Depletion of CPNE7 sensitizes colorectal cancer to 5-fluorouracil by downregulating ATG9B expression.

We aimed to explore the biological function of CPNE7 and determine the impact of CPNE7 on chemotherapy resistance in colorectal cancer (CRC) patients. According to the Gene Expression Profiling Interactive Analysis database and previously published data, CPNE7 was identified as a potential oncogene in CRC. RT-qPCR and Western blotting were performed to verify the expression of CPNE7. Chi-square test was used to evaluate the associations between CPNE7 and clinical features. Cell proliferation, colony formation, cell migration and invasion, cell cycle and apoptosis were assessed to determine the effects of CPNE7. Transcriptome sequencing was used to identify potential downstream regulatory genes, and gene set enrichment analysis was performed to investigate downstream pathways. The effect of CPNE7 on 5-fluorouracil chemosensitivity was verified by half maximal inhibitory concentration (IC50). Subcutaneous tumorigenesis assay was used to examine the role of CPNE7 in sensitivity of CRC to chemotherapy in vivo. Transmission electron microscopy was used to detect autophagosomes. CPNE7 was highly expressed in CRC tissues, and its expression was correlated with T stage and tumour site. Knockdown of CPNE7 inhibited the proliferation and colony formation of CRC cells and promoted apoptosis. Knockdown of CPNE7 suppressed the expression of ATG9B and enhanced the sensitivity of CRC cells to 5-fluorouracil in vitro and in vivo. Knockdown of CPNE7 reversed the induction of the autophagy pathway by rapamycin and reduced the number of autophagosomes. Depletion of CPNE7 attenuated the malignant proliferation of CRC cells and enhanced the chemosensitivity of CRC cells to 5-fluorouracil.

Risk factors for anastomotic leak and postoperative morbidity after right hemicolectomy for colon cancer: results from a prospective, multi-centre, snapshot study in China.

BACKGROUND: Right hemicolectomy is the standard treatment for right-sided colon cancer. There is variation in the technical aspects of performing right hemicolectomy as well as in short-term outcomes. It is therefore necessary to explore best clinical practice following right hemicolectomy in expert centres. METHODS: This snapshot study of right hemicolectomy for colon cancer in China was a prospective, multicentre cohort study in which 52 tertiary hospitals participated. Eligible patients with stage I-III right-sided colon cancer who underwent elective right hemicolectomy were consecutively enrolled in all centres over 10 months. The primary endpoint was the incidence of postoperative 30-day anastomotic leak. RESULTS: Of the 1854 patients, 89.9 per cent underwent laparoscopic surgery and 52.3 per cent underwent D3 lymph node dissection. The overall 30-day morbidity and mortality were 11.7 and 0.2 per cent, respectively. The 30-day anastomotic leak rate was 1.4 per cent. In multivariate analysis, ASA grade > II (P < 0.001), intraoperative blood loss > 50 ml (P = 0.044) and D3 lymph node dissection (P = 0.008) were identified as independent risk factors for postoperative morbidity. Extracorporeal side-to-side anastomosis (P = 0.031), intraoperative blood loss > 50 ml (P = 0.004) and neoadjuvant chemotherapy (P = 0.004) were identified as independent risk factors for anastomotic leak. CONCLUSION: In high-volume expert centres in China, laparoscopic resection with D3 lymph node dissection was performed in most patients with right-sided colon cancer, and overall postoperative morbidity and mortality was low. Further studies are needed to explore the optimal technique for right hemicolectomy in order to improve outcomes further.

LncCMRR Plays an Important Role in Cardiac Differentiation by Regulating the Purb/Flk1 Axis.

