium luciferase Reporter Assay System (Promega) was used to nodules in hMSCs (Fig. 1A). The ALP enzymatic activ - measure the luciferase activity 48 h after transfection. ity was measured as a fold of control and significantly increased on days 7, 14, and 21, with the highest activity Western blot analysis being seen on the 21st day (Fig. 1B). HMSCs were collected and lysed in RIPA buffer (Beyo - The differential expression of LINC00205 in osteo - time) for 30 min. The protein concentration was deter - porosis was assessed using qRT-PCR. The results dem - mined using a BCA Protein Assay Kit (Bio-Rad) and 20 µg onstrated that LINC00205 expression was 1.2- and of protein samples was resolved on a 10% gel by sodium 3.9-fold higher in the bone tissues from patients with dodecyl sulfate polyacrylamide gel electrophoresis OP-no-Frx and OP-Frx, respectively, compared to that (See figure on next page.) Fig. 2 Knockdown of LINC00205 promoted osteogenic differentiation of hMSCs. A The relative expressions of LINC00205 in OP-Frx, OP-no-Frx, and healthy controls as quantified by qRT-PCR. **P < 0.001 and ##P < 0.001. B LINC00205 expressions at 7, 14, and 21 days of the osteoblast differentiation as analyzed by qRT-PCR. ** P < 0.001 when compared to day 0. C Expression levels of LINC00205 in hMSCs, quantified by qRT-PCR, after si-LINC00205 transfection. **P < 0.001 when compared to si-NC. D Viability of hMSCs treated with si-LINC00205 as assessed by the CCK-8 assay. **P < 0.001 when compared to si-NC at the same time. E Relative expression levels of RUNX2, OCN, and ALP mRNAs in hMSCs after treatment with si-LINC00205 were all analyzed using qRT-PCR. **P < 0.001 when compared to si-NC. F The ALP activity in hMSCs transfected with si-LINC00205 via the ALP activity assay. **P < 0.001 when compared to si-NC. G Images of si-LINC00205-transfected hMSCs stained with ARS. All data were from three independent experiments W ang et al. BMC Musculoskeletal Disorders (2023) 24:262 Page 5 of 14 Fig. 2 (See legend on previous page.) Wang et al. BMC Musculoskeletal Disorders (2023) 24:262 Page 6 of 14 in control subjects in the validation cohort (Fig. 2A). differentiation. The binding sites of LINC00205 and Diagnostic potentials of levels of LINC00205 for OP- miR-26b-5p are shown in Fig. 3A. To further verify no-Frx were evaluated by receiver operating character- that LINC00205 is a potential miR-26b-5p sponge, a istic (ROC) curve analysis with patients with OP-no-Frx dual-luciferase reporter system with WT and mutant tissues as true positive cases and control tissues as true LINC00205 was constructed. The luciferase activity negative cases. Area under the curve was 0.825, with remained almost unchanged following co-transfection of standard error of 0.06650 and 95% confidence interval the mutant LINC00205 and miR-26b-5p mimic. In con- (CI) of 0.6943–0.9550. Diagnostic potentials of levels of trast, a 70% reduction in luciferase activity was observed LINC00205 for OP-Frx were evaluated by ROC curve in cells co-transfected with the LINC00205-WT and miR- analysis with patients with OP-Frx tissues as true posi- 26b-5p mimic (Fig. 3B). Additionally, miR-26b-5p levels tive cases and OP-no-Frx tissues as true negative cases. were measured in clinical samples, and qRT-PCR analy- Area under the curve was 0.797, with standard error of sis showed a 60% and 80% decline in miR-26b-5p levels 0.06401 and 95% CI of 0.6714–0.9224 (Supplementary in the bone tissues of patients with OP-no-Frx and OP- Fig. 1A). Moreover, changes in LINC00205 expression Frx, respectively, compared to healthy subjects (Fig. 3C). were observed during the osteogenic differentiation Diagnostic potentials of levels of miR-26b-5p for OP-no- of hMSCs in vitro. Following osteogenic induction of Frx were evaluated by ROC curve analysis with patients hMSCs, LINC00205 levels decreased to 70%, 60%, and with OP-no-Frx tissues as true positive cases and con- 40% on days 7, 14, and 21, respectively, of the original trol tissues as true negative cases. Area under the curve expression level on day 0 (Fig. 2B). Thus, LINC00205 was 0.785, with standard error of 0.06816 and 95% CI of expression was downregulated and the osteogenic differ - 0.6511–0.9183. Diagnostic potentials of levels of miR- entiation period was prolonged, suggesting the involve- 26b-5p for OP-Frx were evaluated by ROC curve analysis ment of LINC00205 in bone formation and that it may with patients with OP-Frx tissues as true positive cases be associated with OP and vertebral fractures. Therefore, and OP-no-Frx tissues as true negative cases. Area under the role of LINC00205 in bone formation was investi- the curve was 0.787, with standard error of 0.06937 and gated further. 