miRNA-21-5p is an important contributor to the promotion of injured peripheral nerve regeneration using hypoxia-pretreated bone marrow-derived neural crest cells.

JOURNAL/nrgr/04.03/01300535-202501000-00035/figure1/v/2024-05-14T021156Z/r/image-tiff Our previous study found that rat bone marrow-derived neural crest cells (acting as Schwann cell progenitors) have the potential to promote long-distance nerve repair. Cell-based therapy can enhance peripheral nerve repair and regeneration through paracrine bioactive factors and intercellular communication. Nevertheless, the complex contributions of various types of soluble cytokines and extracellular vesicle cargos to the secretome remain unclear. To investigate the role of the secretome and extracellular vesicles in repairing damaged peripheral nerves, we collected conditioned culture medium from hypoxia-pretreated neural crest cells, and found that it significantly promoted the repair of sensory neurons damaged by oxygen-glucose deprivation. The mRNA expression of trophic factors was highly expressed in hypoxia-pretreated neural crest cells. We performed RNA sequencing and bioinformatics analysis and found that miR-21-5p was enriched in hypoxia-pretreated extracellular vesicles of neural crest cells. Subsequently, to further clarify the role of hypoxia-pretreated neural crest cell extracellular vesicles rich in miR-21-5p in axonal growth and regeneration of sensory neurons, we used a microfluidic axonal dissociation model of sensory neurons in vitro, and found that hypoxia-pretreated neural crest cell extracellular vesicles promoted axonal growth and regeneration of sensory neurons, which was greatly dependent on loaded miR-21-5p. Finally, we constructed a miR-21-5p-loaded neural conduit to repair the sciatic nerve defect in rats and found that the motor and sensory functions of injured rat hind limb, as well as muscle tissue morphology of the hind limbs, were obviously restored. These findings suggest that hypoxia-pretreated neural crest extracellular vesicles are natural nanoparticles rich in miRNA-21-5p. miRNA-21-5p is one of the main contributors to promoting nerve regeneration by the neural crest cell secretome. This helps to explain the mechanism of action of the secretome and extracellular vesicles of neural crest cells in repairing damaged peripheral nerves, and also promotes the application of miR-21-5p in tissue engineering regeneration medicine.

Application and development of noninvasive biomarkers for colorectal cancer screening: a systematic review.

BACKGROUND: Colorectal cancer (CRC) is the second most common cause of cancer-related death (9.4% of the 9.9 million cancer deaths). However, CRC develops slowly, and early detection and intervention can effectively improve the survival rate and quality of life. Although colonoscopy can detect and diagnose CRC, it is unsuitable for CRC screening in average-risk populations. Some commercial kits based on DNA mutation or methylation are approved for screening, but the low sensitivity for advanced adenoma or early-stage CRC would limit the applications. MAIN RESULTS: Recently, researchers have focused on developing noninvasive or minimally invasive, easily accessible biomarkers with higher sensitivity and accuracy for CRC screening. Numerous reports describe advances in biomarkers, including DNA mutations and methylation, mRNA and miRNA, gut microbes, and metabolites, as well as low-throughput multiomics panels. In small cohorts, the specificity and sensitivity improved when fecal immunochemical testing combined with other biomarkers; further verification in large cohorts is expected. In addition, the continuous improvement of laboratory technology has also improved the sensitivity of detection technology, such as PCR, and the application of CRISPR/Cas technology. Besides, artificial intelligence has extensively promoted the mining of biomarkers. Machine learning was performed to construct a diagnosis model for CRC screening based on the cfDNA fragment features from whole-genome sequencing data. In another study, multiomics markers, including cfDNA, epigenetic, and protein signals, were also discovered by machine learning. Finally, advancements in sensor technology promote the applicability of volatile organic compounds in CRC early detection. CONCLUSION: Here, the authors review advances in early detection and screening of CRC based on different biomarker types. Most studies reported optimistic findings based on preliminary research, and prospective clinical studies are ongoing. These promising biomarkers are expected to more accurately identify early-stage patients with CRC and be applied in the future.

SHANK3 in vagal sensory neurons regulates body temperature, systemic inflammation, and sepsis.