As crucial epigenetic regulators, long noncoding RNAs (lncRNAs) play critical functions in development processes and various diseases. However, the regulatory mechanism of lncRNAs in early heart development is still limited. In this study, we identified cardiac mesoderm-related lncRNA (LncCMRR). Knockout (KO) of LncCMRR decreased the formation potential of cardiac mesoderm and cardiomyocytes during embryoid body differentiation of mouse embryonic stem (ES) cells. Mechanistic analyses showed that LncCMRR functionally interacted with the transcription suppressor PURB and inhibited its binding potential at the promoter region of Flk1, which safeguarded the transcription of Flk1 during cardiac mesoderm formation. We also carried out gene ontology term and signaling pathway enrichment analyses for the differentially expressed genes after KO of LncCMRR, and found significant correlation of LncCMRR with cardiac muscle contraction, dilated cardiomyopathy, and hypertrophic cardiomyopathy. Consistently, the expression level of Flk1 at E7.75 and the thickness of myocardium at E17.5 were significantly decreased after KO of LncCMRR, and the survival rate and heart function index of LncCMRR-KO mice were also significantly decreased as compared with the wild-type group. These findings indicated that the defects in early heart development led to functional abnormalities in adulthood heart of LncCMRR-KO mice. Conclusively, our findings elucidate the main function and regulatory mechanism of LncCMRR in cardiac mesoderm formation, and provide new insights into lncRNA-mediated regulatory network of mouse ES cell differentiation.

Does transanal drainage tubes placement have an impact on the incidence of anastomotic leakage after rectal cancer surgery? a systematic review and meta-analysis.

BACKGROUND: Whether Transanal drainage tubes (TDTs) placement reduces the occurrence of anastomotic leakage (AL) after rectal cancer (RC) surgery remains controversial. Most existing meta-analyses rely on retrospective studies, while the prospective studies present an inadequate level of evidence. METHODS: A systematic review and meta-analysis of prospective studies on TDTs placement in RC patients after surgery was conducted. The main analysis index was the incidence of AL, Grade B AL, and Grade C AL, while secondary analysis index was the incidence of anastomotic bleeding, incision infection, and anastomotic stenosis. A comprehensive literature search was performed utilizing the databases Cochrane Library, Embase, PubMed, and Web of Science. We recorded Risk ratios (RRs) and 95% confidence intervals (CI) for each included study, and a fixed-effect model or random-effect model was used to investigate the correlation between TDTs placement and four outcomes after RC surgery. RESULTS: Seven studies (1774 participants, TDT 890 vs non-TDT 884) were considered eligible for quantitative synthesis and meta-analysis. The meta-analysis revealed that the incidence of AL was 9.3% (83/890) in the TDT group and 10.2% (90/884) in the non-TDT group. These disparities were found to lack statistical significance (P = 0.58). A comprehensive meta-analysis, comprising four studies involving a cumulative sample size of 1259 participants, revealed no discernible disparity in the occurrence of Grade B AL or Grade C AL between the TDT group and the non-TDT group (Grade B AL: TDT 34/631 vs non-TDT 26/628, P = 0.30; Grade C AL: TDT 11/631 vs non-TDT 27/628, P = 0.30). Similarly, the incidences of anastomotic bleeding (4 studies, 876 participants), incision infection (3studies, 713 participants), and anastomotic stenosis (2studies, 561 participants) were 5.5% (24/440), 8.1% (29/360), and 2.9% (8/280), respectively, in the TDT group, and 3.0% (13/436), 6.5% (23/353), and 3.9% (11/281), respectively, in the non-TDT group. These differences were also determined to lack statistical significance (P = 0.08, P = 0.43, P = 0.48, respectively). CONCLUSION: The placement of TDTs does not significantly affect the occurrence of AL, Grade B AL, and Grade C AL following surgery for rectal cancer. Additionally, TDTs placement does not be associated with increased complications such as anastomotic bleeding, incision infection, or anastomotic stenosis. TRIAL REGISTRATION: PROSPERO: CRD42023427914.

Identification of angiogenesis-related subtypes, the development of a prognosis model, and features of tumor microenvironment in colon cancer.

