Xiaochen Qu

Background: Necrotizing enterocolitis (NEC) stands as one of the most lethal conditions afflicting premature infants. There is a close relationship between necroptosis, necroinflammation, and the potential mechanisms of NEC. The purpose of this study was to investigate the mechanism of necroinflammation-associated necroptosis-related genes (NiNRGs) in NEC, identify NiNRGs-related diagnostic markers for NEC, and construct a diagnostic model for NEC through bioinformatics analysis and machine learning. Methods: Differentially expressed NiNRGs (DE-NiNRGs) were identified through differential expression and correlation analysis, followed by gene set enrichment analysis (GSEA) and protein-protein interaction (PPI) network establishment. Three machine learning methods were used to find potential diagnostic biomarkers, evaluated through a receiver operating characteristic (ROC) curve and a nomogram model. Immune infiltration scores for 28 immune cell types in NEC were calculated, along with correlation coefficients for diagnostic marker genes. Various databases predicted interactions between these genes, small molecule drugs, microRNAs, and transcription factors. A single-gene GSEA (sgGSEA) identified significantly enriched signaling pathways associated with diagnostic marker genes in NEC. Results: A total of 29 DE-NiNRGs were identified, linked to 17 pathways, including tumor necrosis factor (TNF), interleukin (IL)-17, and cytosolic DNA-sensing pathways. The PPI network showed close interactions among DE-NiNRGs. Three biomarkers, DAPK1, PARP1, and BIRC3, were selected using machine learning, showing area under the curve (AUC) values ≥0.8 in ROC analysis. The nomogram indicated significant diagnostic score differences between NEC and healthy controls. Type 2 T helper (Th2) cell infiltration differed significantly between NEC and controls, with DAPK1 and BIRC3 expression correlating with immune cells. Transcription factors, microRNAs, and small molecule drugs regulating these markers were identified, and sgGSEA revealed 198, 240, and 217 pathways for DAPK1, PARP1, and BIRC3, respectively. Conclusions: Necroinflammation-induced necroptosis significantly contributes to the progression of NEC. DAPK1, PARP1, and BIRC3 demonstrate substantial diagnostic potential for the condition. Employing bioinformatics to explore potential mechanisms aids in elucidating the genetic pathogenesis of NEC and offers valuable insights for future investigations.

Purpose: Oncostatin M (OSM) is involved in the regulation of osteogenic differentiation and has a major role in the development of heterotopic ossification. The role of OSM in osteogenic differentiation of tendon-derived stem cells (TDSCs) and its mechanism have not been reported. This study aim to investigate the role of OSM in osteogenic differentiation of TDSCs and study the mechanism. Methods: TDSCs were differentiated in osteogenic differentiation medium for 7 days. Recombinant OSM was added to the osteogenic differentiation medium for 7 and 14 days. The effect of Janus kinase 2 (JAK2) inhibitor AZD1480 and signal transducer and activator of transcription 3 (STAT3) inhibitor stattic in the presence of recombinant OSM on osteogenic differentiation of TDSCs was examined after differentiation for 7 and 14 days. Alkaline phosphatase and alizarin red staining were used to assess the effects on early and mid-stage osteogenic differentiation, respectively. Western blotting and qPCR were used to assess the expression of receptor and signalling pathway-related proteins and osteogenic marker genes, respectively. Results: TDSCs were successfully induced to differentiate into osteoblasts. Recombinant OSM promoted osteogenic differentiation of TDSCs to early and mid-stages. After addition of AZD1480 or stattic, decreased alkaline phosphatase and alizarin red staining were observed in the early and mid-stages of osteogenic differentiation. Additionally, decreased expression of receptor and pathway-related proteins, and osteogenic genes was found by western blotting and qPCR, respectively. Conclusion: OSM promotes osteogenic differentiation of TDSCs and the JAK2/STAT3 signalling pathway plays an important role.

