Long noncoding RNA LINC00339 promotes osteoporosis development via modulating of regulator CDC42 by binding PARP1.

Dong-Li Zhu: Writing – original draft, Conceptualization, Writing – review & editing, Methodology, Validation, Software, Data curation. Yan Zhang: Data curation, Methodology, Writing – review & editing, Visualization, Investigation. Xiao-Yu Zhang: Data curation, Methodology, Investigation. Zi-Han Qiu: Formal analysis, Methodology, Data curation. Ke Li: Formal analysis, Visualization, Methodology, Data curation. Xiao-Rong Zhou: Investigation, Methodology, Validation. Zhen-Zhen He: Investigation, Methodology, Formal analysis. Xiao-Feng Chen: Investigation, Data curation, Methodology, Software. Shan-Shan Dong: Methodology, Resources, Formal analysis. Wen Tian: Methodology, Formal analysis, Validation. Ya-Kang Wang: Data curation, Validation, Resources. Tie-Lin Yang: Writing – review & editing, Supervision, Conceptualization. Bo Yang: Resources, Writing – review & editing, Conceptualization, Supervision. Yan Guo: Methodology, Formal analysis, Writing – review & editing, Data curation, Conceptualization, Investigation, Supervision.

This study has been performed in accordance with the Declaration of Helsinki and approved by the institutional review board of Shaanxi Provincial People's Hospital, Hospital (No.2017-052), and written informed consent for participation was obtained from all subjects.

All persons have granted their consent for publication.

The funding for this study was provided by the 10.13039/501100001809 National Natural Science Foundation of China ( 82170896 , 32370653 and 32300486 ); Science Fund for Distinguished Young Scholars of Shaanxi Province ( 2025JC-JCQN-054 ); Innovation Capability Support Program of Shaanxi Province (2022TD-44, 2024RS-CXTD-86); Shannxi Provincial People's Hospital Science and Technology development Incubation Fund ( 2022BJ-11 ); The 10.13039/501100007128 Shaanxi Natural Science Foundation of China (Number 2023-JC-YB-811 ); 10.13039/501100002858 China Postdoctoral Science Foundation ( 2021M692582 ) and the Fundamental Research Funds for the Central Universities.

To investigate the functional involvement of LINC00339 in osteoporosis pathogenesis, we first quantified its expression patterns in clinical specimens. We found a significant elevation of LINC00339 transcript levels in bone tissues from osteoporosis patients compared to age-matched controls ( P  < 0.001; Fig. 1 A, Table S1 ). Notably, this upregulation exhibited a positive correlation with clinical T-scores, a key diagnostic parameter for bone mineral density ( Fig. 1 B). Fig. 1 Osteoblast function is closely correlated with LINC00339 expression. A. The expression of total LINC00339 in the bone tissue of healthy controls (n = 15) and patients with osteoporosis (n = 18). B. Correlation analysis between LINC00339 and T score in bone specimens. C. Analysis of the expression of the osteogenic marker genes OCN after LINC00339 knockdown in primary human osteoblasts. D, E. Analysis of the expression of the osteogenic marker genes RUNX2, ALP and OCN after LINC00339 knockdown and overexpression in U2OS cells. Representative results of three independent experiments are shown. F. ALP staining images and representative Alizarin red staining images of the sh-NC and sh-LINC00339 osteoblasts induced with osteogenic medium for 7 days. Scale bar, 5 mm. G . Quantification of ALP staining areas (Left) and ARS staining areas (Right). H . ALP staining images and representative ARS images of the OE-NC and OE-LINC00339 osteoblasts induced with osteogenic medium for 7 days. Scale bar, 5 mm. I . Quantification of ALP staining areas (Left) and ARS staining areas (Right). n = 3 for each group. Data are represented as mean ± standard deviation. Two-way ANOVA was performed to study the interaction between two independent variables. Significances were determined using two-tailed paired student's t -test between two groups. ∗ P  < 0.05, ∗∗ P  < 0.01, ∗∗∗ P  < 0.001. Fig. 1 Osteoblast function is closely correlated with LINC00339 expression. A. The expression of total LINC00339 in the bone tissue of healthy controls (n = 15) and patients with osteoporosis (n = 18). B. Correlation analysis between LINC00339 and T score in bone specimens. C. Analysis of the expression of the osteogenic marker genes OCN after LINC00339 knockdown in primary human osteoblasts. D, E. Analysis of the expression of the osteogenic marker genes RUNX2, ALP and OCN after LINC00339 knockdown and overexpression in U2OS cells. Representative results of three independent