Angiogenesis is associated with tumor progression, prognosis, and treatment effect. However, the angiogenesis' underlying mechanisms in the tumor microenvironment (TME) still remain unclear. Understanding the dynamic interactions between angiogenesis and TME in colon adenocarcinoma (COAD) is necessary. We downloaded the transcriptome data and corresponding clinical data of colon cancer patients from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases, respectively. We identified two distinct angiogenesis-related molecular subtypes (subtype A and subtype B) and assessed the clinical features, prognosis, and infiltrating immune cells of patients in the two subtypes. According to the prognostic differential genes, we defined two different gene clusters to further explore the correlation between angiogenesis and tumor heterogeneity. Then, we construct the prognostic risk scoring model angiogenesis-related gene (ARG-score) including seven genes (ARMCX2, latent transforming growth factor ß binding protein 1, ADAM8, FABP4, CCL11, CXCL11, ITLN1) using Lasso-multivariate cox method. We analyzed the correlation between ARG-score and prognosis, clinicopathological features, TME, molecular feature, cancer stem cells (CSCs), and microsatellite instability (MSI) status. To assess the application value of ARG-score in clinical treatment, immunophenotype score was used to predict patients' immunotherapy response in colon cancer. We found the mutations of ARGs in TCGA-COAD dataset from genetic levels and discussed their expression patterns based on TCGA and GEO datasets. We observed important differences in clinicopathological features, prognosis, immune feature, molecular feature between the two molecular subgroups. Then, we established an ARG-score for predicting OS and validated its predictive capability. A high ARG-score characterized by higher transcription level of ARGs, suggested lower MSI-high (MSI-H), lower immune score, and worse clinical stage and survival outcome. Additionally, the ARG-score was remarkably related to the CSCs index and immunotherapy sensitivity. We found two new molecular subtypes and two gene clusters based on ARGs and established an ARG-score. Multilayered analysis revealed that ARGs were remarkably correlated to the heterogeneity of colon cancer patients and explained the process of tumorigenesis and progression better. The ARG-score can help us better assess patients' survival outcomes and provide guidance for individualized treatment.

Virtual Monoenergetic Images Facilitate Better Identification of the Arc of Riolan During Splenic Flexure Takedown.

OBJECTIVE: This study aimed to investigate whether virtual monoenergetic images (VMIs) can aid radiologists and surgeons in better identifying the arc of Riolan (AOR) and to determine the optimal kilo electron volt (keV) level. METHODS: Thirty-three patients were included. Conventional images (CIs) and VMI (40-100 keV) were reconstructed using arterial phase spectral-based images. The computed tomography (CT) attenuation and noise of the AOR, the CT attenuation of the erector spinal muscle, and the background noise on VMI and CI were measured, respectively. The signal-to-noise ratio, contrast-to-noise ratio (CNR), and signal intensity ratio were calculated. The image quality of the AOR was evaluated according to a 4-point Likert grade. RESULTS: The CT attenuation, noise, CNR, and signal intensity ratio of the AOR were significantly higher in VMI at 40 and 50 keV compared with CI ( P < 0.001); VMI at 40 keV was significantly higher than 50 keV ( P < 0.05). No significant difference in signal-to-noise ratio, background noise, and CT attenuation of the spinal erector muscle was observed between VMI and CI ( P > 0.05). virtual monoenergetic image at 40 keV produced the best subjective scores. CONCLUSIONS: Virtual monoenergetic image at 40 keV makes it easier to observe the AOR with optimized subjective and objective image quality. This may prompt radiologists and surgeons to actively search for it and encourage surgeons to preserve it during splenic flexure takedown.

Linc1548 Promotes the Transition of Epiblast Stem Cells Into Neural Progenitors by Engaging OCT6 and SOX2.

The transition of embryonic stem cells from the epiblast stem cells (EpiSCs) to neural progenitor cells (NPCs), called the neural induction process, is crucial for cell fate determination of neural differentiation. However, the mechanism of this transition is unclear. Here, we identified a long non-coding RNA (linc1548) as a critical regulator of neural differentiation of mouse embryonic stem cells (mESCs). Knockout of linc1548 did not affect the conversion of mESCs to EpiSCs, but delayed the transition from EpiSCs to NPCs. Moreover, linc1548 interacts with the transcription factors OCT6 and SOX2 forming an RNA-protein complex to regulate the transition from EpiSCs to NPCs. Finally, we showed that Zfp521 is an important target gene of this RNA-protein complex regulating neural differentiation. Our findings prove how the intrinsic transcription complex is mediated by a lncRNA linc1548 and can better understand the intrinsic mechanism of neural fate determination.

Association between tea consumption and colorectal cancer: a systematic review and meta-analysis of a population-based study.

