Human bone marrow mesenchymal stromal cells (hBMSCs) are multipotent stromal cells capable of osteogenic differentiation, making them a promising cell source for bone tissue engineering and regenerative medicine. Identifying key factors that regulate hBMSCs osteogenic differentiation is crucial for enhancing bone regeneration strategies. This study aims to identify target genes underlying impaired osteogenic differentiation of hBMSCs under oxidative stress (OS) through integrated transcriptomic and proteomic approaches, and delineate the role of proenkephalin (PENK) in this process. OS model and impaired osteogenic differentiation model were established in hBMSCs using hydrogen peroxide (H2O2). Cellular oxidative stress levels were assessed using Dihydroethidium (DHE) fluorescent probes and JC-1 staining. Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity and Alizarin Red staining (ARS). Key genes and proteins were predicted via integrated transcriptomic and proteomic analyses. The role of PENK in osteogenic differentiation was validated using lentiviral transfection. This study established 400 µM H2O2 as the optimal concentration for inducing impaired osteogenic differentiation in hBMSCs. Integrated transcriptomic and proteomic analysis identified 18 pivotal regulatory genes that orchestrate impaired osteogenic differentiation of hBMSCs under OS. Among these, PENK was identified as a potential therapeutic target involved in regulating oxidative stress-impaired osteogenic differentiation of hBMSCs. Functional validation confirmed that PENK overexpression promotes osteogenic differentiation in hBMSCs. OS contributes to impaired osteogenic differentiation in hBMSCs. PENK regulates osteogenic differentiation of hBMSCs under OS and holds promise as a novel therapeutic target for bone regeneration and repair.

IntroductionOsteoarthritis (OA), a debilitating joint disorder characterized by synovial inflammation and immune myeloid cell infiltration, currently lacks a comprehensive spatial and transcriptional atlas. This study investigates the spatial dynamics, splicing kinetics, and signaling pathways that drive immune infiltration in OA synovium.MethodsWe integrated single-cell RNA sequencing (scRNA-seq) data from 8 OA and 4 healthy synovial samples with spatial transcriptomics using Spatrio. Spatial transition tensor (STT) analysis decoded multistable spatial homeostasis, while splicing kinetics and non-negative matrix factorization (NMF) identified gene modules. CellPhoneDB and pyLIGER mapped ligand-receptor interactions and transcriptional networks.ResultsRe-annotation of scRNA-seq data resolved synovial cells into 27 subclasses. Spatial analysis revealed OA-specific attractors (8 in OA vs. 6 in healthy samples), including immune myeloid (Attractor3) and lymphoid infiltration (Attractor4). Key genes OLR1 (myeloid homeostasis) and CD69 (T-cell activation) exhibited dysregulated splicing kinetics, driving inflammatory pathways. Myeloid-specific transcription factors (SPI1, MAF, NFKB1) and lymphoid-associated BCL11B were identified as regulators. Computational drug prediction nominated ZILEUTON as a potential inhibitor of ALXN5 to mitigate myeloid infiltration.DiscussionThis study delineates the spatial and transcriptional landscape of OA synovium, linking immune cell dynamics to localized inflammation. The identification of OLR1 and CD69 as spatial homeostasis drivers, alongside dysregulated signaling networks, offers novel therapeutic targets. These findings advance strategies to modulate immune infiltration and restore synovial homeostasis in OA.

Post-traumatic Osteoarthritis (PTOA) is a subtype of osteoarthritis (OA) triggered by mechanical joint injury and is characterized by pathological alterations throughout the joint, including cartilage degradation, subchondral bone remodeling, osteophyte formation, and synovitis. As a highly inflammatory subtype of OA, synovitis contributes to the progression of PTOA, with macrophages playing a pivotal role in the synovial lining. CXCL9 expression is elevated in both synovial fluid and serum of OA patients, and CXCL9 deficiency has been shown to attenuate PTOA progression in mice. However, it remains unclear whether CXCL9 can induce an immunosuppressive phenotype in bone marrow mesenchymal stem cells (BMSCs) and consequently suppress PTOA progression. This study demonstrates that ex vivo pretreatment of BMSCs with CXCL9 robustly induces an enhanced anti-inflammatory phenotype, characterized by the upregulation of key anti-inflammatory mediators. Importantly, these preconditioned BMSCs exhibit significantly enhanced capacity to polarize macrophages toward an anti-inflammatory, pro-reparative M2 phenotype in subsequent co-culture. Mechanistic investigations reveal that this enhanced effect is primarily mediated through activation of the TLR2/TGF-β2 signaling pathway within BMSCs. Finally, in a rat PTOA model, intra-articular injection of CXCL9-preconditioned BMSCs more effectively enhances M2 polarization of synovial macrophages, thereby mitigating disease progression.