95% CI of 0.6505–0.9225 (Supplementary Fig. 1B). In HMSCs were transfected with si-LINC00205, and the addition, miR-26b-5p levels increased over time during levels of LINC00205 decreased by 75% when compared the osteoblast differentiation of hMSCs (Fig. 3D). These to si-NC transfected cells (Fig. 2C). Results from the results demonstrated an opposite trend between the CCK-8 assay revealed that the knockdown of LINC00205 expression of LINC00205 and miR-26b-5p during bone enhanced the viability of the cells approximately 1.8-fold formation. Hence, we speculate that LINC00205 down- (Fig. 2D). The mRNA levels of RUNX2, OCN, and ALP regulates miR-26b-5p in hMSCs via its sponge effect. The were increased approximately 3.3-, 3.6-, and 2.5-fold, co-knockdown of LINC00205 and miR-26b-5p in hMSCs respectively, in the si-LINC00205 group compared to revealed that si-LINC00205 treatment upregulated miR- that in the si-NC group (Fig. 2E). Moreover, the reduced 26b-5p and that miR-26b-5p inhibitor reversed the effect expression of LINC00205 resulted in increased ALP of LINC00205 knockdown (Fig. 3E). These data indi - activity and greater number of calcium nodules in hMSCs cate that miR-26b-5p expression is decreased in OP, and (Fig. 2F and G). These results indicate that LINC00205 LINC00205 acts as a miR-26b-5p sponge. silencing positively modulates osteogenic differentiation in hMSCs. LINC00205 inhibits osteogenic differentiation of hMSCs by sponging miR‑26b‑5p LINC00205 acts as a miR‑26b‑5p sponge Next, we sought to determine the roles of LINC00205 The sequences of LINC00205 and miR-26b-5p were ana - and miR-26b-5p in osteogenic differentiation of hMSCs. lyzed using starBase to elucidate the molecular mecha- Transfection with a miR-26b-5p inhibitor significantly nism through which LINC00205 regulates osteogenic inhibited osteoblast differentiation. In miR-26b-5p (See figure on next page.) Fig. 3 LINC00205 was a miR-26b-5p sponge. A MiR-26b-5p conserved seed sequence in LINC00205 identified using the tool starBase. B The relative luciferase activities of the hMSCs transfected with a NC-mimic or miR-26b-5p mimic plus a dual luciferase vector carrying the wild-type ( WT ) or mutant (MUT ) LINC00205. The reduced luciferase activity confirms the binding between LINC00205 and miR-26b-5p. **P < 0.001 when compared to WT + NC-mimic. C The relative miR-26b-5p expressions among OP-Frx, OP-no-Frx, and healthy controls from the validation set were evaluated via qRT-PCR. **P < 0.001 and ## P < 0.001. D qRT-PCR test of miR-26b-5p expression at 7, 14, and 21 days of osteoblast differentiation. **P < 0.001 when compared to day 0. E Relative miR-26b-5p expressions among hMSCs after transfection were analyzed by qRT-PCR. A notable rise in miR-26b-5p levels was observed after transfection with si-LINC00205. Meanwhile the reverse was observed in miR-26b-5p inhibitor transfected cells. **P < 0.001 when compared to si-NC; ##P < 0.001 when compared to inhibitor-NC; and &&P < 0.001 when compared to si-LINC00205 + inhibitor. All data were from three independent experiments W ang et al. BMC Musculoskeletal Disorders (2023) 24:262 Page 7 of 14 Fig. 3 (See legend on previous page.) inhibitor transfected cells, cell viability was reduced by respectively, and ALP activity was reduced by approxi- approximately 28%, while RUNX2, OCN, and ALP lev- mately 85% (Fig. 4A-E). In addition, miR-26b-5p knock- els were reduced by approximately 80%, 75%, and 70%, down partly eliminated the si-LINC00205-mediated Wang et al. BMC Musculoskeletal Disorders (2023) 24:262 Page 8 of 14 Silencing KMT2C weakens the suppressive effect effects on