Excessive inflammation has been implicated in autism spectrum disorder (ASD), but the underlying mechanisms have not been fully studied. SHANK3 is a synaptic scaffolding protein and mutations of SHANK3 are involved in ASD. Shank3 expression in dorsal root ganglion sensory neurons also regulates heat pain and touch. However, the role of Shank3 in the vagus system remains unknown. We induced systemic inflammation by lipopolysaccharide (LPS) and measured body temperature and serum IL-6 levels in mice. We found that homozygous and heterozygous Shank3 deficiency, but not Shank2 and Trpv1 deficiency, aggravates hypothermia, systemic inflammation (serum IL-6 levels), and sepsis mortality in mice, induced by lipopolysaccharide (LPS). Furthermore, these deficits can be recapitulated by specific deletion of Shank3 in Nav1.8-expressing sensory neurons in conditional knockout (CKO) mice or by selective knockdown of Shank3 or Trpm2 in vagal sensory neurons in nodose ganglion (NG). Mice with Shank3 deficiency have normal basal core temperature but fail to adjust body temperature after perturbations with lower or higher body temperatures or auricular vagus nerve stimulation. In situ hybridization with RNAscope revealed that Shank3 is broadly expressed by vagal sensory neurons and this expression was largely lost in Shank3 cKO mice. Mechanistically, Shank3 regulates the expression of Trpm2 in NG, as Trpm2 but not Trpv1 mRNA levels in NG were significantly reduced in Shank3 KO mice. Our findings demonstrated a novel molecular mechanism by which Shank3 in vagal sensory neurons regulates body temperature, inflammation, and sepsis. We also provided new insights into inflammation dysregulation in ASD.

Mechanisms and treatments of neuropathic itch in a mouse model of lymphoma.

Our understanding of neuropathic itch is limited due to a lack of relevant animal models. Patients with cutaneous T cell lymphoma (CTCL) experience severe itching. Here, we characterize a mouse model of chronic itch with remarkable lymphoma growth, immune cell accumulation, and persistent pruritus. Intradermal CTCL inoculation produced time-dependent changes in nerve innervations in lymphoma-bearing skin. In the early phase (20 days), CTCL caused hyperinnervations in the epidermis. However, chronic itch was associated with loss of epidermal nerve fibers in the late phases (40 and 60 days). CTCL was also characterized by marked nerve innervations in mouse lymphoma. Blockade of C-fibers reduced pruritus at early and late phases, whereas blockade of A-fibers only suppressed late-phase itch. Intrathecal (i.t.) gabapentin injection reduced late-phase, but not early-phase, pruritus. IL-31 was upregulated in mouse lymphoma, whereas its receptor Il31ra was persistently upregulated in Trpv1-expressing sensory neurons in mice with CTCL. Intratumoral anti-IL-31 treatment effectively suppressed CTCL-induced scratching and alloknesis (mechanical itch). Finally, i.t. administration of a TLR4 antagonist attenuated pruritus in early and late phases and in both sexes. Collectively, we have established a mouse model of neuropathic and cancer itch with relevance to human disease. Our findings also suggest distinct mechanisms underlying acute, chronic, and neuropathic itch.

A novel SLC8A1/LINC01913 intergenic region-ALK fusion identified by NGS and validated by IHC and FISH in a stage IIB lung adenocarcinoma patient who remains relapse-free during the treatment of crizotinib: a case report.

In resected non-small cell lung cancer (NSCLC), ALK rearrangements are associated with worse recurrence-free survival (RFS) than other driver genes. In addition, the micropapillary pattern of NSCLC is associated with a poor prognosis. In recent years, crizotinib tyrosine kinase inhibitors (TKIs) have been widely used to treat patients with advanced NSCLC with ALK fusion. Patient survival outcomes have become highly promising, reflecting the necessity of exploring the application of ALK-TKIs in resected, early stage NSCLC with ALK rearrangements. A 60-year-old Chinese man was diagnosed with stage IIB lung adenocarcinoma harboring a novel SLC8A1/LINC01913 intergenic region-ALK fusion identified by NGS and validated by immunohistochemical staining (IHC) and fluorescence in situ hybridization (FISH). Crizotinib (250 mg orally once daily) was administered to the patient following surgery. The patient remained relapse-free after four months and seven months. This report provided a valuable treatment plan for early lung adenocarcinoma patients with high risks to prevent a postoperative recurrence.

