Current Molecular Medicine - Online First

Hemifacial Microsomia (HFM) is the second most common congenital deformity, yet its etiology and pathogenesis remain unclear. Therefore, this study aimed to identify differentially expressed microRNAs (miRNAs) between healthy and affected bone marrow mesenchymal stem cells (BMSCs) from HFM patients, focusing on the functional roles of miR-148a in osteogenesis and osteoclastogenesis.

The specific expression of microRNAs was screened by sequencing and verified by PCR. Through the use of mimics, inhibitors, and knockout technology, we controlled the expression of miR-148a in vivo and in vitro. Osteogenesis and osteoclastogenesis induction, PCR, western blot, ALP staining, alizarin red staining, TRAP staining, micro-CT, and tissue sections were performed to explore the effects of miR-148 on osteogenesis and osteoclastogenesis.

MiR-148a was identified and confirmed through our research as a differentially expressed miRNA in HFM. Overexpression of miR-148a increased osteogenesis-related gene and protein expression and mineralized calcium nodule formation while decreasing osteoclast-related gene and protein levels. Silencing or knockout of miR-148a produced opposite effects. miR-148a knockout mice were smaller than wild-type, with reduced osteogenesis, fewer trabeculae, increased trabecular bone separation in the mandible, and decreased ramus length. Additionally, local overexpression of miR-148a in knockout mice increased local bone mass.

The current study findings demonstrate that miR-148a can influence the bone volume, and its role in chondrogenesis deserves further research. Additionally, further studies on the changes upstream of miR-148a that lead to differences in miR-148a expression between the healthy and affected sides of HFM patients and the differences in bone size are warranted to better understand HFM pathogenesis.

Our findings suggest that the opposing effects of miR-148a on osteogenesis and osteoclastogenesis lead to decreased bone mass in HFM. Local overexpression can reverse bone defects caused by miR-148a, suggesting its promising role in future treatments. We anticipate that further investigations will enhance our understanding and ultimately pave the way for the application of these insights in clinical settings for disease treatment.

Hypothetically, fetal anemic hypoxia causes cellular damage with an increase in oxidative stress levels. This study, using hemoglobin (Hb) Bart’s disease as a study model, aims to compare the levels of oxidative stress and inflammatory markers as well as fetal hemodynamics in anemic fetuses with those of non-anemic fetuses.

Forty pregnancies at risk of fetal Hb Bart’s disease scheduled for cordocentesis at 18 to 22 weeks were recruited into the study. Fetal blood was collected to measure the levels of 8-Isoprostane (8-Isop), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), and hemoglobin as well as for hemoglobin typing.

There was no significant difference in cord blood 8-Isop, TNF-α, and IL-10 levels between the Hb Bart’s disease group and the unaffected group, whereas several hemodynamic parameters, such as cardiac output, cardiac size, cardiac performance, middle cerebral artery – peak systolic velocity, etc., were significantly changed in the fetal Hb Bart’s group. In the subgroup analysis, the level of serum 8-Isop in the severe anemia group tended to increase, though not significantly, compared with the non-anemic group (275.3±141.8 vs. 203.9±49.2 pg/mL; p=0.079).

In response to anemia, fetuses might have a high capacity of hemodynamic adaptation without significant cellular damage, though a trend of an increase in oxidative stress marker was found in severe fetal anemia. Theoretically, intrauterine blood transfusion for fetal anemia during adaptive compensation may result in no residual insults.

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most malignant gastrointestinal tumors. M1 macrophage, a subtype within the Tumor Microenvironment (TME), plays a vital role in the development of cancer. Despite its anti-tumoral functions, the specific mechanisms of its action remain incompletely understood.

The effect of M1 macrophages on the proliferation ability and cell viability of PDAC cells was evaluated by Cell Counting Kit-8 (CCK-8) cell proliferation assay, cell clone formation assay, and flow cytometry. Western blot, qRT-PCR, confocal microscope, RNA-sequencing, and transmission electron microscope were performed to assess lipid peroxidation and ferroptosis level of PDAC cells in the context of M1 macrophage or TNF-α.