PURPOSE: A meta-analysis study was performed to systematically assess the association between tea consumption and CRC risk. METHODS: Cochrane Library, Embase, PubMed, and Web of Science were retrieved to collect articles in English since 24 July 2023. Databases were searched and evaluated by two reviewers independently.We screened the literature based on inclusion and exclusion criteria. After determining the random effect model or fixed utility model based on a heterogeneity test, odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. RESULTS: We included fourteen articles in this meta-analysis. We analyzed the data using a random effect model to explore the association between tea consumption and CRC because of apparent heterogeneity (P < 0.001, I2 = 99.5%). The combined results of all tests showed that there is no statistically significant association between tea consumption and CRC risk (OR = 0.756, 95%CI = 0.470-1.215, P = 0.247). Subsequently, subgroup analysis and sensitivity analysis were performed. Excluding any single study, the overall results ranged from 0.73 (95%CI = 0.44-1.20) to 0.86 (95%CI = 0.53-1.40). It was determined that there was no significant publication bias between tea consumption and CRC risk (P = 0.064) by Egger's tests. CONCLUSIONS: The results indicated that tea consumption may not be significantly associated with the development of CRC. IMPLICATIONS OF KEY FINDINGS: Tea reduces colon cancer risk by 24%, but the estimate is uncertain. The actual effect on risk can range from a reduction of 51% to an increase of 18%, but regional and population differences may cause differences.

Detection of circulating tumor cells in patients with pancreatic cancer by novel nano microfluidic chips and comprehensive nursing intervention.

It was to explore the detection efficiency of novel nano microfluidic chips on circulating tumor cells (CTCs) in pancreatic cancer (PC) patients and the effect of comprehensive nursing intervention on the life indicators of patients after PC surgery. PC, benign pancreas, and healthy volunteers were enrolled. The number and positive rate of CTCs in the subjects, the relationship between the number of CTCs in PC patients and clinical data, the recurrence rate, and the life indicators of patients after intervention were sorted out and analyzed. The positive rate of CTCs was 96.67% in PC group (29/30), 16.67% in benign pancreas group (5/30), and 0% in healthy controls. The positive CTCs in PC patients were correlated with vascular invasion (P<0.05). The recurrence rate after PC resection was 56.67% (17/30). The positive detection rate of nano microfluidic chip was 30/30 (100%), which was higher than against CellSeach 7/30 (23.33%), P<0.05. The physiological function score of intervention group (93.67±1.48) was higher as against controls (60.45±2.03), P<0.05. The social function score of intervention group (91.26±0.44) was higher as against controls (54.92±2.98), P<0.05. The novel nano microfluidic chip has high sensitivity and specificity for the detection of CTCs in PC patients. Postoperative comprehensive nursing intervention can improve the quality of life (QoL) and functional status of PC patients.

PAUPAR and PAX6 sequentially regulate human embryonic stem cell cortical differentiation.

Long noncoding RNAs (lncRNAs) play a wide range of roles in the epigenetic regulation of crucial biological processes, but the functions of lncRNAs in cortical development are poorly understood. Using human embryonic stem cell (hESC)-based 2D neural differentiation approach and 3D cerebral organoid system, we identified that the lncRNA PAUPAR, which is adjacent to PAX6, plays essential roles in cortical differentiation by interacting with PAX6 to regulate the expression of a large number of neural genes. Mechanistic studies showed that PAUPAR confers PAX6 proper binding sites on the target neural genes by directly binding the genomic regions of these genes. Moreover, PAX6 recruits the histone methyltransferase NSD1 through its C-terminal PST enrichment domain, then regulate H3K36 methylation and the expression of target genes. Collectively, our data reveal that the PAUPAR/PAX6/NSD1 complex plays a critical role in the epigenetic regulation of hESC cortical differentiation and highlight the importance of PAUPAR as an intrinsic regulator of cortical differentiation.

Tumor-derived immunoglobulin like transcript 5 induces suppressive immunocyte infiltration in colorectal cancer.