osteogenic differentiation (Fig. 4A-E). of miR‑26b‑5p inhibitor on the osteogenic differentiation ARS staining revealed that the miR-26b-5p inhibitor of hMSCs reduced calcium nodules in hMSCs and reversed the To investigate the effect of miR-26b-5p/KMT2C on osteo - si-LINC00205-induced increase in calcium nodules genic differentiation, we analyzed the viability of hMSCs (Fig. 4F). These results suggest that depletion of miR- using CCK-8 assay following miR-26b-5p or KMT2C 26b-5p reverses the effect of LINC00205 silencing on the knockdown. We observed that si-KMT2C increased osteogenic differentiation of hMSCs. cell viability by 1.4-fold, whereas miR-26b-5p inhibitor reversed the effect of si-KMT2C on cell viability (Fig. 6A). MiR‑26b‑5p targets KMT2C The qRT-PCR results demonstrated that silencing of Next, we sought to elucidate the downstream regulatory KMT2C enhanced RUNX2, OCN, and ALP expression mechanism of miR-26b-5p. TargetScan analysis pre- levels and restored the miR-26b-5p inhibitor-mediated dicted the binding between miR-26b-5p and KMT2C downregulation of RUNX2, OCN, and ALP (Fig. 6B-D). (Fig. 5A). Furthermore, in luciferase assay, the co-treat- ALP activity analysis showed that KMT2C knockdown ment with the miR-26b-5p mimic and KMT2C-WT promoted ALP activity in hMSCs and abolished the effect caused > 60% reduction in luciferase activity compared of miR-26b-5p silencing (Fig. 6E). In addition, ARS staining to co-transfection with the NC mimic and KMT2C- revealed that si-KMT2C promoted calcium nodule forma- WT (Fig. 5B). Next, we analyzed the expression pat- tion in hMSCs and reversed the effects of the miR-26b-5p tern of KMT2C in the clinical samples and found that inhibitor on calcium nodules (Fig. 6F). These data suggest KMT2C was higher in the bone tissues of patients with that low KMT2C expression enhances the osteogenic dif- OP-Frx and OP-no-Frx than that in control subjects ferentiation of hMSCs and attenuates the repressive effect (Fig. 5C). Diagnostic potentials of levels of KMT2C for of miR-26b-5p inhibitor on osteogenic differentiation. OP-no-Frx were evaluated by ROC curve analysis with patients with OP-no-Frx tissues as true positive cases Discussion and control tissues as true negative cases. Area under OP is a bone disorder caused by an imbalance in bone the curve was 0.833 with standard error of 0.06112 and remodeling, which involves bone resorption and forma- 95% CI of 0.7135–0.9532. Diagnostic potentials of lev- tion mediated by osteoclasts and osteoblasts, respectively els of KMT2C for OP-Frx were evaluated by ROC curve [25]. Osteoblasts have a key role in bone formation and a analysis with patients with OP-Frx tissues as true posi- new bone matrix is formed by the proliferation, differen - tive cases and OP-no-Frx tissues as true negative cases. tiation, and matrix mineralization of pre-osteoblasts [26]. Area under the curve was 0.734, with standard error of HMSCs are a source of osteoblasts and play a key role in 0.07401 and 95% CI of 0.5293–0.8795 (Supplementary bone tissue renewal [27]. ALP and OCN are established Fig. 1C). Moreover, KMT2C expression decreased dur- markers of late osteogenic differentiation [28]. RUNX2 ing osteogenic differentiation of hMSCs (Fig. 5D). To has also been reported to be important for osteogenic dif- determine the relationship between KMT2C and miR- ferentiation and bone development [29, 30]. It regulates 26b-5p, hMSCs were transfected with si-KMT2C and downstream molecules, including OCN, and is a crucial miR-26b-5p inhibitors. The treatment with si-KMT2C target in osteogenic differentiation [29, 30]. In this study, was observed to reduce both the mRNA and protein we looked into the role of the LINC00205/miR-26b-5p/ levels of KMT2C, whereas the miR-26b-5p inhibitor KMT2C axis on regulating the osteogenic differentiation restored KMT2C levels (Fig. 5E and F). These results of hMSCs by examining ALP, OCN, RUNX2, and cal- indicate that KMT2C is abnormally expressed in OP cium deposition in them and subsequently the influence and during osteogenic differentiation of hMSCs and on the progression of OP. that it is negatively regulated by miR-26b-5p. (See figure