M1 macrophages inhibited cell proliferation and promoted cell death of PDAC cells, in which ferroptosis played a vital role. Mechanistically, Tumor Necrosis Factor-alpha (TNF-α) released by M1 macrophages binds to the TNFR1 receptor on pancreatic cancer cells, activating the p38 MAPK signaling, which upregulates Acyl-CoA Synthetase Long-chain family member 4 (ACSL4) expression, a critical lipid metabolism enzyme linked to ferroptosis, thereby promoting ferroptosis. Knockdown of ACSL4 or TNFR1 significantly reduced TNF-α-induced ferroptosis.

TNF-α is a major inflammatory cytokine and is mainly generated by macrophages and T lymphocytes. It is involved in many pathological processes, such as inflammatory diseases, autoimmune diseases, and cancer. Studies have shown that the administration of recombinant TNF-α can induce tumor regression in mice with sarcomas. In our study, systemic injection of TNF-α slowed the tumor growth in nude mice, but with no significant difference compared with the control group, which may partially be attributed to its angiogenic activity. TNF-α signals via two distinct membrane-binding receptors, TNFR1 and TNFR2, which regulate various diseases. In pancreatic cancer, the role of TNF-α is complex and poorly understood. In a previous study, they found that exogenous systemic administration of human TNF-α, which interacted with murine TNFR1, significantly increased overall tumor growth in the Panc02-PDAC model. Intriguingly, the loss of TNFR1 led to an impediment of immune cell infiltration into the tumor and impaired immunosurveillance, which accelerated tumor growth. This suggests that TNFR1 exerts both pro-tumoral and anti-tumoral functions in the Panc02-PDAC model, but the overall outcome is likely dependent on the spatiotemporal availability of TNF-α. However, systemic TNF-α injection can lead to severe side effects in animals, limiting its further application. In a recent study, TNFR2 was found to promote tumorigenesis and progression in the KPC-PDAC model. Knockdown of TNFR2 or pretreatment with an anti-TNFR2 antibody could significantly slow the tumor progression and incidence. In our study, TNFR2 was found to have a low expression in pancreatic cancer cells and was barely detected with the failure of knockdown. However, the cell lines used in the former study were established from a KPC mouse model, while our experiments were conducted using human PDAC cell lines. Contrary findings are possible as cell lines originate from two different species. However, we will further investigate the mechanism of this difference.

In summary, this study revealed that M1 macrophages could induce ferroptosis in pancreatic cancer cells through secreting TNF-α, indicating a potential therapeutic option for PDAC.

Obliterative bronchiolitis (OB) is a severe and progressive complication characterized by the fibrotic obliteration of small airways, leading to significant morbidity and mortality, particularly in lung transplant recipients. The pathogenesis of OB involves complex cellular processes, among which epithelial-to-mesenchymal transition (EMT) plays a crucial role. This study investigates the role of mechanosensitive ion channel Piezo1 in promoting OB through Yes-associated protein (YAP)-dependent EMT.

Piezo1-induced signal pathway alterations, fibrosis, and EMT-related features were examined in the mouse OB model and BEAS-2B cells. The efficacy of Piezo1 in EMT and OB was explored and validated both in vitro and in vivo.

Piezo1 was found to be upregulated in OB, and pharmacological inhibition of Piezo1 effectively alleviated EMT and fibrotic deposition. Piezo1 activation stimulated the Ca2+ influx and nuclear translocation of YAP that triggered the transition of epithelial cells into a mesenchymal phenotype, which contributed to airway fibrosis and obstruction. Furthermore, inhibition of YAP or calcium chelation significantly attenuated Piezo1 activation-induced EMT and OB, indicating that YAP and Ca2+ are critical mediators in this process.