Infiltration of immunosuppressive cells in the tumor microenvironment (TME) induced colorectal cancer (CRC) progression and its resistance to immunotherapy. Identification of tumor-specific factors to modulate inhibitory immunocyte infiltration would provide alternative and novel targets for CRC immunotherapy. Immunoglobulin-like transcript (ILT) 5 is a negative regulator of myeloid cell activation. However, its expression and functional role in solid tumors is still unknown. Using human CRC tissues and cell lines, we found that ILT5 was highly expressed in CRC cells compared with normal colorectal epithelial cells. Enriched ILT5 in tumor cells was correlated with advanced tumor stages and poor patient survival. Our subsequent in vitro and in vivo studies revealed that tumor-derived ILT5 inhibited the infiltration of T cells, especially that of CD8+ T cells in the TME, creating suppressive T-cell contexture. Furthermore, ILT5 directed M2-like polarization of tumor-associated macrophages (TAMs). Inhibition of tumor-derived ILT5 restored the immunosuppressive T-cell and TAM contexture, and restricted CRC progression. Our findings identified ILT5 expression in solid tumor cells for the first time and raised ILT5 as a potential immunotarget and prognostic predictor in CRC.

Uncovering potential genes in colorectal cancer based on integrated and DNA methylation analysis in the gene expression omnibus database.

BACKGROUND: Colorectal cancer (CRC) is major cancer-related death. The aim of this study was to identify differentially expressed and differentially methylated genes, contributing to explore the molecular mechanism of CRC. METHODS: Firstly, the data of gene transcriptome and genome-wide DNA methylation expression were downloaded from the Gene Expression Omnibus database. Secondly, functional analysis of differentially expressed and differentially methylated genes was performed, followed by protein-protein interaction (PPI) analysis. Thirdly, the Cancer Genome Atlas (TCGA) dataset and in vitro experiment was used to validate the expression of selected differentially expressed and differentially methylated genes. Finally, diagnosis and prognosis analysis of selected differentially expressed and differentially methylated genes was performed. RESULTS: Up to 1958 differentially expressed (1025 up-regulated and 993 down-regulated) genes and 858 differentially methylated (800 hypermethylated and 58 hypomethylated) genes were identified. Interestingly, some genes, such as GFRA2 and MDFI, were differentially expressed-methylated genes. Purine metabolism (involved IMPDH1), cell adhesion molecules and PI3K-Akt signaling pathway were significantly enriched signaling pathways. GFRA2, FOXQ1, CDH3, CLDN1, SCGN, BEST4, CXCL12, CA7, SHMT2, TRIP13, MDFI and IMPDH1 had a diagnostic value for CRC. In addition, BEST4, SHMT2 and TRIP13 were significantly associated with patients' survival. CONCLUSIONS: The identified altered genes may be involved in tumorigenesis of CRC. In addition, BEST4, SHMT2 and TRIP13 may be considered as diagnosis and prognostic biomarkers for CRC patients.

Critical regulation of a NDIME/MEF2C axis in embryonic stem cell neural differentiation and autism.

A microdeletion within human chromosome 5q14.3 has been associated with the occurrence of neurodevelopmental disorders, such as autism and intellectual disability, and MEF2C haploinsufficiency was identified as main cause. Here, we report that a brain-enriched long non-coding RNA, NDIME, is located near the MEF2C locus and is required for normal neural differentiation of mouse embryonic stem cells (mESCs). NDIME interacts with EZH2, the major component of polycomb repressive complex 2 (PRC2), and blocks EZH2-mediated trimethylation of histone H3 lysine 27 (H3K27me3) at the Mef2c promoter, promoting MEF2C transcription. Moreover, the expression levels of both NDIME and MEF2C were strongly downregulated in the hippocampus of a mouse model of autism, and the adeno-associated virus (AAV)-mediated expression of NDIME in the hippocampus of these mice significantly increased MEF2C expression and ameliorated autism-like behaviors. The results of this study reveal an epigenetic mechanism by which NDIME regulates MEF2C transcription and neural differentiation and suggest potential effects and therapeutic approaches of the NDIME/MEF2C axis in autism.

Downregulation of the paired box gene 3 inhibits the progression of skin cutaneous melanoma by inhibiting c-MET tyrosine kinase : PAX3 downregulation inhibits melanoma progression.