experiments are shown. F. ALP staining images and representative Alizarin red staining images of the sh-NC and sh-LINC00339 osteoblasts induced with osteogenic medium for 7 days. Scale bar, 5 mm. G . Quantification of ALP staining areas (Left) and ARS staining areas (Right). H . ALP staining images and representative ARS images of the OE-NC and OE-LINC00339 osteoblasts induced with osteogenic medium for 7 days. Scale bar, 5 mm. I . Quantification of ALP staining areas (Left) and ARS staining areas (Right). n = 3 for each group. Data are represented as mean ± standard deviation. Two-way ANOVA was performed to study the interaction between two independent variables. Significances were determined using two-tailed paired student's t -test between two groups. ∗ P  < 0.05, ∗∗ P  < 0.01, ∗∗∗ P  < 0.001. To mechanistically interrogate LINC00339's role in osteogenic differentiation, we performed lentiviral-mediated short hairpin RNA (shRNA) knockdown in primary human osteoblasts and U2OS cells. We found that the expression of the osteogenic marker gene OCN (osteocalcin) was significantly increased in primary human osteoblasts after LINC00339 knockdown ( Fig. 1 C). In U2OS cells, genetic silencing of LINC00339 markedly upregulated osteogenic differentiation markers, including RUNX2 (Runt-related transcription factor 2), ALP (alkaline phosphatase), and OCN, at both transcriptional ( Fig. 1 D) and functional levels. Specifically, LINC00339-depleted osteoblasts demonstrated enhanced alkaline phosphatase activity and accelerated matrix mineralization, as evidenced by alizarin red staining ( Fig. 1 F and G). Conversely, ectopic overexpression of LINC00339 in U2OS osteosarcoma cells via lentiviral transduction significantly suppressed the expression of osteogenic markers ( Fig. 1 E) and impaired both ALP activity and calcium nodule formation ( Fig. 1 H and I). These bidirectional genetic manipulations-silencing and overexpression-consistently demonstrated that LINC00339 acts as a negative regulator of osteoblast differentiation. Collectively, our functional genomics approach establishes LINC00339 as a critical epigenetic modulator of osteogenic capacity, with its dysregulation directly linked to impaired bone matrix formation and metabolic homeostasis. To elucidate the molecular mechanisms underlying LINC00339's role in osteoporosis, we first investigated its subcellular localization in osteoblasts. Nuclear-cytoplasmic fractionation combined with RNA fluorescence in situ hybridization (FISH) demonstrated that LINC00339 predominantly resides in the cytoplasm, as evidenced by confocal microscopy analysis ( Fig. 2 A). This cytoplasmic localization suggests that LINC00339 may exert its biological functions primarily through post-transcriptional regulatory mechanisms. Specificity validation of the FISH probe revealed marked reduction of LINC00339 signals in knockdown cells and corresponding signal enhancement in overexpression models ( Fig. S1 ), confirming the reliability of our detection method. Fig. 2 LINC00339 functions by interacting with PARP1. A. RNA FISH showed that LINC00339 was predominantly localized in the cytoplasm. Scale bar, 50 μm. B. Schematic diagram of RNA pull-down experiment. The antisense and sense of LIN00339 were synthesized in vitro. C. Silver staining of biotinylated LIN00339-associated proteins. LC-MS identified the differential RBPs binding to AS-LINC00339 and S-LINC00339. D. Western blot of protein from LINC00339-pulldown assays. Western blot with PARP1 antibody shows only the sense of LINC00339 enrichment PARP1. E. PARP1 RIP assay to analyze interactions between PARP1 and LINC00339 in U2OS osteoblast-like cells. WB shows the PARP1 antibody efficiency of immunoprecipitation. F. RT-qPCR to analyze the enrichment of LINC00339 in RNA-protein complexes. The LINC00339 abundance in anti-PARP1 group was much more than the IgG group. Values were normalized by the input group. ∗∗∗ P  < 0.001 versus IgG, by Student's t -test. (AS-LINC00339: Anti-sense of LINC00339, S-LINC00339: Sense LINC00339). G . IF-RNA FISH experiment was performed in U2OS cells to detect the co-localization of LINC00339 and PARP1. Bar: 10 μm. Data are expressed as the mean ± standard deviation of 3 independent experiments. ∗ P  < 0.05, ∗∗ P  < 0.01 and ∗∗∗ P  < 0.001, two-tailed paired student's t -test. Fig. 2 LINC00339 functions by interacting with PARP1. A. RNA FISH showed that LINC00339 was predominantly localized in the cytoplasm. Scale bar, 50 μm. B. Schematic diagram of RNA pull-down experiment. The antisense and sense of LIN00339 were synthesized