Retraction Notice to: Knockdown of USF1 Inhibits the Vasculogenic Mimicry of Glioma Cells via Stimulating SNHG16/miR-212-3p and linc00667/miR-429 Axis.

[This retracts the article DOI: 10.1016/j.omtn.2018.12.017.].

A Novel LOC101927967 Intergenic Region ALK Fusion Identified by NGS and Validated by IHC and FISH in a Patient with Early Stage Adenocarcinoma of Lung.

Anaplastic lymphoma kinase (ALK) gene rearrangement is an essential driver mutation identified in approximately 5% of non-small cell lung cancers (NSCLCs). The results of clinical trials have demonstrated the impressive efficacy of ALK tyrosine kinase inhibitors (ALK-TKIs). Besides the classic EML4-ALK fusions, a growing list of gene fusion partners for ALK in NSCLC have been identified with heterogeneous clinical responses to ALK-TKIs. However, a LOC101927967-ALK fusion has not been reported in NSCLC. Herein, a novel LOC101927967 downstream intergenic region ALK fusion in an early-stage patient with lung adenocarcinoma was first identified by next-generation sequencing (NGS) and verified by immunohistochemical staining (IHC) and fluorescence in situ hybridization (FISH), which might provide a treatment option for postoperative recurrence.

IL-23 Enhances C-Fiber-Mediated and Blue Light-Induced Spontaneous Pain in Female Mice.

The incidence of chronic pain is especially high in women, but the underlying mechanisms remain poorly understood. Interleukin-23 (IL-23) is a pro-inflammatory cytokine and contributes to inflammatory diseases (e.g., arthritis and psoriasis) through dendritic/T cell signaling. Here we examined the IL-23 involvement in sexual dimorphism of pain, using an optogenetic approach in transgenic mice expressing channelrhodopsin-2 (ChR2) in TRPV1-positive nociceptive neurons. In situ hybridization revealed that compared to males, females had a significantly larger portion of small-sized (100-200 µm2) Trpv1+ neurons in dorsal root ganglion (DRG). Blue light stimulation of a hindpaw of transgenic mice induced intensity-dependent spontaneous pain. At the highest intensity, females showed more intense spontaneous pain than males. Intraplantar injection of IL-23 (100 ng) induced mechanical allodynia in females only but had no effects on paw edema. Furthermore, intraplantar IL-23 only potentiated blue light-induced pain in females, and intrathecal injection of IL-23 also potentiated low-dose capsaicin (500 ng) induced spontaneous pain in females but not males. IL-23 expresses in DRG macrophages of both sexes. Intrathecal injection of IL-23 induced significantly greater p38 phosphorylation (p-p38), a marker of nociceptor activation, in DRGs of female mice than male mice. In THP-1 human macrophages estrogen and chemotherapy co-application increased IL-23 secretion, and furthermore, estrogen and IL-23 co-application, but not estrogen and IL-23 alone, significantly increased IL-17A release. These findings suggest a novel role of IL-23 in macrophage signaling and female-dominant pain, including C-fiber-mediated spontaneous pain. Our study has also provided new insight into cytokine-mediated macrophage-nociceptor interactions, in a sex-dependent manner.

IL-23/IL-17A/TRPV1 axis produces mechanical pain via macrophage-sensory neuron crosstalk in female mice.

Although sex dimorphism is increasingly recognized as an important factor in pain, female-specific pain signaling is not well studied. Here we report that administration of IL-23 produces mechanical pain (mechanical allodynia) in female but not male mice, and chemotherapy-induced mechanical pain is selectively impaired in female mice lacking Il23 or Il23r. IL-23-induced pain is promoted by estrogen but suppressed by androgen, suggesting an involvement of sex hormones. IL-23 requires C-fiber nociceptors and TRPV1 to produce pain but does not directly activate nociceptor neurons. Notably, IL-23 requires IL-17A release from macrophages to evoke mechanical pain in females. Low-dose IL-17A directly activates nociceptors and induces mechanical pain only in females. Finally, deletion of estrogen receptor subunit α (ERα) in TRPV1+ nociceptors abolishes IL-23- and IL-17-induced pain in females. These findings demonstrate that the IL-23/IL-17A/TRPV1 axis regulates female-specific mechanical pain via neuro-immune interactions. Our study also reveals sex dimorphism at both immune and neuronal levels.