BACKGROUND: The PAX3 (paired box gene 3) gene is highly expressed in several cancer types. However, its underlying mechanism of action in skin cutaneous melanoma (SKCM) remains unknown. METHODS: In this study, we used the GEPIA database and western blotting to analyze the expression of PAX3. We performed the Kaplan-Meier survival analysis to evaluate the prognostic value of PAX3 in SKCM. Next, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed to evaluate the function of PAX3-related co-expressed genes. Additionally, the function and potential mechanism of action of PAX3 in SKCM were studied through functional experiments. Western blotting was used to detect the changes in the levels of epithelial-mesenchymal transition (EMT)-related and MET (c-MET tyrosine kinase) proteins following PAX3 knockdown. Finally, we assessed the correlation between PAX3 expression and the infiltration of CD4+/CD8+ T cells using the TISIDB database. RESULTS: We found that PAX3 was overexpressed in the SKCM tissues and that these levels were indicative of a poor prognosis of SKCM. The KEGG pathway enrichment analysis showed that PAX3-related co-expressed genes were mainly associated with the oncogenic pathways. Knocking down PAX3 significantly inhibited the proliferation, invasion, and migration of SK-MEL-28 cells. The PAX3 expression was related significantly to the immune infiltration level of CD4+/CD8+ T cells. CONCLUSIONS: Our findings demonstrated that PAX3 knockdown could reverse the EMT of tumor cells, inhibit the growth, and progression of SKCM cells. Therefore, PAX3 may have implications as a potential therapeutic target and promising prognostic biomarker for SKCM.

Opioid-free anesthesia compared to opioid anesthesia for laparoscopic radical colectomy with pain threshold index monitoring: a randomized controlled study.

BACKGROUND: Few studies have investigated the depth of intraoperative analgesia with non-opioid anesthesia. This study evaluated whether opioid-free anesthesia can provide an effective analgesia-antinociception balance monitored by the / pain threshold index in laparoscopic radical colectomy. METHODS: We enrolled 102 patients undergoing laparoscopic radical colectomy with general anesthesia. Participants were randomly allocated into two groups to receive opioid-free anesthesia (group OFA) with dexmedetomidine (loading dose with 0.6 µg·kg-1 for 10 min and then 0.5 µg·kg-1·h-1 continuous infusion) and sevoflurane plus bilateral paravertebral blockade (0.2 µg·kg-1 dexmedetomidine and 0.5% ropivacaine 15 ml per side) or opioid-based anesthesia (group OA) with remifentanil, sevoflurane, and bilateral paravertebral blockade (0.5% ropivacaine 15 ml per side). The primary outcome variable was pain intensity during the operation, as assessed by the pain threshold index with the multifunction combination monitor HXD- I. Results were analyzed using repeated measures analysis of variance and Student's t-test. The secondary outcomes were wavelet index, lactic levels, and blood glucose concentration during the operation. The visual analog scale (VAS), rescue analgesic consumption, and side-effects of opioids after surgery were further assessed. RESULTS: One hundred and one patients were included in the analysis. Analysis revealed that the intraoperative pain threshold index readings were not significantly different between the groups from incision to the end of the operation (P = 0.06). Furthermore, similar changes in the brain wavelet index readings were observed in the OFA and OA groups. There was no statistical difference in VAS scores between the groups (P > 0.05); however, non-opioid anesthesia did reduce the rescue analgesic consumption after operation (P < 0.05). In the OFA group, the blood glucose levels increased by 20% compared to baseline and were significantly higher than those in the OA group (P < 0.001). The incidences of postoperative nausea and vomiting, urine retention, intestinal paralysis and pruritus were not significantly different from those in the OA group (P > 0.05). CONCLUSIONS: This study suggests that compared to the opioid anesthesia regimen, our opioid-free anesthesia regimen achieved an equally effective intraoperative pain threshold index in laparoscopic radical colectomy. The incidence of opioid-related adverse reactions was not different between regimens, and intraoperative blood glucose levels were higher with opioid-free anesthesia. TRIAL REGISTRATION: ChiCTR1900021223, 02/02/2019, Title: " Opioid-free anesthesia in laparoscopic surgery: a randomized controlled trial ". Website: hppts:// www.chictr.ogr.cn.