Stress-induced apoptosis presents an obstacle to bone marrow mesenchymal stem cell (BMSC) transplantation to repair steroid-induced osteonecrosis of the femoral head (SONFH). Thus, appropriate intervention strategies should be explored to mitigate this. In our previous study, we discovered a new subgroup of BMSCs—the oxidative stress-resistant BMSCs (OSR-BMSCs)—which can survive the oxidative stress microenvironment in the osteonecrotic area, through a mechanism that currently remains unclear. In this study, we found that B-lymphoid tyrosine kinase (BLK) may be the crucial factor regulating the oxidative stress resistance of OSR-BMSCs, as it is highly expressed in these cells. Knockdown of BLK eliminated oxidative stress resistance, aggravated oxidative stress-induced apoptosis, reduced the survival of OSR-BMSCs in the oxidative stress microenvironment of the osteonecrotic area, and greatly weakened the transplantation efficacy of OSR-BMSCs for SONFH. By contrast, BLK was weakly expressed in oxidative stress-sensitive BMSCs (OSS-BMSCs). Overexpression of BLK in susceptible OSS-BMSCs allowed them to acquire oxidative stress resistance, inhibited oxidative stress-induced apoptosis, promoted their survival in the osteonecrotic area, and improved the transplantation efficacy of OSS-BMSCs for SONFH. Mechanistically, BLK concurrently activates redox and apoptotic signaling networks through its tyrosine kinase activity, which confers oxidative stress resistance to BMSCs and inhibits their stress-induced apoptosis of BMSCs. Herein, we report that OSR-BMSCs have intrinsic oxidative stress resistance that is conferred and mediated by BLK. This finding provides a potential new intervention strategy for improving the survival of transplanted BMSCs and the therapeutic efficacy of BMSC transplantation for SONFH.

Stress-induced apoptosis presents an obstacle to bone marrow mesenchymal stem cell (BMSC) transplantation to repair steroid-induced osteonecrosis of the femoral head (SONFH). Thus, appropriate intervention strategies should be explored to mitigate this. In our previous study, we discovered a new subgroup of BMSCs-the oxidative stress-resistant BMSCs (OSR-BMSCs)-which can survive the oxidative stress microenvironment in the osteonecrotic area, through a mechanism that currently remains unclear. In this study, we found that B-lymphoid tyrosine kinase (BLK) may be the crucial factor regulating the oxidative stress resistance of OSR-BMSCs, as it is highly expressed in these cells. Knockdown of BLK eliminated oxidative stress resistance, aggravated oxidative stress-induced apoptosis, reduced the survival of OSR-BMSCs in the oxidative stress microenvironment of the osteonecrotic area, and greatly weakened the transplantation efficacy of OSR-BMSCs for SONFH. By contrast, BLK was weakly expressed in oxidative stress-sensitive BMSCs (OSS-BMSCs). Overexpression of BLK in susceptible OSS-BMSCs allowed them to acquire oxidative stress resistance, inhibited oxidative stress-induced apoptosis, promoted their survival in the osteonecrotic area, and improved the transplantation efficacy of OSS-BMSCs for SONFH. Mechanistically, BLK concurrently activates redox and apoptotic signaling networks through its tyrosine kinase activity, which confers oxidative stress resistance to BMSCs and inhibits their stress-induced apoptosis of BMSCs. Herein, we report that OSR-BMSCs have intrinsic oxidative stress resistance that is conferred and mediated by BLK. This finding provides a potential new intervention strategy for improving the survival of transplanted BMSCs and the therapeutic efficacy of BMSC transplantation for SONFH.