Improvement of sensory neuron growth and survival via negatively regulating PTEN by miR-21-5p-contained small extracellular vesicles from skin precursor-derived Schwann cells.

BACKGROUND: Patients with peripheral nerve injury (PNI) often suffer from hypoxic ischemic impairments, in particular when combined with vascular damage, causing neuronal dysfunction and death. Increasing attention has been paid on skin precursor-derived Schwann cells (SKP-SCs), and previous study has shown that SKP-SCs could promote sensory recovery after cell therapy for PNI, resembling the effect of naive SCs, and SKP-SC-derived extracellular vesicles (SKP-SC-EVs) are putatively supposed to be promising therapeutic agents for neural regeneration. METHODS: SKPs were induced to differentiate towards SCs with cocktail factors (N2, neuregulin-1ß, and forskolin) in vitro. SKP-SC-EVs were isolated by exoEasy Maxi Kit and characterized by morphology and phenotypic markers of EVs. Rat sensory neurons from dorsal root ganglions (DRGs) were primarily cultured in regular condition or exposed to oxygen-glucose-deprivation (OGD) condition. SKP-SC-EVs were applied to DRGs or sensory neurons, with LY294002 (a PI3K inhibitor) added; the effect on neurite outgrowth and cell survival was observed. Moreover, microRNA (miR) candidate contained in SKP-SC-EVs was screened out, and miR-mimics were transfected into DRG neurons; meanwhile, the negative regulation of PTEN/PI3K/Akt axis and downstream signaling molecules were determined. RESULTS: It was shown that SKP-SC-EVs could improve the neurite outgrowth of DRGs and sensory neurons. Furthermore, SKP-SC-EVs enhanced the survival of sensory neurons after OGD exposure by alleviating neuronal apoptosis and strengthening cell viability, and the expression of GAP43 (a neuron functional protein) in neurons was upregulated. Moreover, the neuro-reparative role of SKP-SC-EVs was implicated in the activation of PI3K/Akt, mTOR, and p70S6k, as well as the reduction of Bax/Bcl-2 ratio, that was compromised by LY294002 to some extent. In addition, transferring miR-21-5p mimics into sensory neurons could partly protect them from OGD-induced impairment. CONCLUSIONS: Sum up, SKP-SC-EVs could improve neurite outgrowth of DRG sensory neurons in physiological and pathological condition. Moreover, the in vitro therapeutic potential of SKP-SC-EVs on the survival and restoration of OGD-injured sensory neurons was evidenced to be associated with miR-21-5p contained in the small EVs and miR-21-5p/PTEN/PI3K/Akt axis.

lncRNA LINC00665 Stabilized by TAF15 Impeded the Malignant Biological Behaviors of Glioma Cells via STAU1-Mediated mRNA Degradation.

Glioma is a brain cancer characterized by strong invasiveness with limited treatment options and poor prognosis. Recently, dysregulation of long non-coding RNAs (lncRNAs) has emerged as an important component in cellular processes and tumorigenesis. In this study, we demonstrated that TATA-box binding protein associated factor 15 (TAF15) and long intergenic non-protein coding RNA 665 (LINC00665) were both downregulated in glioma tissues and cells. TAF15 overexpression enhanced the stability of LINC00665, inhibiting malignant biological behaviors of glioma cells. Both metal regulatory transcription factor 1 (MTF1) and YY2 transcription factor (YY2) showed high expression levels in glioma tissues and cells, and their knockdown inhibited malignant progression. Mechanistically, overexpression of LINC00665 was confirmed to destabilize MTF1 and YY2 mRNA by interacting with STAU1, and knockdown of STAU1 could rescue the MTF1 and YY2 mRNA degradation caused by LINC00665 overexpression. G2 and S-phase expressed 1 (GTSE1) was identified as an oncogene in glioma, and knockdown of MTF1 or YY2 decreased the mRNA and protein expression levels of GTSE1 through direct binding to the GTSE1 promoter region. Our study highlights a key role of the TAF15/LINC00665/MTF1(YY2)/GTSE1 axis in modulating the malignant biological behaviors of glioma cells, suggesting novel mechanisms by which lncRNAs affect STAU1-mediated mRNA stability, which can inform new molecular therapies for glioma.