Macrophage Infiltration Initiates RIP3/MLKL-Dependent Necroptosis in Paclitaxel-Induced Neuropathic Pain.

Paclitaxel (PTX) is a commonly used antitumor drug. Approximately 80% of all patients receiving PTX chemotherapy develop chemotherapy-induced peripheral neuropathy (CIPN), limiting the use of PTX. Moreover, CIPN responds poorly to conventional analgesics. Experimental evidence suggests that the neuroinflammatory response plays an essential role in paclitaxel-induced peripheral neuropathy (PIPN). Previous studies have confirmed that dorsal root ganglion (DRG) neuron necroptosis and accompanying inflammation are linked with PIPN; however, the potential upstream regulatory mechanisms remain unclear. Preclinical studies have also established that macrophage infiltration in the DRG is associated with PIPN. TNF-α released by activated macrophages is the primary regulatory signal of necroptosis. In this study, we established a rat model of PIPN via quartic PTX administration (accumulated dose: 8 mg/kg, i.p.). The regulatory effect of macrophage infiltration on necroptosis in PIPN was observed using a macrophage scavenging agent (clodronate disodium). The results showed that PTX increased macrophage infiltration and the levels of TNF-α and IL-1ß in the DRG. PTX also upregulated the levels of necroptosis-related proteins, including receptor-interacting protein kinase (RIP3) and mixed-lineage kinase domain-like protein (MLKL) in DRG neurons and promoted MLKL phosphorylation, resulting in neuronal necrosis and hyperalgesia. In contrast, clodronate disodium effectively removed macrophages, reduced the levels of RIP3, MLKL, and pMLKL, and decreased the number of necrotic cells in the DRG of PIPN rats, alleviating the behavioral pain abnormalities. These results suggest that PTX promotes macrophage infiltration, which results in the release of TNF-α and IL-1ß in the DRG and the initiation of neuronal necroptosis via the RIP3/MLKL pathway, ultimately leading to neuropathic pain.

Paclitaxel induces cognitive impairment via necroptosis, decreased synaptic plasticity and M1 polarisation of microglia.

CONTEXT: Paclitaxel (PTX) leads to chemotherapy brain (chemo-brain) which is characterised by cognitive impairment. It has been reported that necroptosis is associated with cognitive impairment in some neurodegenerative diseases, but it is not clear whether it is related to the development of chemo-brain. OBJECTIVE: To investigate the role of necroptosis and related changes in PTX-induced cognitive impairment. MATERIALS AND METHODS: C57bl/6n mice were randomly divided into five groups: control, vehicle, and different concentrations of PTX (6, 8, 10 mg/kg). Two additional groups received pre-treatment with Gdcl3 or PBS through Intracerebroventricular (ICV) injection before PTX-treatment. Cognitive function, necroptosis, synaptic plasticity and microglia polarisation were analysed. RESULTS: PTX (10 mg/kg) induced significant cognitive impairment, accompanied by changes in synaptic plasticity, including decreased density of PSD95 (0.65-fold), BDNF (0.44-fold) and dendritic spines (0.57-fold). PTX induced necroptosis of 53.41% (RIP3) and 61.91% (MLKL) in hippocampal neurons, with high expression of RIP3 (1.58-fold) compared with the control group. MLKL (1.87-fold) exhibited the same trend, reaching a peak on the 14th day. The increased expression of iNOS (1.63-fold) and inflammatory factors such as TNF-α (1.85-fold) and IL-ß (1.89-fold) compared to the control group suggests that M1 polarisation of microglia is involved in the process of cognitive impairment. Pre-treatment with Gdcl3 effectively reduced the number of microglia (0.50-fold), inhibited the release of TNF-α (0.73-fold) and IL-ß (0.56-fold), and improved cognitive impairment. CONCLUSION: We established a stable animal model of PTX-induced cognitive impairment and explored the underlying pathophysiological mechanism. These findings can guide the future treatment of chemo-brain.

DUOX2 promotes the progression of colorectal cancer cells by regulating the AKT pathway and interacting with RPL3.