in vitro. C. Silver staining of biotinylated LIN00339-associated proteins. LC-MS identified the differential RBPs binding to AS-LINC00339 and S-LINC00339. D. Western blot of protein from LINC00339-pulldown assays. Western blot with PARP1 antibody shows only the sense of LINC00339 enrichment PARP1. E. PARP1 RIP assay to analyze interactions between PARP1 and LINC00339 in U2OS osteoblast-like cells. WB shows the PARP1 antibody efficiency of immunoprecipitation. F. RT-qPCR to analyze the enrichment of LINC00339 in RNA-protein complexes. The LINC00339 abundance in anti-PARP1 group was much more than the IgG group. Values were normalized by the input group. ∗∗∗ P  < 0.001 versus IgG, by Student's t -test. (AS-LINC00339: Anti-sense of LINC00339, S-LINC00339: Sense LINC00339). G . IF-RNA FISH experiment was performed in U2OS cells to detect the co-localization of LINC00339 and PARP1. Bar: 10 μm. Data are expressed as the mean ± standard deviation of 3 independent experiments. ∗ P  < 0.05, ∗∗ P  < 0.01 and ∗∗∗ P  < 0.001, two-tailed paired student's t -test. To identify potential molecular partners of LINC00339, we performed RNA pull-down assays followed by mass spectrometry analysis. Following in vitro transcription to generate sense and antisense RNA probes ( Fig. 2 B), silver staining detected multiple distinct protein bands specifically associated with the sense strand ( Fig. 2 C). Subsequent LC-MS analysis identified 51 RNA-binding proteins (RBPs) with specific affinity for LINC00339. Applying stringent filtering criteria (Unique Peptides >2), we narrowed this list to 7 high-confidence candidates ( Tables S3-S4 ). Among these, PARP1 emerged as a prime target due to its established regulatory role in osteoblast function [ 14 , 15 ]. Validation experiments confirmed the specific interaction between LINC00339 and PARP1. Immunoblot analysis of RNA-protein pull-down assays revealed PARP1 enrichment exclusively with the sense strand ( Fig. 2 D). Reciprocal RNA immunoprecipitation (RIP) assays using anti-PARP1 antibody demonstrated significant enrichment of LINC00339 compared to IgG controls ( Fig. 2 E), with immunoprecipitation efficiency verified by Western blot ( Fig. 2 F). Immunofluorescence (IF)-RNA FISH revealed that LINC00339 and PARP1 had co-localization in cells ( Fig. 2 G). These complementary approaches provide compelling evidence for a direct molecular interaction between LINC00339 and PARP1, suggesting their potential cooperation in bone metabolism regulation. As a key regulator of bone metabolism [ 16 ], CDC42 has been shown to be essential for skeletal development, with Cdc42-deficient mice exhibiting severe skeletal abnormalities [ 17 , 18 ]. We found a significant decrease in CDC42 expression levels in osteoporosis patients ( P  < 0.001) ( Fig. 3 A). To delineate the mechanistic relationship between LINC00339 and CDC42 regulation, we established stable cell lines with lentivirus-mediated LINC00339 overexpression or knockdown. We found that LINC00339 knockdown promoted the expression of CDC42 ( Fig. 3 B–D). Strikingly, LINC00339 overexpression significantly suppressed both mRNA and protein levels of CDC42 ( Fig. 3 C–E). This regulatory pattern was mirrored in PARP1 manipulation experiments: PARP1 knockdown via shRNA elevated CDC42 expression ( Fig. 4 A–C), whereas PARP1 overexpression inhibited CDC42 transcription, resulting in concomitant reductions at both transcriptional and translational levels ( Fig. 4 B–D). These complementary findings suggest that LINC00339 and PARP1 may coordinately regulate CDC42 protein homeostasis, potentially through modulation of CDC42 stability. Fig. 3 LINC00339 regulates the expression of CDC42. A. The expression of total CDC42 in the bone tissue of healthy controls (n = 15) and patients with osteoporosis (n = 18). B. Effect of LINC00339 knockdown on mRNA of CDC42. C. Effect of LINC00339 overexpression on mRNA of CDC42. D. Effect of LINC00339 knockdown in protein level on CDC42. E. Effect of LINC00339 overexpression in protein level on CDC42. Data shown are the quantification of protein levels. Data are expressed as the mean ± standard deviation of 3 independent experiments. ∗ P  < 0.05, ∗∗ P  < 0.01 and ∗∗∗ P  < 0.001, two-tailed paired student's t -test. Fig. 3 Fig. 4 PARP1 played important roles in LINC00339-mediated target gene CDC42. A. Effect of PARP1 knockdown on expression levels of CDC42. B. Effect of PARP1 overexpression on expression level of CDC42. C. Effect of PARP1 knockdown on protein level of CDC42. D. Effect of PARP1 overexpression on protein level of CDC42. E, F. Co-IP