Role of ANKHD1/LINC00346/ZNF655 Feedback Loop in Regulating the Glioma Angiogenesis via Staufen1-Mediated mRNA Decay.

Accumulating evidence shows that long noncoding RNA (lncRNA) dysregulation plays a critical role in tumor angiogenesis. Glioma is characterized by abundant angiogenesis. Herein, we investigated the expression and function of LINC00346 in the regulation of glioma angiogenesis. The present study first demonstrated that ANKHD1 (ankyrin repeat and KH domain-containing protein 1) and LINC00346 were significantly increased in glioma-associated endothelial cells (GECs), whereas ZNF655 (zinc finger protein 655) was decreased in GECs. Meanwhile, ANKHD1 inhibition, LINC00346 inhibition, or ZNF655 overexpression impeded angiogenesis of GECs. Moreover, ANKHD1 targeted LINC00346 and enhanced the stability of LINC00346. In addition, LINC00346 bound to ZNF655 mRNA through their Alu elements so that LINC00346 facilitated the degradation of ZNF655 mRNA via a STAU1 (Staufen1)-mediated mRNA decay (SMD) mechanism. Futhermore, ZNF655 targeted the promoter region of ANKHD1 and formed an ANKHD1/LINC00346/ZNF655 feedback loop that regulated glioma angiogenesis. Finally, knockdown of ANKHD1 and LINC00346, combined with overexpression of ZNF655, resulted in a significant decrease in new vessels and hemoglobin content in vivo. The results identified an ANKHD1/LINC00346/ZNF655 feedback loop in the regulation of glioma angiogenesis that may provide new targets and strategies for targeted therapy against glioma.

Anti-PD-1 treatment impairs opioid antinociception in rodents and nonhuman primates.

Emerging immunotherapies with monoclonal antibodies against programmed cell death protein-1 (PD-1) have shown success in treating cancers. However, PD-1 signaling in neurons is largely unknown. We recently reported that dorsal root ganglion (DRG) primary sensory neurons express PD-1 and activation of PD-1 inhibits neuronal excitability and pain. Opioids are mainstay treatments for cancer pain, and morphine produces antinociception via mu opioid receptor (MOR). Here, we report that morphine antinociception and MOR signaling require neuronal PD-1. Morphine-induced antinociception after systemic or intrathecal injection was compromised in Pd1 -/- mice. Morphine antinociception was also diminished in wild-type mice after intravenous or intrathecal administration of nivolumab, a clinically used anti-PD-1 monoclonal antibody. In mouse models of inflammatory, neuropathic, and cancer pain, spinal morphine antinociception was compromised in Pd1 -/- mice. MOR and PD-1 are coexpressed in sensory neurons and their axons in mouse and human DRG tissues. Morphine produced antinociception by (i) suppressing calcium currents in DRG neurons, (ii) suppressing excitatory synaptic transmission, and (iii) inducing outward currents in spinal cord neurons; all of these actions were impaired by PD-1 blockade in mice. Loss of PD-1 also enhanced opioid-induced hyperalgesia and tolerance and potentiates opioid-induced microgliosis and long-term potentiation in the spinal cord in mice. Last, intrathecal infusion of nivolumab inhibited intrathecal morphine-induced antinociception in nonhuman primates. Our findings demonstrate that PD-1 regulates opioid receptor signaling in nociceptive neurons, leading to altered opioid-induced antinociception in rodents and nonhuman primates.

Circular RNA USP1 regulates the permeability of blood-tumour barrier via miR-194-5p/FLI1 axis.