Dual oxidase 2 (DUOX2) is an important regulatory protein in the organic process of thyroid hormone iodine. Mounting evidence suggests that DUOX2 plays a crucial role in the occurrence and development of cancers. However, the function and mechanism of DUOX2 in colorectal cancer (CRC) have not been fully clarified. In the present study, the relationship between the expression of DUOX2 and the clinicopathological features and prognosis of CRC patients was analyzed. Furthermore, the effects of DUOX2 on proliferation and invasion in vitro and in vivo were examined. DUOX2-associated proteins were identified by immunoprecipitation (IP). Next-generation sequencing detection was performed to illustrate the mechanism of DUOX2 in CRC cells. It was found that the expression levels of DUOX2 in metastatic sites were significantly higher than those in primary tumor tissues, and this was demonstrated to be associated with poor prognosis. The knockdown of DUOX2 inhibited the invasion and migration of CRC cells. Furthermore, DUOX2 regulated the stability of ribosomal protein uL3 (RPL3) by affecting the ubiquitination status of RPL3, and the invasion and migration ability of DUOX2 can be reversed by the overexpression of RPL3. The downregulation of DUOX2 can affect the expression level of a large number of genes, and a number of these are enriched in the PI3K-AKT pathway. Some of the changes caused by DUOX2 can be reversed by RPL3. In summary, DUOX2 exhibits a significantly higher expression in CRC tumor samples, and facilitates the invasion and metastasis ability of CRC cells by interacting with RPL3.

Extracellular vesicles isolated by size-exclusion chromatography present suitability for RNomics analysis in plasma.

BACKGROUND: Extracellular vesicles (EVs), known as cell-derived membranous structures harboring a variety of biomolecules, have been widely used in liquid biopsy. Due to the complex biological composition of plasma, plasma RNA omics analysis (RNomics) is easily affected, thus it is necessary to select an optimal strategy from exiting methods according to the performance for intended application. METHODS: In this study, four different strategies for EVs isolation were performed and compared (i.e. ultracentrifugation (UC), size exclusion chromatography (SEC), and two most frequently-used commercially available isolation kit (ExoQuick and exoEasy). We compared the yield, purity, PCR quantification of RNAs, miRNA-seq analyses and mRNA-seq analyses of RNAs from EVs isolated using four methods. RESULTS: The results showed that the lowest miRNA binding protein AGO2 (Argonaute-2) and the highest EVs-specific miRNA and lncRNA were observed in EVs obtained through SEC, meanwhile the content of the non-specific miRNA was the lowest. Further RNA-Seq data revealed that RNAs obtained via SEC presented more useful reads for both miRNA and mRNA. Furthermore, the mRNA delivered via SEC tended to have a concentration comparable to the ideal FPKM (Fragments Per Kilobase Million) value. CONCLUSIONS: SEC shall be used as an optimal strategy for the isolation of EVs in plasma RNomics analysis.

MiR-184 directly targets Wnt3 in cardiac mesoderm differentiation of embryonic stem cells.

Embryonic stem (ES) cells have the property of self-renewal and multi-directional differentiation, and provide an ideal model for studying early embryo development in vitro. Wnt3, as Wnt family member 3, plays a vital role during ES cell differentiation. However, the exact regulatory mechanism of Wnt3 remains to be elucidated. MicroRNAs can directly regulate gene expression at the post-transcriptional level and play critical function in cell fate determination. Here, we found the expression level of miR-184 decreased when ES cells differentiated into cardiac mesoderm then increased during the process as differentiated into cardiomyocytes, which negatively correlated with the expression of Wnt3. Overexpression of miR-184 during the process of ES cell differentiation into cardiac mesoderm repressed cardiac mesoderm differentiation and cardiomyocyte formation. Bioinformatics prediction and mechanism studies showed that miR-184 directly bound to the 3'UTR region of Wnt3 and inhibited the expression level of Wnt3. Consistently, knockdown of Wnt3 mimicked the effects of miR-184-overexpression on ES cell differentiation into cardiac mesoderm, whereas overexpression of Wnt3 rescued the inhibition effects of miR-184 overexpression on ES cell differentiation. These findings demonstrated that miR-184 is a direct regulator of Wnt3 during the differentiation process of ES cells, further enriched the epigenetic regulatory network of ES cell differentiation into cardiac mesoderm and cardiomyocytes.