Mesenchymal stem cells possess not only multilineage differentiation and self-renewal capabilities but also immunomodulatory functions. Macrophages are a critical component of innate immunity and can undergo polarization upon stimulation, with M1 and M2 macrophages exhibiting distinct surface markers, cytokine secretion profiles, and functional properties. Mesenchymal stem cells regulate macrophage polarization through multiple mechanisms, among which paracrine effects play a predominant role. In this review, we discuss the mechanisms underlying mesenchymal stem cells-mediated regulation of macrophage polarization, with a particular focus on paracrine signaling, as well as preconditioning strategies-Especially proinflammatory cytokine pretreatment-That influence the ability of mesenchymal stem cells to modulate macrophage polarization.

Further study of the mechanism of glucocorticoid (GC)-induced osteoblast (OB) apoptosis is highly important for the prevention and treatment of GC-induced osteoporosis and osteonecrosis. Serine/arginine-rich splicing factor 1 (Srsf1) expression was downregulated in a dose-dependent manner during GC-induced OB apoptosis. Knockdown of Srsf1 significantly promotes GC-induced OB apoptosis, while overexpression of Srsf1 significantly inhibits GC-induced OB apoptosis. Mechanistically, GC induces the up-regulation of histone deacetylase 4 (Hdac4) in OB, and inhibits the expression of transcription activator forkhead box C1 (Foxc1) by reducing the levels of histone H3 lysine 9 acetylation (H3K9ac) and H3K27ac in the promoter region of Foxc1, thereby down-regulating Srsf1. Next, SRSF1 regulates GC-induced OB apoptosis by regulating Bcl-2 modifying factor (Bmf) alternative splicing. From the perspective of alternative splicing, this study demonstrates that Srsf1 and its regulatory mechanism may serve as a new target for the prevention and treatment of GC-induced osteoporosis and osteonecrosis.

Secondary brain injury (SBI) is a noticeable contributor to the high mortality and morbidity rates associated with intracerebral hemorrhage (ICH), and effective treatment options remain limited. Cystatin C (CysC) emerges as a novel candidate for SBI intervention. The therapeutic effects and underlying mechanisms of CysC in mitigating SBI following ICH were explored in the current research. An in vivo ICH rat model was established by injecting autologous blood into the right caudate nucleus. Western blotting (WB) was utilized to assess the levels of CysC, cathepsin B (CTSB), and the NLRP3 inflammasome. Subsequently, the ICH rat model was treated with exogenous CysC supplementation or CysC knockdown plasmids. Various parameters, including Evans blue (EB) extravasation, brain water content, and neurological function in rats, were examined. RT-qPCR and WB were employed to determine the expression levels of CTSB and the NLRP3 inflammasome. The co-expression of CTSB, CysC, and NLRP3 inflammasome with GFAP, NeuN, and Iba1 was assessed through double-labeled immunofluorescence. The interaction between CysC and CTSB was investigated using double-labeled immunofluorescence and co-immunoprecipitation. The findings revealed an elevation of CysC expression level, particularly at 24 h after ICH. Exogenous CysC supplementation alleviated severe brain edema, neurological deficit scores, and EB extravasation induced by ICH. Conversely, CysC knockdown produced opposite effects. The expression levels of CTSB and the NLRP3 inflammasome were significantly risen following ICH, and exogenous CysC supplement attenuated their expression levels. Double-labeled immunofluorescence illustrated that CysC, CTSB, and the NLRP3 inflammasome were predominantly expressed in microglial cells, and the interaction between CysC and CTSB was evidenced. CysC exhibited potential in ameliorating SBI following ICH via effectively suppressing the activation of the NLRP3 inflammasome mediated by CTSB specifically in microglial cells. These findings underscore the prospective therapeutic efficacy of CysC in the treatment of ICH-induced complications.