on next page.) Fig. 4 LINC00205 hindered the osteogenic differentiation of hMSCs by serving as miR-26b-5p sponge. A The CCK-8 assay revealed a lower viability in miR-26b-5p inhibitor transfected hMSCs compared to those that had si-LINC00205 transfection. **P < 0.001 when compared to si-NC; ##P < 0.001 when compared to inhibitor-NC; and &&P < 0.001 when compared to si-LINC00205 + inhibitor at the same time. B-D The relative expressions of RUNX2 (B), OCN (C), and ALP (D) mRNAs in hMSCs after si-LINC00205 or miR-26b-5p inhibitor transfection as quantified by qRT-PCR. The expressions of the three mRNAs are the highest among cells transfected with si-LINC00205, while the opposite is true for miR-26b-5p inhibitor transfected cells. **P < 0.001 when compared to si-NC; ##P < 0.001 when compared to inhibitor-NC; and &&P < 0.001 when compared to si-LINC00205 + inhibitor. E The ALP activity assay revealed that the ALP activity was the highest in hMSCs transfected with si-LINC00205, while was the lowest in hMSCs transfected with miR-26b-5p inhibitor. **P < 0.001 when compared to si-NC; ##P < 0.001 when compared to inhibitor-NC; and &&P < 0.001 when compared to si-LINC00205 + inhibitor. F Images of the si-LINC00205 and miR-26b-5p inhibitor transfected hMSCs stained with ARS. All data were from three independent experiments W ang et al. BMC Musculoskeletal Disorders (2023) 24:262 Page 9 of 14 Fig. 4 (See legend on previous page.) Wang et al. BMC Musculoskeletal Disorders (2023) 24:262 Page 10 of 14 LncRNAs regulate osteogenic differentiation; thus, osteogenic differentiation of hMSCs. This study is the they are important for the development of OP [31]. For first to reveal that LINC00205 regulates the osteogenic example, Tang et al. showed that lncRNA IGF2-AS pro- differentiation of hMSCs by sponging miR-26b-5p. motes osteogenic differentiation of hMSCs as evidenced KMT2C promotes the synthesis of enhancer RNA and by increased ARS staining and levels of ALP, osterix transcription promoters. It is also crucial in the activa- (Osx), OCN, and RUNX2 [32]. Several studies have tion of enhancers and cell type specific gene expression revealed that LINC00205 is involved in the regulation of during cell differentiation [36, 37]. In addition, KMT2C various biological functions in cells. Xie et al. [33] found may affect the enhancer activity of genes that influence that LINC00205 was elevated in lung cancer and that its invasive and metastatic properties in osteosarcoma [20, knockdown inhibited the growth and migration of can- 38]. However, there are limited reports on the role of cer cells and increased apoptosis. Similarly, Zhang et al. KMT2C in osteogenic differentiation and development [12] showed that depletion of LINC00205 reduced the of OP. Bioinformatics analysis revealed that miR-26b-5p proliferation and metastasis of retinoblastoma cells and targets and represses KMT2C expression. Moreover, promoted apoptosis. We examined LINC00205 levels in KMT2C expression was examined in the bone tissues of OP-no-Frx and OP-Frx. Although LINC00205 was highly patients with OP and in hMSCs during osteogenic dif- expressed in OP, its level was higher in OP-Frx suggest- ferentiation. A higher level of KMT2C was observed ing that LINC00205 may worsen OP. Hence, we further in OP-no-Frx and OP-Frx patients compared to con- looked into the role of LINC00205 in regulating the oste- trol subjects. The levels of KMT2C were also higher in ogenic differentiation of hMSCs. In vitro experiments hMSCs, which decreased with the progression of osteo- showed that LINC00205 was under-expressed during genic differentiation. It has been suggested that KMT2C osteogenic differentiation, and its knockdown promoted may be used to treat vertebral fractures and OP. This ALP, OCN, and RUNX2 expression, ALP activity, and study revealed that silencing KMT2C promotes RUNX2, calcium deposition. These results indicate the involve - OCN, and ALP mRNA levels, ALP activity, and calcium ment of LINC00205 in the osteogenic differentiation of nodule formation in hMSCs in vitro. Moreover, KMT2C hMSCs. partially attenuated miR-26b-5p interference-mediated The target-mimetic and sponge/decoy