Background: Tumor cells exhibit a heightened susceptibility to lysosomal-dependent cell death (LCD) compared to normal cells. However, the role of LCD-related genes (LCD-RGs) in Osteosarcoma (OS) remains unelucidated. This study aimed to elucidate the role of LCD-RGs and their mechanisms in OS using several existing OS related datasets, including TCGA-OS, GSE16088, GSE14359, GSE21257 and GSE162454. Results: Analysis identified a total of 8,629 DEGs1, 2,777 DEGs2 and 21 intersection genes. Importantly, two biomarkers (ATP6V0D1 and HDAC6) linked to OS prognosis were identified to establish the prognostic model. Significant differences in risk scores for OS survival were observed between high and low-risk cohorts. Additionally, scores of dendritic cells (DC), immature DCs and γδT cells differed significantly between the two risk cohorts. Cell annotations from GSE162454 encompassed eight types (myeloid cells, osteoblastic OS cells and plasma cells). ATP6V0D1 was found to be significantly over-expressed in myeloid cells and osteoclasts, while HDAC6 was under-expressed across all cell types. Moreover, single-cell trajectory mapping revealed that myeloid cells and osteoclasts differentiated first, underscoring their pivotal role in patients with OS. Furthermore, ATP6V0D1 expression progressively decreased with time. Conclusions: A new prognostic model for OS, associated with LCD-RGs, was developed and validated, offering a fresh perspective for exploring the association between LCD and OS.

Musculoskeletal deficits are among the most common extra-intestinal manifestations and complications of inflammatory bowel disease (IBD). This study aimed to identify crosstalk genes between IBD and osteoporosis (OP) and potential relationships between crosstalk and neutrophil extracellular traps (NETs)-related genes. Three common hub genes from different compared groups are actually the same, namely HDAC6, IL-8, and PPIF. ROC showed that the combined diagnostic value of HDAC6, IL-8, and PPIF was higher than each of the three key hub genes. Immune infiltration results showed that HDAC6 and IL-8 key genes negatively correlated with CD65 bright natural killer cells. USF1 was the common upstream TFs between HDAC6 and PPIF, and MYC was the common upstream TFs between IL-8 and PPIF in RegNetwork. Taken together, this study shows a linked mechanism between IBD and OP via NETs and crosstalk genes. These findings may show light on better diagnosis and treatment of IBD complicated with OP.

Background: M1 macrophages (Mφs) are involved in osteogenic differentiation of ligamentum flavum (LF) cells and play an important role in heterotopic ossification. However, the mechanism by which M1 Mφs influence osteogenic differentiation of LF cells has not been studied. Methods: The effect of conditioned medium including secretions of M1 Mφs (CM‐M1) on LF cells was analyzed by GeneChip profiling and ingenuity pathway analysis (IPA). THP‐1 cells were polarized into M1 Mφs and CM‐M1 was used to induce LF cells. In addition, LF cells were induced by CM‐M1 in the presence of cyclooxygenase 2 (COX‐2) inhibitors or oncostatin M (OSM)‐neutralizing antibodies. Based on the presence of OSM, knockout of OSMR or GP130 receptors, or addition of the Janus kinase 2 (JAK2) inhibitor AZD1480 or signal transducer and activator of transcription 3 (STAT3) inhibitor Stattic were examined for effects on osteogenic differentiation of LF cells. OSM secretion was quantified by ELISA, while qPCR and western blot were used to evaluate expression of osteogenic genes and receptor and signaling pathway‐related proteins, respectively. Results: GeneChip and IPA results indicate that the OSM signaling pathway and its downstream signaling molecules JAK2 and STAT3 are significantly activated. ELISA results indicate that OSM is highly expressed in cells treated with CM‐M1 and lowly expressed in cells treated with CM‐M1 and a COX‐2 inhibitor. Besides, CM‐M1 induces osteogenic differentiation of LF cells, which is weakened when COX‐2 inhibitors or OSM‐neutralizing antibody are added to it. Recombinant OSM could induce osteogenic differentiation of LF cells and upregulate expression of OSMR, GP130, phosphorylated (P)‐JAK2, and P‐STAT3. Upon knockdown of OSMR or GP130, or the addition of AZD1480 or Stattic, P‐JAK2 and P‐STAT3 expression were decreased and osteogenic differentiation was reduced. Conclusion: M1 Mφ‐derived OSM induces osteogenic differentiation of LF cells and the JAK2/STAT3 signaling pathway plays an important role.