Bone marrow mesenchymal stem cell (BMSC) transplantation is a promising regenerative therapy; however, the survival rate of BMSCs after transplantation is low. Oxidative stress is one of the main reasons for the high apoptosis rate of BMSCs after transplantation, so there is an urgent need to explore the mechanism of oxidative stress-induced apoptosis of BMSCs. Our previous transcriptome sequencing results suggested that the expression of P53-induced nuclear protein 1 (TP53INP1) and the tumor suppressor P53 (P53) was significantly upregulated during the process of oxidative stress-induced apoptosis of BMSCs. The present study further revealed the role and mechanism of TP53INP1 and P53 in oxidative stress-induced apoptosis in BMSCs. Overexpression of TP53INP1 induced apoptosis of BMSCs, knockdown of TP53INP1 alleviated oxidative stress apoptosis of BMSCs. Under oxidative stress conditions, P53 is regulated by TP53INP1, while P53 can positively regulate the expression of TP53INP1, so the two form a positive feedback loop. To clarify the mechanism of feedback loop formation. We found that TP53INP1 inhibited the ubiquitination and degradation of P53 by increasing the phosphorylation level of P53, leading to the accumulation of P53 protein. P53 can act on the promoter of the TP53INP1 gene and increase the expression of TP53INP1 through transcriptional activation. This is the first report on a positive feedback loop formed by TP53INP1 and P53 under oxidative stress. The present study clarified the formation mechanism of the positive feedback loop. The TP53INP1–P53 positive feedback loop may serve as a potential target for inhibiting oxidative stress-induced apoptosis in BMSCs.

Hypoxia-induced apoptosis of bone marrow mesenchymal stem cells (BMSCs) limits the efficacy of their transplantation for steroid-induced osteonecrosis of the femoral head (SONFH). As apoptosis and RNA methylation are closely related, exploring the role and mechanism of RNA methylation in hypoxic apoptosis of BMSCs is expected to identify new targets for transplantation of BMSCs for SONFH and enhance transplantation efficacy. We performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) combined with RNA-seq on a hypoxia-induced apoptosis BMSC model and found that the RNA methyltransferase-like 3 (METTL3) is involved in hypoxia-induced BMSC apoptosis. The expression of METTL3 was downregulated in BMSCs after hypoxia and in BMSCs implanted in osteonecrosis areas. Knockdown of METLL3 under normoxic conditions promoted apoptosis of BMSCs. In contrast, overexpression of METTL3 promoted the survival of BMSCs under hypoxic conditions, and overexpression of METTL3 promoted the survival of BMSCs in the osteonecrosis area and the repair of the osteonecrosis area. Regarding the mechanism, the m6A levels of the mRNAs of anti-apoptotic genes Bcl-2, Mcl-1, and BIRC5 were significantly increased upon the overexpression of METTL3 under hypoxic conditions, which promoted the binding of Bcl-2, Mcl-1, and BIRC5 mRNAs to IGF2BP2, enhanced the mRNA stability, and increased the protein expression of the three anti-apoptotic genes. In conclusion, overexpression of METTL3 promoted m6A modification of mRNAs of Bcl-2, Mcl-1, and BIRC5, promoted the binding of IGF2BP2 to the above-mentioned mRNAs, enhanced mRNA stability, inhibited hypoxia-induced BMSC apoptosis, and promoted repair of SONFH, thereby providing novel targets for transplantation of BMSCs for SONFH.

The osteonecrotic area of steroid-induced avascular necrosis of the femoral head (SANFH) is a hypoxic microenvironment that leads to apoptosis of transplanted bone marrow mesenchymal stem cells (BMSCs). However, the underlying mechanism remains unclear. Here, we explore the mechanism of hypoxic-induced apoptosis of BMSCs, and use the mechanism to improve the transplantation efficacy of BMSCs. Our results show that the long non-coding RNA AABR07053481 (LncAABR07053481) is downregulated in BMSCs and closely related to the degree of hypoxia. Overexpression of LncAABR07053481 could increase the survival rate of BMSCs. Further exploration of the downstream target gene indicates that LncAABR07053481 acts as a molecular “sponge” of miR-664-2-5p to relieve the silencing effect of miR-664-2-5p on the target gene Notch1 . Importantly, the survival rate of BMSCs overexpressing LncAABR07053481 is significantly improved after transplantation, and the repair effect of BMSCs in the osteonecrotic area is also improved. This study reveal the mechanism by which LncAABR07053481 inhibits hypoxia-induced apoptosis of BMSCs by regulating the miR-664-2-5p/ Notch1 pathway and its therapeutic effect on SANFH.