Piezo1 expression was found to be upregulated in OB, and its activation induced the epithelial-to-mesenchymal transition (EMT) process via a YAP-dependent pathway. Piezo1 could accelerate EMT and the occlusion rate of grafts via Ca2+ influx-dependent YAP activation in OB, suggesting a direct role in facilitating EMT and subsequent fibrotic remodeling in OB.

The present results highlight that Piezo1 promotes OB through a YAP-dependent EMT pathway, suggesting Piezo1 as a novel therapeutic strategy for treating OB and potentially improving outcomes of lung transplant recipients.

Studies have shown that abnormal stress is a significant inducer of Intervertebral Disc Degeneration (IVDD). Although traction force is commonly used to delay IVDD, its effects on Nucleus Pulposus Cells (NPCs) and their secreted exosomes remain unclear. In addition, this study systematically revealed the relationship between miR-8485 and IVDD for the first time.

Cellular experiments were performed using a Flexcell cell stretching platform to apply traction force to NPCs. After optimizing loading parameters, NPC-derived exosomes (NPCs-exo) were isolated and subjected to miRNA high-throughput sequencing. Differentially expressed miRNAs were identified, and their regulatory effects on the Wnt/β-catenin pathway were investigated. Ex vivo rabbit spinal samples were used to validate the cellular experimental results under traction force loading.

NPCs-exo were found to be internalized by NPCs, and traction force promoted NPCs-exo secretion. High-throughput sequencing and differential expression analysis identified miR-8485 as a differentially expressed miRNA in NPCs-exo secreted under Cyclic Mechanical Tension (CMT) conditions. Dual-luciferase reporter assays confirmed the targeted regulatory relationship between miR-8485 and GSK-3β, as well as its involvement in the Wnt/β-catenin pathway-mediated regulation of NPCs degeneration. Ex vivo experiments, including morphological and immunofluorescence analyses, revealed that the traction group exhibited better morphology than the pressure group, with a more organized AF, NP, and higher NPCs content, though some loss persisted. Both groups showed significant differences in ECM markers (Collagen II, Aggrecan, MMP3) compared to the control (p < 0.05). Additionally, the traction group had significantly higher Collagen II and Aggrecan levels than the pressure group (p < 0.05).

CMT can promote the secretion of NPCs-exo, which are internalized by the NPCs. Through the delivery of miR-8485, NPCs-exo target and regulate GSK-3β, thereby enhancing Wnt/β-catenin pathway activity. This mechanism increases NPCs viability and extracellular matrix synthesis while suppressing apoptosis, ultimately delaying IVDD progression. Immunofluorescence staining in animal experiments confirmed that traction force effectively improves extracellular matrix expression in the IVD and mitigates stress-induced morphological alterations of the IVD.

This study aimed to compare CD82 methylation patterns in peripheral blood among patients with rheumatoid arthritis [RA], inflammatory arthritis, and healthy controls [HC] and to assess their clinical associations with hypertension in RA.

In this cross-sectional study, CD82 methylation at positions 44596705-44596865 on chromosome 11 was analyzed using targeted methylation techniques in peripheral blood from patients with RA, psoriatic arthritis [PsA], ankylosing spondylitis [AS], gout, and HC.

CD82 cg22143324 methylation levels were significantly different between RA patients and healthy controls [P<0.0001], PsA [P=0.0281], and AS [P=0.0360]. In RA subgroups, individuals negative for both rheumatoid factor [RF] and cyclic citrullinated peptide [CCP] [RA-DN], as well as those positive for both [RA-DP], exhibited significantly different methylation levels compared to HC [P=0.0355 and P<0.0001, respectively]. ROC analysis indicated a promising diagnostic potential for CD82 cg22143324 methylation, especially with the TTT haplotype. Correlation analysis revealed significant associations between CD82 methylation and CCP levels, as well as hypertension in RA patients.

The analysis conducted revealed altered CD82 cg22143324 methylation in RA, with potential utility in distinguishing seronegative patients from healthy controls. An association between lower methylation levels and comorbid hypertension in RA patients was also observed, warranting further investigation.