Study Design. Basic experimental study. Objective. To clarify the role of macrophages (Mϕs) in the osteogenic differentiation of ligamentum flavum (LF) cells. Summary of Background Data. Mϕs and secreted factors are involved in the regulation of cell osteogenic differentiation, and play an important role in the process of heterotopic ossification. Whether Mϕs are involved in the development of ossification of the ligamentum flavum (OLF) have not been reported. Methods. The expression of CD68+ Mϕs in ossified LF tissue was identified by immunohistochemical staining. THP-1 cells were polarized to M1 and M2, and identified by flow cytometry and immunofluorescence. The alkaline phosphatase activity and osteogenic differentiation-related gene expression in LF cells were evaluated following incubation with each Mϕs conditioned medium (CM). ELISA was used to detect the pro-inflammatory cytokines in the supernatants, and qPCR was used to detect the expression of the corresponding receptors in the LF cells after incubation with the CM. LF cells were induced with CM-M1 in the presence of neutralizing antibodies to further test whether cytokines secreted by M1 Mϕs impacted their osteogenic differentiation. Results. CD68+ Mϕs were found on the OLF samples. THP-1 cells were polarized into M1 and M2, and both M1 and M2 Mϕs promoted the osteogenic differentiation of LF cells. The concentrations of TNF-α, IL-1β, and IL-6 in M1 Mϕ supernatants were greater than those in M2, and greater levels of these cytokine receptors were observed in LF cells induced with CM-M1 than those with CM-M2. Osteogenic differentiation of LF cells induced by CM-M1 decreased after IL-6 was neutralized; however, not after IL-1β and TNF-α were neutralized. Conclusion. M1 Mϕ-derived IL-6 promotes the osteogenic differentiation of LF cells, which may be a pathway in which Mϕs regulate the osteogenic differentiation of LF cells. Level of Evidence: N/A

Purpose Genetic factors play a crucial role in thoracic ossification of the ligamentum flavum (TOLF). This study aimed to better understand the association between single nucleotide polymorphisms (SNP) in functional regions of the collagen VI, alpha 1 gene (COL6A1) and TOLF, and to confirm COL6A1 as a TOLF susceptibility gene. Methods Ten tag SNPs in COL6A1 were genotyped using the SNaPshot assay, and allele and genotype frequencies were compared between TOLF patients and control individuals. The function of SNPs associated with disease was studied. For COL6A1 promoter SNPs, the transcriptional activity of each haplotype was determined by luciferase reporter assays. For COL6A1 exonic SNPs, the effect of nucleotide substitutions on COL6A1 expression was determined by western blotting. COL6A1 mRNA expression in ligamentum flavum tissues from TOLF patients with different genotypes was examined using reverse transcription real-time PCR. Results Four SNPs were associated or possibly associated with TOLF, with higher pathogenic allele and genotype frequencies seen in TOLF patients compared with controls. The rs17551710/rs7671-GG/GG genotype appeared to be related to disease severity. Nucleotide substitutions at rs17551710 and rs7671 increased COL6A1 transcriptional activity and nucleotide substitutions at rs1053312 and rs13051496 increased COL6A1 protein expression. COL6A1 mRNA expression was significantly up-regulated in individuals with rs17551710/rs7671-GG/GG and rs1053312/rs13051496-AA+AG/CC genotypes compared with other genotypes. Conclusion SNPs in the COL6A1 promoter and exonic regions are associated with TOLF in the Chinese Han population, and lead to up-regulated COL6A1 expression. We confirmed COL6A1 as a TOLF susceptibility gene that may be involved in TOLF pathology.

Ossification of the ligamentum flavum (OLF) is a disorder of heterotopic ossification of spinal ligaments and is the main cause of thoracic spinal canal stenosis. Previous studies suggested that miR-132-3p negatively regulates osteoblast differentiation. However, whether miR-132-3p is involved in the process of OLF has not been investigated. In this study, we investigated the effect of miR-132-3p and its target genes forkhead box O1 (FOXO1), growth differentiation factor 5 (GDF5) and SRY-box 6 (SOX6) on the osteogenic differentiation of ligamentum flavum (LF) cells. We demonstrated that miR-132-3p was down-regulated during the osteogenic differentiation of LF cells and negatively regulated the osteoblast differentiation. Further, miR-132-3p targeted FOXO1, GDF5 and SOX6 and down-regulated the protein expression of these genes. Meanwhile, FOXO1, GDF5 and SOX6 were up-regulated after osteogenic differentiation and the down-regulation of endogenous FOXO1, GDF5 or SOX6 suppressed the osteogenic differentiation of LF cells. In addition, we also found FOXO1, GDF5 and SOX6 expression in the ossification front of OLF samples. Overall, these results suggest that miR-132-3p inhibits the osteogenic differentiation of LF cells by targeting FOXO1, GDF5 and SOX6.