目的 探讨急性脑出血患者入院低血钾发生情况、影响因素及其与短期预后的关系.方法 采用回顾性统计方法,以纳入该院2019年1月至2020年12月收治的急性脑出血患者359例作为脑出血组,纳入同时期性别、年龄相匹配的健康人群294例作为健康对照组,比较2组患者的血钾水平.进一步将急性脑出血患者根据血钾水平分为正常血钾组(血钾3.5～5.5 mmol/L)和低血钾组(血钾<3.5 mmol/L),分析影响急性脑出血患者低血钾的相关因素.追踪脑出血患者30 d预后,以改良Rankin量表(mRS)评分将患者分为预后不良组(3～6分)和预后良好组(0～<3分),分析急性脑出血患者血钾水平与短期预后的关系.结果 急性脑出血患者血钾水平明显低于健康对照组[(3.72±0.49)mmol/L vs.(4.06±0.34)mmol/L,P<0.05];急性脑出血患者中低血钾组108例,正常血钾组251例,低血钾发生率30.1％,二元logistic回归分析表明,性别[OR=0.440,95％CI(0.293,0.777)]、入院NIHSS评分[(OR=1.043,95％CI(1.019,1.068)]、尿素氮[(OR=0.849,95％CI(0.753,0.958)]为急性脑出血患者发生低血钾的独立影响因素;预后良好组与预后不良组血钾水平比较[(3.73±0.43)mmol/L vs.(3.69±0.44)mmol/L],差异无统计学意义(P>0.05),发病30 d死亡组与存活组血钾水平比较[(3.57±0.44)mmol/L vs.(3.73±0.43)mmol/L],差异有统计学意义(P<0.05),二元logistic回归分析发现低血钾不是急性脑出血患者发病30 d死亡的独立危险因素.结论 急性脑出血患者易出现低钾血症,女性、入院NIHSS评分越高、尿素氮水平越低的急性脑出血患者低血钾发生率越高;发病30 d死亡的急性脑出血患者血钾水平较存活患者更低,但低血钾不能预测急性脑出血患者短期不良预后.

We explored the association between the serum level of cystatin C (CysC) at admission and short-term functional outcome in patients with hypertensive intracerebral hemorrhage (HICH) without chronic kidney disease (CKD). A total of 555 patients with HICH were consecutively recruited after admission and were followed-up for 3 months after admission. The primary outcome was poor functional outcome (modified Rankin Scale [mRS] score ≥ 3). The median serum CysC level in our cohort was 1.03 mg/L (interquartile range, .89-1.20). Patients were categorized into four groups according to the serum CysC quartiles. Multivariate logistic regression analysis revealed a negative association between serum CysC and poor functional outcome at 3-month follow-up (quartile [Q]1 vs. Q4: adjusted odds ratio [OR] = .260, 95% confidence interval [CI] = .098, .691, p < .001). The negative association between serum CysC and poor functional outcome at 3 months was more pronounced in subgroups with smaller hematoma volume (≤ 30 mL), and absence of secondary intraventricular hemorrhage (IVH). Addition of serum CysC to a model containing conventional risk factors improved the model performance with net reclassification index (NRI) of .426% (p < .001) and integrated discrimination improvement (IDI) of .043% (p < .001) for poor functional outcome. Serum CysC was found to be a negative predictor of poor short-term functional outcome in HICH patients independent of renal function.