Recent studies indicate circular RNAs are related to dysregulation of vascular endothelial cell function, yet the underlying mechanisms have remained elusive. Here, we characterized the functional role of circular RNA USP1 (circ-USP1) in the regulation of the blood-tumour barrier (BTB) permeability and the potential mechanisms. In the current study, the circ-USP1 expressing level was up-regulated in glioma cerebral microvascular endothelial cells (GECs) of the BTB model in vitro. Knockdown of circ-USP1 disrupted the barrier integrity, increased its permeability as well as reduced tight junction-related protein claudin-5, occludin and ZO-1 expressions in GECs. Bioinformatic prediction and luciferase assay indicated that circ-USP1 bound to miR-194-5p and suppressed its activity. MiR-194-5p contributed to circ-USP1 knockdown-induced increase of BTB permeability via targeting and down-regulating transcription factor FLI1. Furthermore, FLI1 regulated the expressions of claudin-5, occludin and ZO-1 in GECs through binding to their promoter regions. Single or combined treatment of circ-USP1 and miR-194-5p effectively promoted anti-tumour drug doxorubicin across BTB to induce apoptosis of glioma cells. Overall, this present study identified the crucial regulation of circ-USP1 on BTB permeability via miR-194-5p/FLI1 axis-mediated regulation of tight junction proteins, which might facilitate the development of therapeutics against human gliomas.

Structural Insights into Electrophile Irritant Sensing by the Human TRPA1 Channel.

Transient receptor potential channel subfamily A member 1 (TRPA1) is a Ca2+-permeable cation channel that serves as one of the primary sensors of environmental irritants and noxious substances. Many TRPA1 agonists are electrophiles that are recognized by TRPA1 via covalent bond modifications of specific cysteine residues located in the cytoplasmic domains. However, a mechanistic understanding of electrophile sensing by TRPA1 has been limited due to a lack of high-resolution structural information. Here, we present the cryoelectron microscopy (cryo-EM) structures of nanodisc-reconstituted ligand-free TRPA1 and TRPA1 in complex with the covalent agonists JT010 and BITC at 2.8, 2.9, and 3.1 Å, respectively. Our structural and functional studies provide the molecular basis for electrophile recognition by the extraordinarily reactive C621 in TRPA1 and mechanistic insights into electrophile-dependent conformational changes in TRPA1. This work also provides a platform for future drug development targeting TRPA1.

Bone marrow-derived neural crest precursors improve nerve defect repair partially through secreted trophic factors.

BACKGROUND: Emerging evidence suggests that neural crest-derived cells (NCCs) present important functions in peripheral nerve regeneration to correct the insufficiency of autogenous Schwann cells. Postmigratory NCCs have been successfully isolated from adult rat bone marrow in our previous work. In this study, we aim to provide neural crest-derived Schwann cell precursors (SCPs) for repair of nerve defects in adult rats, and partially reveal the mechanisms involved in neuroregeneration of cell therapy. METHODS: A clonal cell line of neural crest precursors of rat bone marrow origin (rBM-NCPs) with SCP identity was expanded in adherent monolayer culture to ensure the stable cell viability of NCPs and potentiate the repair of nerve defects after rBM-NCPs implantation based on tissue engineering nerve grafts (TENG). Here the behavioral, morphological, and electrophysiological detection was performed to evaluate the therapy efficacy. We further investigated the treatment with NCP-conditioned medium (NCP-CM) to sensory neurons after exposure to oxygen-glucose-deprivation (OGD) and partially compared the expression of trophic factor genes in rBM-NCPs with that in mesenchymal stem cells of bone marrow origin (rBM-MSCs). RESULTS: It was showed that the constructed TENG with rBM-NCPs loaded into silk fibroin fiber scaffolds/chitosan conduits repaired 10-mm long sciatic nerve defects more efficiently than conduits alone. The axonal regrowth, remyelination promoted the reinnervation of the denervated hind limb muscle and skin and thereby alleviated muscle atrophy and facilitated the rehabilitation of motor and sensory function. Moreover, it was demonstrated that treatment with NCP-CM could restore the cultured primary sensory neurons after OGD through trophic factors including epidermal growth factor (EGF), platelet-derived growth factor alpha (PDGFα), ciliary neurotrophic factor (CNTF), and vascular endothelial growth factor alpha (VEGFα). CONCLUSIONS: In summary, our findings indicated that monolayer-cultured rBM-NCPs cell-based therapy might effectively repair peripheral nerve defects partially through secreted trophic factors, which represented the secretome of rBM-NCPs differing from that of rBM-MSCs.