detection of the interaction of PARP1 and CDC42. G . IF staining was performed in U2OS cells to detect the co-localization of CDC42 and PARP1. Bar: 10 μm. Data are expressed as the mean ± standard deviation of 3 independent experiments. ∗ P  < 0.05, ∗∗ P  < 0.01, and ∗∗∗ P  < 0.001, two-tailed paired student's t -test. Fig. 4 LINC00339 regulates the expression of CDC42. A. The expression of total CDC42 in the bone tissue of healthy controls (n = 15) and patients with osteoporosis (n = 18). B. Effect of LINC00339 knockdown on mRNA of CDC42. C. Effect of LINC00339 overexpression on mRNA of CDC42. D. Effect of LINC00339 knockdown in protein level on CDC42. E. Effect of LINC00339 overexpression in protein level on CDC42. Data shown are the quantification of protein levels. Data are expressed as the mean ± standard deviation of 3 independent experiments. ∗ P  < 0.05, ∗∗ P  < 0.01 and ∗∗∗ P  < 0.001, two-tailed paired student's t -test. PARP1 played important roles in LINC00339-mediated target gene CDC42. A. Effect of PARP1 knockdown on expression levels of CDC42. B. Effect of PARP1 overexpression on expression level of CDC42. C. Effect of PARP1 knockdown on protein level of CDC42. D. Effect of PARP1 overexpression on protein level of CDC42. E, F. Co-IP detection of the interaction of PARP1 and CDC42. G . IF staining was performed in U2OS cells to detect the co-localization of CDC42 and PARP1. Bar: 10 μm. Data are expressed as the mean ± standard deviation of 3 independent experiments. ∗ P  < 0.05, ∗∗ P  < 0.01, and ∗∗∗ P  < 0.001, two-tailed paired student's t -test. To further investigate the molecular interplay between these components, co-immunoprecipitation (Co-IP) assays revealed direct physical interaction between PARP1 and CDC42 ( Fig. 4 E and F), providing structural basis for their functional association. IF staining revealed that CDC42 and PARP1 had co-localization in cells ( Fig. 4 G). Moreover, we found in primary human osteoblasts that the expression of PARP1 was also significantly decreased after LINC00339 knockdown ( Fig. 5 A). Notably, while LINC00339 overexpression upregulated PARP1 expression levels ( Fig. 5 B). Crucially, the suppressive effects of LINC00339 overexpression on CDC42 protein abundance and activity were effectively rescued by PARP1 knockdown ( Fig. 5 C and D), establishing PARP1 as an essential mediator of LINC00339's regulatory function. Further, we reintroduced PARP1 into LINC00339-knockdown cells to investigate their combined effects on CDC42 expression. Our findings revealed that in LINC00339-deficient cells. Our results show that in the cells with LINC00339 knockdown, the expression of CDC42 increases ( Fig. 5 E and F). However, restoring the expression of PARP1 in LINC00339 knockdown cells leads to a decrease in the expression of CDC42 ( Fig. 5 E and F). Collectively, our data support a model wherein cytoplasmic LINC00339 forms a functional complex with PARP1, which subsequently targets CDC42. This LINC00339-PARP1-CDC42 regulatory axis ultimately suppresses osteoblast differentiation through coordinated post-transcriptional regulation of CDC42 expression. Fig. 5 LINC00339–PARP1 complex co-regulates the expression of CDC42. A. The effects of LINC00339 knockdown on PARP1 mRNA expression were detected in primary human osteoblasts. B. Effect of LINC00339 overexpression on mRNA and protein expression of PARP1 in U2OS cells. C. Effect of PARP1 knockdown on LINC00339 overexpression-induced mRNA expression of CDC42. D. Effect of PARP1 knockdown on LINC00339 overexpression-induced protein expression of CDC42. E, F. Effect of PARP1 overexpression on LINC00339 knockdown-induced mRNA and protein expressions of CDC42. Data are expressed as the mean ± standard deviation of 3 independent experiments. ∗ P  < 0.05, ∗∗ P  < 0.01, and ∗∗∗ P  < 0.001 versus vector, by Student's t -test. Fig. 5 LINC00339–PARP1 complex co-regulates the expression of CDC42. A. The effects of LINC00339 knockdown on PARP1 mRNA expression were detected in primary human osteoblasts. B. Effect of LINC00339 overexpression on mRNA and protein expression of PARP1 in U2OS cells. C. Effect of PARP1 knockdown on LINC00339 overexpression-induced mRNA expression of CDC42. D. Effect of PARP1 knockdown on LINC00339 overexpression-induced protein expression of CDC42. E, F. Effect of PARP1 overexpression on LINC00339 knockdown-induced mRNA and protein expressions of CDC42. Data are expressed as the mean ± standard deviation of 3 independent experiments. ∗ P  < 0.05, ∗∗ P  < 0.01, and ∗∗∗ P  < 0.001 versus vector, by Student's t -test.

