[Bioinformatics Analysis on Key Genes and Immune Infiltration of Osteosarcoma].

Objective To screen the potential key genes of osteosarcoma by bioinformatics methods and analyze their immune infiltration patterns. Methods The gene expression profiles GSE16088 and GSE12865 associated with osteosarcoma were obtained from the Gene Expression Omnibus(GEO),and the differentially expressed genes(DEGs)related to osteosarcoma were screened by bioinformatics tools.Gene Ontology(GO)annotation,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment,and analysis of immune cell infiltration were then carried out for the DEGs.The potential Hub genes of osteosarcoma were identified by protein-protein interaction network,and the expression of Hub genes in osteosarcoma and normal tissue samples was verified via the Cancer Genome Atlas(TCGA). Results A total of 108 DEGs were screened out.GO annotation and KEGG pathway enrichment revealed that the DEGs were mainly involved in integrin binding,extracellular matrix (ECM) structural components,ECM receptor interactions,and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway.Macrophages were the predominant infiltrating immune cells in osteosarcoma.Secreted phosphoprotein 1(SPP1),matrix metallopeptidase 2(MMP2),lysyl oxidase(LOX),collagen type V alpha(II)chain(COL5A2),and melanoma cell adhesion molecule(MCAM)presented differential expression between osteosarcoma and normal tissue samples(all P<0.05). Conclusions SPP1,MMP2,LOX,COL5A2,and MCAM are all up-regulated in osteosarcoma,which may serve as potential biomarkers of osteosarcoma.Macrophages are the key infiltrating immune cells in osteosarcoma,which may provide new perspectives for the treatment of osteosarcoma.

CTSD upregulation as a key driver of spinal ligament abnormalities in spinal stenosis.

Spinal stenosis (SS) is frequently caused by spinal ligament abnormalities, such as ossification and hypertrophy, which narrow the spinal canal and compress the spinal cord or nerve roots, leading to myelopathy or sciatic symptoms; however, the underlying pathological mechanism is poorly understood, hampering the development of effective nonsurgical treatments. Our study aims to investigate the role of co-expression hub genes in patients with spinal ligament ossification and hypertrophy. To achieve this, we conducted an integrated analysis by combining RNA-seq data of ossification of the posterior longitudinal ligament (OPLL) and microarray profiles of hypertrophy of the ligamentum flavum (HLF), consistently pinpointing CTSD as an upregulated hub gene in both OPLL and HLF. Subsequent RT-qPCR and IHC assessments confirmed the heightened expression of CTSD in human OPLL, ossification of the ligamentum flavum (OLF), and HLF samples. We observed an increase in CTSD expression in human PLL and LF primary cells during osteogenic differentiation, as indicated by western blotting (WB). To assess CTSD's impact on osteogenic differentiation, we manipulated its expression levels in human PLL and LF primary cells using siRNAs and lentivirus, as demonstrated by WB, ALP staining, and ARS. Our findings showed that suppressing CTSD hindered the osteogenic differentiation potential of PLL and LF cells, while overexpressing CTSD activated osteogenic differentiation. These findings identify CTSD as a potential therapeutic target for treating spinal stenosis associated with spinal ligament abnormalities.

Integrated DNA methylation analysis reveals a potential role for PTPRN2 in Marfan syndrome scoliosis.

Background: Marfan syndrome (MFS) is a rare genetic disorder caused by mutations in the Fibrillin-1 gene (FBN1) with significant clinical features in the skeletal, cardiopulmonary, and ocular systems. To gain deeper insights into the contribution of epigenetics in the variability of phenotypes observed in MFS, we undertook the first analysis of integrating DNA methylation and gene expression profiles in whole blood from MFS and healthy controls (HCs). Methods: The Illumina 850K (EPIC) DNA methylation array was used to detect DNA methylation changes on peripheral blood samples of seven patients with MFS and five HCs. Associations between methylation levels and clinical features of MFS were analyzed. Subsequently, we conducted an integrated analysis of the outcomes of the transcriptome data to analyze the correlation between differentially methylated positions (DMPs) and differentially expressed genes (DEGs) and explore the potential role of methylation-regulated DEGs (MeDEGs) in MFS scoliosis. The weighted gene co-expression network analysis was used to find gene modules with the highest correlation coefficient with target MeDEGs to annotate their functions in MFS. Results: Our study identified 1253 DMPs annotated to 236 genes that were primarily associated with scoliosis, cardiomyopathy, and vital capacity. These conditions are typically associated with reduced lifespan in untreated MFS. We calculated correlations between DMPs and clinical features, such as cobb angle to evaluate scoliosis and FEV1% to assess pulmonary function. Notably, cg20223687 (PTPRN2) exhibited a positive correlation with cobb angle of scoliosis, potentially playing a role in ERKs inactivation. Conclusions: Taken together, our systems-level approach sheds light on the contribution of epigenetics to MFS and offers a plausible explanation for the complex phenotypes that are linked to reduced lifespan in untreated MFS patients.

