Identification and Validation of SLC9A2 as A Potential Tumor Suppressor in Colorectal Cancer: Integrating Bioinformatics Analysis with Experimental Confirmation.

OBJECTIVE: To uncover the mechanisms underlying the development of colorectal cancer (CRC), we applied bioinformatic analyses to identify key genes and experimentally validated their possible roles in CRC onset and progression. METHODS: We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis on differentially expressed genes (DEGs), constructed a protein-protein interaction (PPI) network to find the top 10 hub genes, and analyzed their expression in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ). We also studied the correlation between these genes and immune cell infiltration and prognosis and validated the expression of SLC9A2 in CRC tissues and cell lines using qRT-PCR and Western blotting. Functional experiments were conducted in vitro to investigate the effects of SLC9A2 on tumor growth and metastasis. RESULTS: We found 130 DEGs, with 45 up-regulated and 85 down-regulated in CRC. GO analysis indicated that these DEGs were primarily enriched in functions related to the regulation of cellular pH, zymogen granules, and transmembrane transporter activity. KEGG pathway analysis revealed that the DEGs played pivotal roles in pancreatic secretion, rheumatoid arthritis, and the IL-17 signaling pathway. We identified 10 hub genes: CXCL1, SLC26A3, CXCL2, MMP7, MMP1, SLC9A2, SLC4A4, CLCA1, CLCA4, and ZG16. GO enrichment analysis showed that these hub genes were predominantly involved in the positive regulation of transcription. Gene expression analysis revealed that CXCL1, CXCL2, MMP1, and MMP7 were highly expressed in CRC, whereas CLCA1, CLCA4, SLC4A4, SLC9A2, SLC26A3, and ZG16 were expressed at lower levels. Survival analysis revealed that 5 key genes were significantly associated with the prognosis of CRC. Both mRNA and protein expression levels of SLC9A2 were markedly reduced in CRC tissues and cell lines. Importantly, SLC9A2 overexpression in SW480 cells led to a notable inhibition of cell proliferation, migration, and invasion. Western blotting analysis revealed that the expression levels of phosphorylated ERK (p-ERK) and phosphorylated JNK (p-JNK) proteins were significantly increased, whereas there were no significant changes in the expression levels of ERK and JNK following SLC9A2 overexpression. Correlation analysis indicated a potential link between SLC9A2 expression and the MAPK signaling pathway. CONCLUSION: Our study suggests that SLC9A2 acts as a tumor suppressor through the MAPK pathway and could be a potential target for CRC diagnosis and therapy.

Ralstonia solanacearum Suppresses Tomato Root Growth by Downregulation of a Wall-Associated Receptor Kinase.

The root architecture of a range of host plants is altered in response to Ralstonia solanacearum infection. This work aimed to identify host genes involved in root development during R. solanacearum infection. A deficient mutant of the type III secretion system regulator hrpB was created in R. solanacearum GMI1000. The hrpB mutant was impaired in virulence but showed a similar suppressive effect as wild-type GMI1000 on tomato root development. Based on comparative transcriptome analysis, 209 genes were found that showed the same changed expression pattern in GMI1000 and hrpB mutant infected roots relative to uninoculated roots. Among them, the wall-associated receptor kinase WAKL20 was substantially downregulated in GMI1000 and hrpB mutant infected roots. Knockdown of WAKL20 led to a shorter primary root length and fewer lateral roots in tomato as well as in Nicotiana benthamiana. The WAKL20 is a pivotal target suppressed by R. solanacearum to shape the altered root development during infection.

Multivariate logistic regression analysis of clinical characteristics and risk factors of cognitive impairment after cerebral ischemic stroke: implications for clinical treatment.