In this study, Human Embryonic Kidney 293T (HEK293T) cells and the U2OS cells were utilized, both of which were purchased from the American Type Culture Collection (ATCC). HEK293T cells were cultured in 1 × DMEM (GIBCO, USA), while U2OS cells were cultured in RPMI-1640 medium. Both mediums were supplemented with 10 % fetal bovine serum (FBS) (Invitrogen, USA), 100 U mL − 1 penicillin and 100 μg mL − 1 streptomycin. The cells were maintained in 5 % CO 2 at 37 °C incubator. To induce osteoblast differentiation, U2OS cells were cultured for 7 days in the presence of 50 μg mL −1 ascorbic acid (1043003, Sigma-Aldrich) and 10 mM β-glycerophosphate (G9422, Sigma-Aldrich). Human primary osteoblasts were isolated from cancellous bone tissue obtained from lumbar vertebrae of surgical patients (vertebral compression fractures or lumbar disc herniation). Cells were cultured in MEM-α medium (HyClone, USA) at 37 °C under 5 % CO 2 . All donor samples were provided by Shaanxi Provincial People's Hospital, China. The study protocol was approved by the institutional ethics committees of the participating institutions, and written informed consent was obtained from all participants. Both normal and osteoporotic bone tissue samples were clinically obtained from their lumbar spine (Lumbar L1-L5) with an average age of 60 years old who underwent surgical procedures at the Xi'an Jiaotong University Shaanxi Provincial People's Hospital (Xi'an, China) ( Table S1 ). Bone mineral density (BMD) was measured using dual energy X-ray absorptiometry (DXA). Based on the information provided by the National Osteoporosis Foundation, T score ≤ −2.5 is considered as osteoporosis, whereas T score ≥ −1.0 is considered as normal. The study was approved by the Ethics Committee of Xi'an Jiaotong University Shaanxi Provincial People's Hospital. Prior to their participation, written consent from all participants was obtained after they were fully informed. The shRNA plasmids targeting LINC00339, PARP1, and a negative control (shNC) were constructed, respectively. The overexpression plasmids were created by cloning the cDNA of LINC00339 or PARP1 into the pcDNA3.1 vector using PCR, with the corresponding empty vectors as the control. The aforementioned plasmids were transfected into HEK293T cells with the transfection reagent of polyethylenimine (PEI) (Polysciences, USA), along with lentivirus packaging plasmids, specifically psPAX2 (Addgene #12259) and pCMV-VSVG (Addgene #8454). The lentiviruses generated and released into the supernatant of the cultured HEK293T cells, these were subsequently collected and concentrated, then add condensed lentiviruses into U2OS and human primary osteoblasts cells in the presence of polybrene (Solarbio, China). After 48 h, puromycin (1 mg mL − 1 ) (Beyotime, China) was used to screen the positive cells. The primers used in this study are provided in Table S2 . Total RNA was extracted from U2OS and human primary osteoblasts cells with Fast 200 reagent kit (Fastagen, China) and then converted to cDNA with the PrimeScript RT reagent kit (TakaRa, Japan). RT-PCR reactions were performed with the QuantiTect SYBR Green RT-PCR Kit (QIAGEN, USA) on the Bio-Rad CFX Connect PCR machine (Bio-Rad, USA). GAPDH served as an internal control. All PCR amplification reactions were replicated three times. Total proteins were extracted from the cells utilizing the lysis buffer. Subsequently, the proteins were separated according to their molecular weight using a using a 10 % SDS-PAGE gel. The separated proteins were then transferred from the gel to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, USA) employing a semidry transfer method. Following this, the membrane was incubated in the blocking buffer for 1 h. A diluted primary antibody targeting CDC42, PARP1 and β-Actin (1:2000; Santa Cruz) was applied to the membrane and incubated overnight at 4 °C. After washing three times with TBST, a diluted secondary antibody was applied to the membrane and incubated for 1 h at room temperature. The proteins were subsequently detected and visualized using a chemiluminescence detection kit (ECL, Millipore, USA). The results were normalized against β-Actin. Allowing for a comparative analysis of the expression levels across different experimental groups. In this study, RNA FISH was conducted using a FISH kit (RiboBio, Guangzhou, China). In summary, the cells were fixed using 4 % paraformaldehyde. After fixation, the cells were hybridized overnight at a probe