The specific pathogenesis of steroid-induced osteonecrosis of the femoral head (SONFH) is still not fully understood, and there is currently no effective early cure. Understanding the role and mechanism of long noncoding RNAs (lncRNAs) in the pathogenesis of SONFH will help reveal the pathogenesis of SONFH and provide new targets for its early prevention and treatment. In this study, we first confirmed that glucocorticoid (GC)-induced apoptosis of bone microvascular endothelial cells (BMECs) is a pre-event in the pathogenesis and progression of SONFH. Then, we identified a new lncRNA in BMECs via lncRNA/mRNA microarray, termed Fos-associated lincRNA ENSRNOT00000088059.1 (FAR591). FAR591 is highly expressed during GC-induced BMEC apoptosis and femoral head necrosis. Knockout of FAR591 effectively blocked the GC-induced apoptosis of BMECs, which then alleviated the damage of GCs to the femoral head microcirculation and inhibited the pathogenesis and progression of SONFH. In contrast, overexpression of FAR591 significantly promoted the GC-induced apoptosis of BMECs, which then aggravated the damage of GCs to the femoral head microcirculation and promoted the pathogenesis and progression of SONFH. Mechanistically, GCs activate the glucocorticoid receptor, which translocates to the nucleus and directly acts on the FAR591 gene promoter to induce FAR591 gene overexpression. Subsequently, FAR591 binds to the Fos gene promoter (–245∼–51) to form a stable RNA:DNA triplet structure and then recruits TATA-box binding protein associated factor 15 and RNA polymerase II to promote Fos expression through transcriptional activation. Fos activates the mitochondrial apoptotic pathway by regulating the expression of Bcl-2 interacting mediator of cell death (Bim) and P53 upregulated modulator of apoptosis (Puma) to mediate GC-induced apoptosis of BMECs, which leads to femoral head microcirculation dysfunction and femoral head necrosis. In conclusion, these results confirm the mechanistic link between lncRNAs and the pathogenesis of SONFH, which helps reveal the pathogenesis of SONFH and provides a new target for the early prevention and treatment of SONFH.

Oxidative stress damage is a common problem in bone marrow mesenchymal stem cell (BMSC) transplantation. Under stress conditions, the mitochondrial function of BMSCs is disrupted, which accelerates senescence and apoptosis of BMSCs, ultimately leading to poor efficacy. Therefore, improving mitochondrial function and enhancing the antioxidative stress capacity of BMSCs may be an effective way of improving the survival rate and curative effect of BMSCs. In the present study, we have confirmed that overexpression of nicotinamide mononucleotide adenylyl transferase 3 (NMNAT3) improves mitochondrial function and resistance to stress-induced apoptosis in BMSCs. We further revealed the mechanism of NMNAT3-mediated resistance to stress-induced apoptosis in BMSCs. We increased the level of nicotinamide adenine dinucleotide (NAD+) by overexpressing NMNAT3 in BMSCs and found that it could significantly increase the activity of silent mating type information regulation 2 homolog 3 (Sirt3) and significantly decrease the acetylation levels of Sirt3-dependent deacetylation-related proteins isocitrate dehydrogenase 2 (Idh2) and Forkhead-box protein O3a (FOXO3a). These findings show that NMNAT3 may increase the activity of Sirt3 by increasing NAD(+) levels. Our results confirm that the NMNAT3-NAD(+)-Sirt3 axis is a potential mechanism for improving mitochondrial function and enhancing antioxidative stress capacity of BMSCs. In the present study, we take advantage of the role of NMNAT3 in inhibiting stress-induced apoptosis of BMSCs and provide new methods and ideas for breaking through the bottleneck of transplantation efficacy of BMSCs in the clinic.

