Function and regulation of nuclear factor 1 X-type on chondrocyte proliferation and differentiation.

Nuclear factor 1 X-type (Nfix) is a transcription factor related to mental and physical development. However, very few studies have reported the effects of Nfix on cartilage. This study aims to reveal the influence of Nfix on the proliferation and differentiation of chondrocytes, and to explore its potential action mechanism. We isolated primary chondrocytes from the costal cartilage of newborn C57BL/6 mice and with Nfix overexpression or silencing treatment. We used Alcian blue staining and found that Nfix overexpression significantly promoted ECM synthesis in chondrocytes while silencing inhibited ECM synthesis. Using RNA-seq technology to study the expression pattern of Nfix in primary chondrocytes. We found that Nfix overexpression significantly up-regulated genes that are related to chondrocyte proliferation and extracellular matrix (ECM) synthesis and significantly down-regulated genes related to chondrocyte differentiation and ECM degradation. Nfix silencing, however, significantly up-regulated genes associated with cartilage catabolism and significantly down-regulated genes associated with cartilage growth promotion. Furthermore, Nfix exerted a positive regulatory effect on Sox9, and we propose that Nfix may promote chondrocyte proliferation and inhibit differentiation by stimulating Sox9 and its downstream genes. Our findings suggest that Nfix may be a potential target for the regulation of chondrocyte proliferation and differentiation.

Identification of copper death-associated molecular clusters and immunological profiles in rheumatoid arthritis.

Objective: An analysis of the relationship between rheumatoid arthritis (RA) and copper death-related genes (CRG) was explored based on the GEO dataset. Methods: Based on the differential gene expression profiles in the GSE93272 dataset, their relationship to CRG and immune signature were analysed. Using 232 RA samples, molecular clusters with CRG were delineated and analysed for expression and immune infiltration. Genes specific to the CRGcluster were identified by the WGCNA algorithm. Four machine learning models were then built and validated after selecting the optimal model to obtain the significant predicted genes, and validated by constructing RA rat models. Results: The location of the 13 CRGs on the chromosome was determined and, except for GCSH. LIPT1, FDX1, DLD, DBT, LIAS and ATP7A were expressed at significantly higher levels in RA samples than in non-RA, and DLST was significantly lower. RA samples were significantly expressed in immune cells such as B cells memory and differentially expressed genes such as LIPT1 were also strongly associated with the presence of immune infiltration. Two copper death-related molecular clusters were identified in RA samples. A higher level of immune infiltration and expression of CRGcluster C2 was found in the RA population. There were 314 crossover genes between the 2 molecular clusters, which were further divided into two molecular clusters. A significant difference in immune infiltration and expression levels was found between the two. Based on the five genes obtained from the RF model (AUC = 0.843), the Nomogram model, calibration curve and DCA also demonstrated their accuracy in predicting RA subtypes. The expression levels of the five genes were significantly higher in RA samples than in non-RA, and the ROC curves demonstrated their better predictive effect. Identification of predictive genes by RA animal model experiments was also confirmed. Conclusion: This study provides some insight into the correlation between rheumatoid arthritis and copper mortality, as well as a predictive model that is expected to support the development of targeted treatment options in the future.

Comparative transcriptome analysis provides insight into the molecular targets and signaling pathways of deer TGF-1 regulating chondrocytes proliferation and differentiation.

BACKGROUND: Chondrocytes are the only cell components in the cartilage, which has the poor regeneration ability. Thus, repairing damaged cartilage remains a huge challenge. Sika deer antlers are mainly composed of cartilaginous tissues that have an astonishing capacity for repair and renewal. Our previous study has demonstrated the transforming growth factor ß (TGF-ß1) is considered to be a key molecule involved in rapid growth, with the strongest expression in the cartilage layer. However, it remains to be clarified whether deer TGF-ß1 has significantly different function from other species such as mouse, and what is the molecular mechanism of regulating cartilage growth. METHODS: Primary chondrocytes was collected from new born mouse rib cartilage. The effect of TGF-ß1 on primary chondrocytes viability was elucidated by RNA sequencing (RNA-seq) technology combined with validation methods such as quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence assay (IFA). Differential expression genes were identified using the DEGseq package. RESULTS: Our results demonstrated that the overexpression of deer TGF-ß1 possibly promoted chondrocyte proliferation and extracellular matrix (ECM) synthesis, while simultaneously suppressing chondrocyte differentiation through regulating transcription factors, growth factors, ECM related genes, proliferation and differentiation marker genes, such as Comp, Fgfr3, Atf4, Stat1 etc., and signaling pathways such as the MAPK signaling pathway, inflammatory mediator regulation of TRP channels etc. In addition, by comparing the amino acid sequence and structures between the deer TGF-ß1 and mouse TGF-ß1, we found that deer TGF-ß1 and mouse TGF-ß1 proteins are mainly structurally different in arm domains, which is the main functional domain. Phenotypic identification results showed that deer TGF-ß1 may has stronger function than mouse TGF-ß1. CONCLUSION: ​These results suggested that deer TGF-ß1 has the ability to promote chondrogenesis by regulating chondrocyte proliferation, differentiation and ECM synthesis. This study provides insights into the molecular mechanisms underlying the effects of deer TGF-ß1 on chondrocyte viability.

Novel insights into the effect of deer IGF-1 on chondrocyte viability and IL-1ß-induced inflammation response.