concentration of 5 mM. The FISH probes specifically targeting LINC00339 were uniquely labeled with CY3 and custom-synthesized by RiboBio Company. Finally, the cells were subjected to blocking using an HRP Blocker, and the nuclei were counterstained with DAPI. Furthermore, we also performed immunofluorescence (IF) staining and IF-RNA FISH experiments respectively in U2OS cells to detect the co-localization of CDC42 and PARP1, LINC00339 and PARP1. The FISH probe in IF-RNA FISH was obtained by using the T7 high yield RNA transcription kit (Beyotime, China), and the concentrations of anti-CDC42 and anti-PARP1 (Santa cruz) were both 1:150. The TSA fluorescence system kit (Gzjnbio, China) is used for fluorescence signal amplification. All images were obtained and visualized utilizing the confocal microscope (Leica, Germany). The sense and antisense strands of LINC00339 were synthetized via in vitro transcription with the MEGAscript T7 Transcription kit (Thermo Fisher Scientific, USA). Full-length LINC00339 was producted in vitro using biotinylated nucleotides. The biotinylated LINC00339, along with antisense LINC00339 serving as a negative control, was subsequently incubated with total cell lysates from cells exhibiting high expression levels of LINC00339. Afterward, we isolated and precipitated the complexes using streptavidin. The biotin-tagged RNAs were combined with streptavidin agarose beads (Life Technologies, USA) and allowed to incubate at 4 °C overnight. Freshly prepared total cell lysates were introduced into each reaction, along with a Protease/Phosphatase Inhibitor and RNase inhibitor. Subsequently, the mixture was incubated at 4 °C with rotation for 1 h. At last, the eluted proteins were separated through SDS-PAGE and identified by mass spectrometry. RIP assay was performed utilizing the Magna RIP kit (Millipore, USA), adhering strictly to the manufacturer's instructions, and purified rabbit IgG from Millipore (USA) was used concurrently as a negative control. The quantities of PARP1 and mRNAs present in the immunoprecipitated (IP) PARP1-mRNA or IgG-mRNA complexes, as well as in the original extracts from which these complexes were immunoprecipitated, were identified using RT-qPCR and Western blot. The cells underwent three washes utilizing 1 × PBS, and subsequently, proteins were extracted utilizing 2 mL of IP lysis buffer specifically designed for Western blot and IP procedures (Solarbio, China). After centrifugation, the soluble supernatant was collected, and 1 μg of the primary antibody was added to the cell lysate. The collected supernatant was utilized for Co-IP, with 50 μL reserved for input. The supernatant was incubated overnight at 4 °C with rotation, using normal mouse IgG, PARP1 and CDC42 antibody. To precipitate the antibody-protein complexes, the Protein A/G Magnetic Beads (Thermo Fisher Scientific, USA) were utilized in this assay. The immunoprecipitated proteins were isolated by suspending the beads and heating them for 5 min to boiling point. Then samples were used for Western blot. For ALP staining, cells were fixed with 4 % paraformaldehyde fixative solution (Beyotime, China) for 30 min at room temperature, then rinsed three times with 1 × PBS. The cells were stained using a BCIP/NBT ALP color development kit (Beyotime, China) according to the manufacturer's instructions. We fixed the cells with 4 % paraformaldehyde fixative solution (Beyotime, China) for 30 min, followed by rinsing them three times with 1 × PBS to remove residual fixative. Then, added the ARS solution (Solarbio, China) to the fixed cells and incubated for 1 h at room temperature. Remove the excess stain by rinsing with distilled water or PBS until the background was clear. Captured and visualized images for the stained cells under a microscope to detect mineralized nodules or calcium deposits. The results were normalized to the control group. The data were presented in the form of mean values ± standard deviation, representing the results of three independent experimental replicates. To determine significant differences in mean values between two groups, a two-tailed Student's t -test was employed. One-way analysis of variance (ANOVA) was utilized to assess three or more distinct groups. Subsequently, Bonferroni's post-hoc comparisons were carried out. All data analyses and graphical representation were conducted utilizing the GraphPad Prism 9.0. A P -value of less than 0.05 was deemed to indicate a statistically significant result.