Circulating miR-107 as a diagnostic biomarker of osteoporotic vertebral compression fracture increases bone formation in vitro and in vivo.

AIMS: This study aimed to examine the key circulating microRNAs (miRNAs) in the plasma of patients with osteoporotic vertebral compression fracture and assess their potential role as diagnostic biomarkers and explore their function in vitro and in vivo. METHODS: Weighted gene co-expression network analysis (WGCNA) was applied to identify hub miRNAs for subsequent analysis. The candidate miRNAs were tested using plasma from 144 patients and the results were applied to construct receiver operating characteristic (ROC) curves to assess their diagnostic value. In addition, the function of the target miRNA was validated in MC3T3-E1 cells, human bone marrow-derived mesenchymal stromal cells (BMSCs), and an ovariectomized (OVX) mouse model. KEY FINDINGS: Seven modules were obtained by WGCNA analysis. The expression levels of circulating miR-107 in the red module were significantly lower in osteoporotic patients than in healthy controls. In addition, miR-107 provided discrimination with an AUC > 85 % by ROC analyses to differentiate women osteoporosis patients from healthy controls and differentiate women osteoporotic patients with vertebral compression fractures from osteoporotic patients without vertebral compression fractures. In vitro experiments revealed that miR-107 levels were increased in osteogenically induced MC3T3-E1 cells and BMSCs and transfection with synthetic miR-107 could promote bone formation. Lastly, the bone parameters were improved by miR-107 upregulation in OVX mice. SIGNIFICANCE: Our findings show that circulating miR-107 plays an essential role in facilitating osteogenesis and may be a useful diagnostic biomarker and therapeutic target in osteoporosis.

Full-Endoscopic Lumbar Discectomy for Lumbar Disc Herniation with Posterior Ring Apophysis Fracture: A Retrospective Study.

OBJECTIVE: The present retrospective study evaluated the clinical results of full-endoscopic lumbar discectomy (FELD) for the treatment of lumbar disc herniation (LDH) with lumbar posterior ring apophysis fracture (PRAF) using an interlaminar or a transforaminal approach at an inpatient surgery center. METHODS: Patients with single-level LDH with type III PRAF who had undergone FELD using an interlaminar or a transforaminal approach from January 2010 to December 2015 were enrolled. The general data recorded included sex, age, location, surgical approach, operative time, hospital stay, perioperative complications, and recurrence. The presence of mobile and immobile fragments was documented. The clinical outcomes were evaluated using a visual analog scale for low back and leg pain. The Oswestry Disability Index was used for the functional assessment and the modified MacNab criteria for patient satisfaction. RESULTS: FELD was performed successfully in all cases and no serious perioperative complications were observed. A mobile fragment of PRAS was present in 18 patients and an immobile fragment in 15 patients. Complications occurred in 2 of the 33 included patients; 1 dual tear (3.0%) and 1 transient dysesthesia (3.0%) that did not require further treatment. Recurrence developed in 1 patient (3.0%) and required reoperation. The visual analog scale and Oswestry Disability Index scores had significantly improved postoperatively at 3, 6, and 12 months and the final follow-up visit (P < 0.05). Using the modified MacNab criteria, an excellent or good rate of 93.4% was achieved. CONCLUSIONS: FELD is a safe and effective minimally invasive approach for the treatment of LDH with type III PRAF. Sufficient preparation and experience are required to achieve satisfactory results.