functions of inhibition of osteogenic differentiation. This suggests that lncRNAs have recently been uncovered [34]. Several miR-26b-5p directly targets KMT2C to regulate osteo- reports have revealed the role of miR-26b-5p in bone dis- genic differentiation of hMSCs. eases. MiR-26b-5p accelerates chondrocyte aging, carti- Admittedly, this study has several limitations. In vivo lage degeneration, and osteoarthritis progression [35]. experiments to investigate the crosstalk between Furthermore, miR-26b-5p induces calcium deposition LINC00205/miR-26b-5p/KMT2C were not carried out and promotes the expression of osteogenic genes such in this study. This issue needs to be addressed in future as ALP, OPN, OCN, and COL1A1 in rat bone marrow- studies to further elucidate the effects of LINC00205 on derived mesenchymal stem cells (BMSCs) [19]. Because OP and vertebral fractures in animals. In addition, the miR-26b-5p induces osteogenic differentiation, we ana - downstream mechanism of LINC00205/miR-26b-5p/ lyzed its expression in OP in this study. The results KMT2C axis and the miR-26b gene cluster including revealed low expression of miR-26b-5p in OP-Frx and miR-26b-3p merit further investigation. OP-no-Frx, and further knockdown inhibited the osteo- In conclusion, this is the first report of the mechanism genic differentiation of hMSCs. Furthermore, targeting and expression of LINC00205/miR-26b-5p/KMT2C in analysis revealed that LINC00205 sponges miR-26b-5p. OP and vertebral fractures. LINC00205 may worsen OP Rescue experiments demonstrated that miR-26b-5p by reducing hMSCs viability, levels of osteogenic differ - interference reversed LINC00205 knockdown-induced entiation marker genes, and calcium nodule formation. (See figure on next page.) Fig. 5 MiR-26b-5p targeted the KMT2C gene. A The miR-26b-5p conserved seed sequence in KMT2C as predicted by TargetScan. B The relative luciferase activities of hMSCs transfected with a NC-mimic or miR-26b-5p mimic plus a dual luciferase vector carrying the WT or MUT KMT2C. The reduced luciferase activity confirms the KMT2C and miR-26b-5p binding. **P < 0.001 when compared to WT + NC mimics. C Relative KMT2C expressions among OP-Frx, OP-no-Frx, and healthy controls from the validation set were evaluated via qRT-PCR. **P < 0.001 and ##P < 0.001. D KMT2C expressions analyzed by qRT-PCR at 7, 14, and 21 days of osteoblast differentiation. **P < 0.001 when compared to day 0. E Relative KMT2C mRNA expression in hMSCs after treatment with si-KMT2C or miR-26b-5p inhibitor, as analyzed by qRT-PCR. **P < 0.001 when compared to inhibitor-NC; ##P < 0.001 when compared to si-NC; and &&P < 0.001 when compared to inhibitor + si-KMT2C. F Protein bands (left) and relative protein expressions of KMT2C evaluated by Western blotting analysis of hMSCs after si-KMT2C or miR-26b-5p inhibitor transfection. **P < 0.001 when compared to inhibitor-NC; ##P < 0.001 when compared to si-NC; and &&P < 0.001 when compared to inhibitor + si-KMT2C. All data were from three independent experiments W ang et al. BMC Musculoskeletal Disorders (2023) 24:262 Page 11 of 14 Fig. 5 (See legend on previous page.) Wang et al. BMC Musculoskeletal Disorders (2023) 24:262 Page 12 of 14 Fig. 6 Silencing KMT2C attenuated the repressive effect of miR-26b-5p inhibitor on the osteogenic differentiation of hMSCs. A The CCK-8 assay demonstrated a lower viability in miR-26b-5p inhibitor transfected hMSCs, whereas a higher viability was observed after si-KMT2C transfection. **P < 0.001 when compared to si-NC; ##P < 0.001 when compared to inhibitor-NC; and &&P < 0.001 when compared to si-KMT2C + inhibitor at the same time. B-D The relative expressions of RUNX2 (B), OCN (C), and ALP (D) mRNAs in hMSCs after treatment with si-KMT2C or miR-26b-5p inhibitor as measured by qRT-PCR. **P < 0.001 when compared to si-NC; ##P < 0.001 when compared to inhibitor-NC; and &&P < 0.001 when compared to si-KMT2C + inhibitor. 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Journal
BMC Musculoskeletal Disorders – Springer Journals
Published: Apr 4, 2023
Keywords: lncRNA LINC00205; Osteoporosis; Spinal fracture; miR-26b-5p; KMT2C; Human mesenchymal stem cells