Emerging evidence underscores the critical regulatory roles of long non-coding RNAs (lncRNAs) in diverse physiological and pathological processes [ 19 ]. In this study, we identified significant upregulation of LINC00339 in osteoporotic bone tissue compared to healthy controls. Functional analyses revealed that elevated LINC00339 levels substantially suppressed the expression of key osteogenic markers, including ALP, RUNX2, and OCN, whereas LINC00339 knockdown enhanced their expression. These findings were corroborated by diminished alkaline phosphatase (ALP) activity and reduced Alizarin Red S (ARS)-stained mineralized nodules in LINC00339-overexpressing cells. Mechanistically, LINC00339 exerts its inhibitory effects on osteogenic differentiation by forming a cytoplasmic complex with PARP1, which destabilizes CDC42 protein. Collectively, our data delineate a novel regulatory axis in which LINC00339 impairs osteoblast differentiation through PARP1-mediated suppression of CDC42, as schematically summarized in Fig. 6 . This pathway provides mechanistic insight into the dysregulation of bone formation in osteoporosis and highlights potential therapeutic targets for bone metabolic disorders. Fig. 6 Proposed model for LINC00339-mediated regulation of the differentiation in osteoblast. Fig. 6 Proposed model for LINC00339-mediated regulation of the differentiation in osteoblast. During the differentiation of osteoblasts, several LncRNAs have been evidenced to exhibit an inhibitory effect on bone formation. Zhu et al. reported that LncRNA ANCR is significantly downregulated in osteogenic differentiation [ 20 ]. Mechanistically, the upregulation of LncRNA ANCR suppresses RUNX2 gene expression Through the enlistment of EZH2, resulting in the suppression of osteogenic differentiation [ 20 ]. Additionally, the suppression of LncRNA ANCR elevates the expression levels of osteogenic marker genes in a human bone-marrow stromal cell line. The enhancement is facilitated by the p38 MAPK signaling pathway, as evidenced by the decrease in the phosphorylated form of p38 following ANCR overexpression [ 21 ]. In vitro, Silva, A. M. et al. reported the regulatory roles of lncRNA in osteoclastogenesis [ 22 ]. The lncRNA GM15416 exerts inhibitory effects on osteoblast apoptosis and functions as a preventive factor against osteoporosis through specifically targeting the c-Fos/Fas pathway. [ 23 ]. The upregulation of LncRNA AK077216 enhances the expression of NFATc1, thus promoting osteoclast differentiation [ 24 ]. Lately, the significance of lncRNAs regulatory roles in maintaining bone homeostasis has been acknowledged [ 25 , 26 ]. For example, Bmncr [ 25 ], ORLNC1 [ 26 ], and lnc-ob1 [ 27 ] are known to be involved in bone formation. In our study, we identified the LINC00339, which we found to have high-than-normal expression levels in osteoblast from patients with osteoporosis. To evaluate the role of LINC00339 in osteoporosis, we performed overexpression and knockdown experiments. Overexpression of LINC00339 reduced the differentiation of osteoblast, and knock-down of LINC00339 increased the differentiation of osteoblast. These data imply that LINC00339 negatively regulates the differentiation of osteoblast, resulting in osteoporosis. LINC00339 is located on chromosome 1p36 and is 1.1 kb. It has been documented to possess diverse biological functions in cancer. LINC00339's role in various diseases has been reported. such as endometriosis [ 28 ]. hepatocellular carcinoma [ 29 ], colorectal cancer [ 30 ] and so on. The prognosis of most cancers was significantly improved by inhibiting LINC00339. In our study, we observed that LINC00339 overexpression suppressed the activation of CDC42, which might have resulted from a LINC00339 overexpression-induced reduction of CDC42 protein levels. Furthermore, the decrease in CDC42 protein expression and activity, induced by LINC00339 overexpression, was counteracted by the suppression of PARP1 through shRNA-mediated knockdown. In other words, LINC00339 and PARP1 might modulate the mRNA translation of CDC42. Further investigations are required to elucidate the precise mechanisms through PARP1 influences the mRNA metabolism of CDC42. Taken together, our findings provide evidence that LINC00339 bound specifically to PARP1 and formed a functional LINC00339-PARP1 complex. Nevertheless, we cannot exclude the possibility that additional, yet unidentified factors may contribute to the regulation of CDC42 activation by LINC00339. In this study, we addressed the question of whether PARP1 is required for LINC00339-mediated differentiation of osteoblast. First, we found that an increase in LINC00339 expression caused a reduction in both the expressions of CDC42. Interestingly, LINC00339 overexpression affected expression levels of PARP1. Second, Co-IP analysis revealed that PARP1 bound to CDC42. Third, the knockdown of PARP1 was able to counteract the decrease in CDC42 proteins caused by the overexpression of LINC00339. Overall, our discoveries indicate that the formation of a stable LINC00339-PARP1 complex within the cytoplasm is a necessary condition for PARP1 to interact with and regulate CDC42.