Activation of the phosphatidylinositol 3-kinase/protein kinase Akt pathway mediates CD151-induced endothelial cell proliferation and cell migration.

We previously reported that CD151 promotes neovascularization and improves blood perfusion in rat hind-limb ischemia model, but the precise mechanism is still unclear. Endothelial cell proliferation and cell migration play critical roles in angiogenesis. Many growth factors and hormones have been shown to regulate cell proliferation, cell migration and angiogenesis, including the activation of eNOS activity, via the PI3K/Akt signaling pathway. Whether CD151 induces cell proliferation and cell migration via PI3K/Akt signaling pathway is not known. Here we showed that CD151 promotes human umbilical vein endothelial cell (HUVEC) proliferation, migration and tube formation in vitro, accompanied by increased phosphorylation of Akt and eNOS, leading to increased eNOS activity and nitric oxide (NO) levels after rAAV-CD151 infection, whereas infection with rAAV-anti-CD151 attenuated the effects of CD151, which suggested that CD151 can activate PI3K/Akt pathway. Moreover, inhibitors of PI3K (LY294002) and eNOS (l-NAME) can attenuate CD151-induced cell proliferation and cell migration. The results suggested that activation of PI3K/Akt signaling pathway mediates CD151-induced cell proliferation and migration.

CT-1 induces angiogenesis by regulating the ADMA/DDAH Pathway.

BACKGROUND: Cardiotrophin-1 (CT-1), a member of the IL-6 superfamily, is elevated in the serum of patients with ischemic and valvular heart disease. In this study, we hypothesized that CT-1 induces endothelial cell angiogenesis and that the ADMA/DDAH pathway plays an important role in the process. METHODS: pEGFP-N1-CTF1-GFP and pEGFP-N1 were constructed and used to transiently transfect to HUVECs, mediated by LipofectamineTM 2000. After transfection, the expression of CT-1 was examined by qRT-PCR and western blotting. Endothelial cell proliferation assay was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) method. Migration assay was performed using transwell, tube formation test was examined on Matrigel, eNOSmRNA expression was assayed by qRT-PCR, DDAH I, DDAHII and VEGF expression were detected by western blotting, the level of ADMA and the activity of DDAH were measured by High Performance Liquid Chromatography, NOS activity and the concentration of NO were assayed by L-[3H] citrulline production from L-[3H]arginine. RESULTS: Overexpression of CT-1, increased endothelial cell proliferation, migration and formation of blood vessels, upregulated the expression of eNOSmRNA, DDAHI, DDAHII and VEGF, elevated the activity of DDAH and NOS, decreased the level of ADMA and promoted NO synthesis. In contrast, ADMA partially inhibited the effects of CT-1 induction. CONCLUSIONS: Overexpression of CT-1 increases cell proliferation, migration and formation of blood vessels. This result also suggests that CT-1 may regulate angiogenesis through the ADMA/DDAH pathway.

CD151 gene delivery increases eNOS activity and induces ECV304 migration, proliferation and tube formation.

AIM: To investigate the effects of CD151 on the activity of endothelial NO synthase (eNOS), and ECV304 migration, proliferation and tube formation. METHODS: pAAV-CD151 and pAAV-anti-CD151 were constructed and used to transiently transfect ECV304 mediated with Lipofectamine 2000. After transfection, the expression of CD151 was measured by Western blotting. Cell migration assay was performed using Boyden transwell; proliferation assay was evaluated using the 3- [4,5-dimethylthiazol-2-yl]-2,5, diphenyltetrazolium bromide (MTT) method, and tube formation test was examined on matrigel. eNOS activity was assayed by L- [3H]citrulline production from L-[3H]arginine. The involvement of eNOS was explored using an eNOS inhibitor (L-NAME) and the effects in the process were observed. RESULTS: CD151 promotes cell migration, proliferation and tube formation. In addition, CD151 increases eNOS activity. Moreover, cell migration, proliferation and tube formation induced by CD151 are inhibited when L-NAME is used, which indicates that there is an involvement of eNOS in CD151-induced cell migration, cell proliferation and tube formation. CONCLUSION: CD151 promotes ECV304 migration, proliferation and tube formation. The mechanism is that CD151 increases eNOS activity. This result also suggests that eNOS is involved in the angiogenic effects of CD151.