Background: Stroke ranks first among disease fatalities, and those who do survive stroke are prone to cognitive impairment. The aim of this study was to explore the clinical characteristics of post stroke cognitive impairment (PSCI) and the risk factors of PSCI using multivariate logistic regression. Methods: January 2018 to January 2021, the clinical data of 120 patients treated for cerebral ischemic stroke (CIS) at Chengde Central Hospital were retrospectively analyzed. In this study, patients were divided into 2 groups: a control group and a cognitive impairment group. The clinical characteristics of cognitive impairment following CIS were determined using multivariate logistic regression analysis to examine the risk factors and identify clinical implications. Results: This study included the assessment of overall cognitive function and daily living activities of 120 participants, 68 of whom experienced cognitive impairment, representing an incidence of 57%, while 43% patients represented no cognitive impairment after CIS. After the careful analysis of the data, there were remarkable differences in age, sex, education level, stroke history, infarction area, and infarction location (P<0.05). There was no remarkable difference in the history of hypertension, diabetes, atrial fibrillation, carotid intima thickness, smoking, or drinking (P>0.05). The degree of white matter degeneration, brain atrophy, and dominant hemisphere involvement was higher in the cognitive impairment group (P<0.05). The results of multivariate logistic regression analysis indicated that sex, age, education level, stroke history, infarction size, and infarction location were the main risk factors for cognitive impairment after CIS (P<0.05). Conclusions: Patients with cognitive impairment after CIS have imaging features of white matter degeneration, brain atrophy, and involvement of dominant hemispheres. The results of multivariate logistic regression analysis indicated that sex, age, education level, stroke history, infarct size, and infarct location were main risk factors of cognitive impairment after CIS.

ZIC2 induces pro-tumor macrophage polarization in nasopharyngeal carcinoma by activating the JUNB/MCSF axis.

Nasopharyngeal carcinoma (NPC) is a common malignant epithelial tumor of the head and neck that often exhibits local recurrence and distant metastasis. The molecular mechanisms are understudied, and effective therapeutic targets are still lacking. In our study, we found that the transcription factor ZIC2 was highly expressed in NPC. Although ZIC family members play important roles in neural development and carcinogenesis, the specific mechanism and clinical significance of ZIC2 in the tumorigenesis and immune regulation of NPC remain elusive. Here, we first reported that high expression of ZIC2 triggered the secretion of MCSF in NPC cells, induced M2 polarization of tumor-associated macrophages (TAMs), and affected the secretion of TAM-related cytokines. Mechanistically, ChIP-seq and RNA-seq analyses identified JUNB as a downstream target of ZIC2. Furthermore, ZIC2 was significantly enriched in the promoter site of JUNB and activated JUNB promoter activity, as shown by ChIP-qPCR and luciferase assays. In addition, JUNB and MCSF participated in ZIC2-induced M2 TAMs polarization. Thus, blocking JUNB and MCSF could reverse ZIC2-mediated M2 TAMs polarization. Moreover, Kaplan-Meier survival analyses indicated that high expression of ZIC2, JUNB, and CD163 was positively associated with a poor prognosis in NPC. Overexpression of ZIC2 induced tumor growth in vivo, with the increase of JUNB, MCSF secretion, and CD163. In summary, our study implies that ZIC2 induces M2 TAM polarization, at least in part through regulation of JUNB/MCSF and that ZIC2, JUNB, and CD163 can be utilized as prognostic markers for NPC and as therapeutic targets for cancer immunotherapy.

Study of 43 SLE patients with autoimmune liver cirrhosis: emphasis on clinical features and differences from lupus without cirrhosis.

OBJECTIVE: To investigate the clinical characteristics of systemic lupus erythematosus accompanied by autoimmune liver cirrhosis (SLE-ALC) patients and differences from the non-cirrhosis group. METHODS: Forty-three patients with SLE-ALC were enrolled in this study from 2653 patients with SLE in Peking University People's Hospital. A descriptive case-control study was performed between SLE-ALC patients and the entry time-matched non-cirrhosis group. RESULTS: Among the 43 SLE-ALC patients, 41 (95.3%) were female. Eight patients (18.6%) were first found to have cirrhosis and then diagnosed with SLE. Eighteen patients (41.9%) had jaundice and 27 (62.8%) had esophageal and gastric varices. The age of SLE-ALC patients was 51.1 ± 17.2 years, which was significantly older than the non-cirrhosis group (P < 0.001). Lung involvement was more common as initial manifestations in SLE-ALC patients during the SLE course (P=0.027). Compared with the non-cirrhosis group, SLE-ALC patients had worse liver function. A significantly higher rate of hematological system involvement (anemia, leucopenia, and thrombocytopenia) and a higher level of immunoglobulins were observed in SLE-ALC patients (P<0.05). Moreover, SLE-ALC patients displayed a lower positive rate of anti-double-stranded DNA and anti-ribosomal P protein (P<0.05). The most common radiologic manifestations are ascitic fluid (72.1%) and splenomegaly (71.4%) in SLE-ALC patients. Six SLE-ALC patients underwent liver biopsy, and interface hepatitis was present in all patients. CONCLUSIONS: Cirrhosis is rare in SLE patients but is manifested as a unique pattern of clinical features characterized by late-onset age, lung involvement, high immunoglobulins, and impaired liver function.