In summary, our research furnishes evidence that LINC00339 facilitate osteoporosis development and progression. The silencing of LINC00339 enhances osteogenic differentiation, demonstrated by heightened ALP activity, while its overexpression impedes osteoblast proliferation in vitro. Mechanistically, our study suggests that upregulation of LINC00339 represses CDC42 gene expression by binding PARP1, thereby causing the inhibition of osteogenic differentiation. Therefore, LINC00339 could potentially be a diagnostic biomarker for patients suffering from osteoporosis, moreover, targeting the LINC00339-PARP1-CDC42 axis may present new perspectives in the prevention or treatment strategies for osteoporosis.

Osteoporosis is a prevalent condition affecting the entire skeletal system, leading to the weakening of bones and a heightened risk of fractures [ 1 ]. Alarmingly, its global prevalence of osteoporosis is increasing in the ageing population, over 20 % of women aged 40 years or older had osteoporosis [ 2 ]. As global life expectancies continue to rise, osteoporosis is expected to pose a significant economic burden on most nations. It is anticipated that the worldwide economic cost of this condition will surge to reach an estimated $131.5 billion by the year 2050 [ 3 ]. Long noncoding RNAs (lncRNAs) have surfaced as crucial modulators of gene expression and protein functionality, employing diverse molecular mechanisms at both transcriptional and posttranscriptional levels [ 4 , 5 ]. The length of LncRNAs over 200 nucleotides and play essential roles acting as signals, decoys, guides, and scaffolds in a variety of central biological processes, encompassing epigenetics, gene transcription, splicing and translation [ 6 , 7 ]. Notably, a select subset of lncRNAs holds immense promise as potential biomarkers for diagnosing a wide range of diseases [ 8 , 9 ]. Prior studies have illuminated that lncRNAs are involved in numerous human diseases, osteoporosis being a noteworthy example among them [ 10 , 11 ]. Nonetheless, our current comprehension of the intricate mechanisms underlying the regulation of bone metabolism remains constrained. LINC00339 expression is significantly associated with several clinical characteristics. The expression of LINC00339 has been proven to increase in human diseases and has been recognized as a factor that contributes to the onset and progression of these conditions [ 12 ]. In 2018, our research has shown that an intergenic SNP rs6426749, act as a regulatory enhancer, modulating the expression levels of LINC00339 [ 13 ]. This discovery underscores the fundamental interplay between SNPs and LINC00339, opening new avenues for research. Further, our comprehensive bioinformatics analysis and functional experiments have pointed to a novel role for LINC00339 in negatively regulating the expression of cell division cycle 42 (CDC42), a crucial gene intricately involved in bone metabolism. This finding highlights the potential of LINC00339 as a key regulator in bone health and underscores the need for further investigations to unravel the precise molecular mechanisms underlying its involvement in the emergence and progression of osteoporosis. While the role of LINC00339 as a regulator in cancer has been thoroughly investigated, its function in modulating the CDC42 protein in the context of osteoporosis remains largely unexplored. Thus, future research endeavors should focus on elucidating these mechanisms, which could pave the way for the development of novel therapeutic strategies for osteoporosis and other bone-related disorders. In the course of our ongoing investigation, we have uncovered compelling evidence that LINC00339 is significantly upregulated in the bone tissue of individuals afflicted with osteoporosis. This upregulation serves a pivotal role in negatively modulating the differentiation process of osteoblasts, the cells responsible for bone formation. Intriguingly, our study further reveals an intricate interaction between LINC00339 and poly (ADP-Ribose) polymerase 1 (PARP1), a crucial enzyme involved in DNA repair and cellular metabolism. This interaction results in the suppression of CDC42, a key regulator of cellular migration, adhesion, and cytoskeletal organization. Our findings underscore the importance of the LINC00339-PARP1-CDC42 axis in the progression of osteoporosis, positioning it as a promising therapeutic target for intervention. By illuminating this novel pathway, we offer fresh perspectives into the intricate mechanisms that underlie the development and progression of this debilitating bone disease. This discovery holds immense potential to facilitate the development of targeted therapeutic strategies aimed at reversing or mitigating the effects of osteoporosis.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The datasets supporting the conclusions of this article can be obtained from the corresponding authors.