Clinical treatment of Osteoarthritis (OA) remains a challenge due to the poor self-regeneration ability of cartilage. Deer antler is the only cartilage tissue that can completely regenerate each year. Insulin-like growth factor 1 (IGF-1) is one of the major active components in the deer antler that participate in regulating the rapid regeneration of deer antler cartilage. This has led us to speculate that deer IGF-1 might potentially become a candidate drug for reducing damage and inflammation of OA. Thus, we aimed to explore the underlying mechanism of deer IGF-1 in chondrocyte proliferation, differentiation, and inflammation response. Deer, mouse, and human IGF-1 amino acid sequences and protein structures were aligned using CLUSTAL and PSIPRED. The underlying molecular mechanism of deer IGF-1 on primary chondrocytes was investigated by RNA-sequencing (RNA-seq) technology combined with various experiments. Cytokine interleukin-1ß (IL-1ß) was used to induce the inflammation response of primary chondrocytes. We found that deer IGF-1 was more similar to human IGF-1 than mouse IGF-1. qRT-PCR and immunofluorescence assay indicated that deer IGF-1 had stronger effects than mouse IGF-1. We also found that the deer IGF-1 enhanced the expression of cell proliferation, differentiation, and extracellular matrix (ECM)-related genes, but decreased the expression of ECM-degrading genes. Deer IGF-1 also attenuated the IL-1ß-induced inflammatory and ECM degradation in chondrocytes. This study provides insight into the molecular mechanisms of deer IGF-1 on primary chondrocyte viability and presents a candidate for combatting inflammatory responses in OA development.

Transcriptional sequencing analysis reveals the potential use of deer antler for "tonifying the kidney and strengthening bone".

BACKGROUND: It is recorded in the Chinese Pharmacopoeia that deer antlers can be used to tonify the kidney and strengthen bone. Although numerous studies have demonstrated that deer antler has protective effects on the kidney and bone, its molecular mechanisms remain to be elucidated. The aim of this study was to explore the molecular mechanism underlying its effects on the bone and kidney. METHODS: Water extract of pilose antler was prepared and then filtered through a 0.45 µm Hollow Fiber Cartridge (GE Healthcare, USA). The filtrate was freeze-dried by a Heto PowerDry LL3000 Freeze Dryer (Thermo, USA) and stored at - 80 °C. Rats were treated with deer antler extract (DAE) prepared in advance, and gene regulatory network in the kidney and bone was detected by RNA-Seq technique. Micro-CT was used to detect bone trabecular formation, bone mineral density (BMD) and bone volume fraction (BV/TV). RESULTS: The results demonstrate that DAE could jointly heighten renal function by maintaining renal homeostasis, combating renal fibrosis, and reducing renal inflammation by regulating ion transport. Furthermore, DAE can strengthen the bone system by stimulating osteoblast differentiation and regulating bone regeneration and the bone marrow microenvironment. Micro-CT results confirmed that DAE can promote bone trabecular formation and increase BMD and BV/TV. We also identified many genes that can regulate both the kidney and bone simultaneously, which explained the theory of "kidney governing bone" at the molecular level and provided possible strategies for further application of this theory to treat diseases. CONCLUSIONS: DAE enhances renal function, maintains renal homeostasis, positively regulates skeletal system development, and increases bone mineral density. The underlying mechanism involves improving the expression levels of functional genes involved in renal function and regulation and repair, as well as genes that positively regulate skeletal system development.

Comprehensive RNA sequencing in primary murine keratinocytes and fibroblasts identifies novel biomarkers and provides potential therapeutic targets for skin-related diseases.

BACKGROUND: Keratinocytes and fibroblasts represent the major cell types in the epidermis and dermis of the skin and play a significant role in maintenance of skin homeostasis. However, the biological characteristics of keratinocytes and fibroblasts remain to be elucidated. The purpose of this study was to compare the gene expression pattern between keratinocytes and fibroblasts and to explore novel biomarker genes so as to provide potential therapeutic targets for skin-related diseases such as burns, wounds, and aging. METHODS: Skin keratinocytes and fibroblasts were isolated from newborn mice. To fully understand the heterogeneity of gene expression between keratinocytes and fibroblasts, differentially expressed genes (DEGs) between the two cell types were detected by RNA-seq technology. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the known genes of keratinocytes and fibroblasts and verify the RNA-seq results. RESULTS: Transcriptomic data showed a total of 4309 DEGs (fold-change > 1.5 and q-value < 0.05). Among them, 2197 genes were highly expressed in fibroblasts and included 10 genes encoding collagen, 16 genes encoding transcription factors, and 14 genes encoding growth factors. Simultaneously, 2112 genes were highly expressed in keratinocytes and included 7 genes encoding collagen, 14 genes encoding transcription factors, and 8 genes encoding growth factors. Furthermore, we summarized 279 genes specifically expressed in keratinocytes and 33 genes specifically expressed in fibroblasts, which may represent distinct molecular signatures of each cell type. Additionally, we observed some novel specific biomarkers for fibroblasts such as Plac8 (placenta-specific 8), Agtr2 (angiotensin II receptor, type 2), Serping1 (serpin peptidase inhibitor, clade G, member 1), Ly6c1 (lymphocyte antigen 6 complex, locus C1), Dpt (dermatopontin), and some novel specific biomarkers for keratinocytes such as Ly6a (lymphocyte antigen 6 complex, locus A) and Lce3c (late cornified envelope 3C), Ccer2 (coiled-coil glutamate-rich protein 2), Col18a1 (collagen, type XVIII, alpha 1) and Col17a1 (collagen type XVII, alpha 1). In summary, these data provided novel identifying biomarkers for two cell types, which can provide a resource of DEGs for further investigations.