EDIL3 and VEGF Synergistically Affect Angiogenesis in Endothelial Cells.

Background: Angiogenesis is one of the histologically predominant characteristics of psoriasis. Vascular endothelial growth factor (VEGF) and epidermal growth factor-like repeats and discoidin I-like domains 3 (EDIL3) have critical effects on angiogenesis. Both these proteins are vital proangiogenic factors in tumor occurrence and progression; however, the relationship between EDIL3 and VEGF with psoriasis remains unclear. Objective: We aimed to elucidate the role of EDIL3 and VEGF and the involved mechanisms in psoriasis-associated angiogenesis. Methods: EDIL3 and VEGF expression in cutaneous tissue was determined by immunohistochemical assay. The effects of EDIL3 on VEGF, VEGFR2, and the growth, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) were analyzed by Western blotting assay, cell counting kit-8 assay, Transwell assay, and Matrigel tube formation assay. Results: EDIL3 and VEGF levels in psoriatic lesions significantly increased as compared to those in normal individuals and showed a positive relationship with the Psoriasis Area and Severity Index. The downregulation of EDIL3 decreased VEGF and VEGFR2 expression in HUVECs. Moreover, the decreased expression of EDIL3 and VEGF reduced the growth, invasion, and tube formation abilities of HUVECs, while EDIL3 resistance to VEGF and VEGFR2 was restored by using the EDIL3 recombinant protein. Conclusion: These results suggest that psoriasis is also characterized by EDIL3 and VEGF-mediated angiogenesis. Thus, EDIL3 and VEGF could serve as novel targets for treating psoriasis.

Hematological and neurological expressed 1 (HN1) activates c-Myc signaling by inhibiting ubiquitin-mediated proteasomal degradation of c-Myc in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a poor prognosis due to the usually advanced stage at diagnosis. Sustained activation of the MYC oncogene is implicated in the development of HCC; however, the molecular mechanisms of MYC deregulation in HCC are poorly understood. Here, real-time PCR and western blotting were used to measure the expression of hematological and neurological expressed 1 (HN1) in HCC cells. Expression of HN1 and MYC in clinical specimens was analyzed using immunohistochemistry. The role of HN1 in HCC proliferation, migration, and invasion was explored in vitro and in vivo. MYC expression was measured using real-time PCR and western blotting. MYC transcriptional activity was assessed using a luciferase reporter system. Expression of MYC target genes was quantified using real-time PCR. Protein interaction between MYC and HN1 was assessed using co-immunoprecipitation and western blotting. We identified HN1 as a novel regulatory factor of the glycogen synthase kinase (GSK) 3ß-MYC axis. HN1 expression is elevated in liver tumor tissues and cells, and significantly correlates with poor survival in HCC patients. Upregulation of HN1 promotes, and silencing of HN1 represses, the proliferation and metastasis of liver cancer cells in vitro and in vivo. Moreover, our results demonstrate that HN1 sustains stabilization and persistent activity of MYC via interaction with GSK3ß in HCC. Importantly, the tumor-promoting effects of HN1 on HCC cells were attenuated by suppressing MYC. In conclusion, constitutive activation of MYC by HN1 promotes the progression of HCC; therefore, HN1 might be a novel therapeutic target for HCC.

The hypoxia-related signature predicts prognosis, pyroptosis and drug sensitivity of osteosarcoma.