Objective:To investigate the effect and mechanism of LOC103693069 on improving bone marrow mesenchymal stem cells (BMSCs) hypoxic apoptosis.Methods:Primary cells were extracted from SD rats and provided by the Experimental Animal Center of Guizhou Medical University in April 2020. We can use the whole bone marrow adherent culture method to isolate and culture BMSCs in vitro. Then we culture BMSCs in different hypoxia concentrations: normoxia, 5% O 2, 1% O 2, 0% O 2. After culturing for 48 hours, and determine the optimal hypoxia concentration. Construction of LOC103693069 lentivirus-transfected BMSCs, divided into 4 groups: BMSCs group, BMSCs+ Lv-NC group, BMSCs+ Lv-LOC103693069 group, BMSCs+ Lv-LOC103693069-RNAi group. The effect of anti-hypoxic apoptosis was evaluated and its mechanism was explored by Combined with Cells by Biosynthesis and Luciferase Experiment Functional Experiments etc. One-way analysis of variance was used for comparison between multiple groupsand LSD- t test was used for pairwise comparison between groups. Results:The cultured BMSCs were identified as positive for osteogenic, cartilage, and adipogenic differentiation, and it was determined that 0% O2 culture for 48 hours was a model condition. Through gene chip detection combined with real-time fluorescent quantitative polymerase chain reaction (qPCR). After verification, it was finally determined that LOC103693069 was the target gene. After hypoxia treatment, the apoptosis rate of BMSCs+ Lv-LOC103693069 group was (47.00±1.23)% lower than that of BMSCs+ Lv-NC group (70.20±1.56)%, the difference was statistically significant ( t=4.56, P<0.05). The downstream pathway was predicted to be THY1/Notch by biosynthesis. We designed a blocking experiment. Firstly, the apoptosis rate of cells the overexpression of LOC103693069 after hypoxia treatment was (41.00±1.23)%. On this basis, THY1 was inhibited, resulting in a lower apoptosis rate (74.30±2.13)% than that of the overexpression LOC103693069 group. The difference was statistically significant ( t=4.35, P<0.05). The apoptosis rate of LOC103693069 cells was inhibited was (72.60±1.72)%, which was higher than that of overexpressed THY1 cells (42.50±1.12)%, in this case, to do the rescue experiment, the difference was statistically significant ( t=4.12, P<0.05). Subsequently, luciferase experiments confirmed that LOC103693069 and THY1 mRNA 3’ uncompiled region sequence (3’ UTR) have the same binding site on miR-140-5p. Cell function experiments also have confirmed that LOC103693069 can regulate the expression level of THY1 mRNA. However, it can be reversed by miR-140-5p. It have confirmed that LOC103693069 competitively binding miR-140-5p can regulate THY1/Notch pathway. Conclusion:LOC103693069 regulates the THY1/Notch pathway by competitively binding miR-140-5P to improve the hypoxic apoptosis of BMSCs.

Bone marrow mesenchymal stem cells (BMSCs) have strong regenerative potential and show good application prospects for treating clinical diseases. However, in the process of BMSC transplantation for treating ischemic and hypoxic diseases, BMSCs have high rates of apoptosis in the hypoxic microenvironment of transplantation, which significantly affects the transplantation efficacy. Our previous studies have confirmed the key role of long non-coding RNA Tmem235 (LncRNA Tmem235) in the process of hypoxia-induced BMSC apoptosis and its downstream regulatory mechanism, but the upstream mechanism by which hypoxia regulates LncRNA Tmem235 expression to induce BMSC apoptosis is still unclear. Under hypoxic conditions, we found that the level of LncRNA Tmem235 promoter histone H3 lysine 27 trimethylation modification (H3K27me3) was significantly increased by CHIP-qPCR. Moreover, H3K27me3 cooperated with LncRNA Tmem235 promoter DNA methylation to inhibit the expression of LncRNA Tmem235 and promote apoptosis of BMSCs. To study the mechanism of hypoxia-induced modification of LncRNA Tmem235 promoter H3K27me3 in the hypoxia model of BMSCs, we detected the expression of H3K27 methylase and histone demethylase and found that only histone methylase enhancer of zeste homolog 2 (EZH2) expression was significantly upregulated. Knockdown of EZH2 significantly decreased the level of H3K27me3 modification in the LncRNA Tmem235 promoter. The EZH2 promoter region contains a hypoxia-responsive element (HRE) that interacts with hypoxia-inducible factor-1alpha (HIF-1α), which is overexpressed under hypoxic conditions, thereby promoting its overexpression. In summary, hypoxia promotes the modification of the LncRNA Tmem235 promoter H3K27me3 through the HIF-1α/EZH2 signaling axis, inhibits the expression of LncRNA Tmem235, and leads to hypoxic apoptosis of BMSCs. Our findings improve the regulatory mechanism of LncRNA Tmem235 during hypoxic apoptosis of BMSCs and provide a more complete theoretical pathway for targeting LncRNA to inhibit hypoxic apoptosis of BMSCs.