Osteosarcoma (OS) is one of the most common types of solid sarcoma with a poor prognosis. Solid tumors are often exposed to hypoxic conditions, while hypoxia is regarded as a driving force in tumor recurrence, metastasis, progression, low chemosensitivity and poor prognosis. Pytoptosis is a gasdermin-mediated inflammatory cell death that plays an essential role in host defense against tumorigenesis. However, few studies have reported relationships among hypoxia, pyroptosis, tumor immune microenvironment, chemosensitivity, and prognosis in OS. In this study, gene and clinical data from Therapeutically Applicable Research to Generate Effective Treatments (TARGET) and Gene Expression Omnibus (GEO) databases were merged to develop a hypoxia risk model comprising four genes (PDK1, LOX, DCN, and HMOX1). The high hypoxia risk group had a poor prognosis and immunosuppressive status. Meanwhile, the infiltration of CD8+ T cells, activated memory CD4+ T cells, and related chemokines and genes were associated with clinical survival outcomes or chemosensitivity, the possible crucial driving forces of the OS hypoxia immune microenvironment that affect the development of pyroptosis. We established a pyroptosis risk model based on 14 pyroptosis-related genes to independently predict not only the prognosis but also the chemotherapy sensitivities. By exploring the various connections between the hypoxic immune microenvironment and pyroptosis, this study indicates that hypoxia could influence tumor immune microenvironment (TIM) remodeling and promote pyroptosis leading to poor prognosis and low chemosensitivity.

EDIL3 influenced the αvß3-FAK/MEK/ERK axis of endothelial cells in psoriasis.

One of the earliest events in the development of psoriatic lesion is a vascular network expansion. The abnormal vascular network is associated with increased endothelial cells (ECs) survival, proliferation, adhesion, migration, angiogenesis and permeability in psoriatic lesion. Our previous study demonstrated that epidermal growth factor-like repeats and discoidin I-like domains 3 (EDIL3) derived from psoriatic dermal mesenchymal stem cells (DMSCs) promoted cell-cell adhesion, migration and angiogenesis of ECs, but the molecular mechanism of upstream or downstream has not been explored. So, this study aimed to explore the association between EDIL3 derived from DMSCs (DMSCs-derived EDIL3) and psoriasis-associated angiogenesis. We injected recombinant EDIL3 protein to mouse model of psoriasis to confirm the roles of EDIL3 in psoriasis. Besides, we employed both short-interference RNA (si-RNA) and lentiviral vectors to explore the molecular mechanism of EDIL3 promoting angiogenesis in psoriasis. In vivo, this research found that after injected recombination EDIL3 protein, the epidermis thickness and microvessel density were both elevated. EDIL3 accelerated the process of psoriasis in the IMQ-induced psoriasis-like mouse model. Additionally, we confirmed that in vitro DMSCs-derived EDIL3 is involved in the tube formation of ECs via αvß3-FAK/MEK/ERK signal pathway. This suggested that DMSCs-derived EDIL3 and αvß3-FAK/MEK/ERK signal pathway in ECs play an important role in the pathogenesis of psoriasis. And the modification of DMSCs, EDIL3 and αvß3-FAK/MEK/ERK signal pathway will provide a valuable therapeutic target to control the angiogenesis in psoriasis.

The effect of serum IL-2 levels on the prognosis of primary biliary cholangitis-related liver failure and the preliminary exploration of its mechanism.

Background: In primary biliary cholangitis (PBC), the levels of serum IL-2 were involved in liver inflammation and immune changes. This study aimed to investigate the prognostic significance of serum IL-2 combined with total bilirubin (TBIL) in liver failure and cytokine changes during the disease. Methods: A total of 160 PBC patients treated with UDCA were included. Parameters at admission were collected, and the COX regression model was used to predict independent risk factors associated with PBC disease progression. We identified the optimal cut-off values and prognosis effects of serum IL-2 and TBIL based on the time-dependent receiver operating characteristic (ROC) curve. We also analyzed the incidence of liver failure with Kaplan-Meier survival analysis. In addition, the changes of cytokines (mainly IL-2) in liver tissues and blood samples from 11 patients with end-stage PBC liver failure and five healthy controls were examined. Results: Age, IL-2, ALB, γ-GT, ALP, TBIL, Hb, TBA, WBC, and PLT, as well as anti-Sp100, were found to be independent risk factors in PBC patients with liver failure. Patients with decreased serum IL-2 levels and increased TBIL levels have a significantly higher incidence of liver failure and a worse prognosis. Patients with advanced PBC liver failure after liver transplantation exhibited a significant decrease in levels of serum IL-2 and a relatively immunosuppressed status. Conclusions: The combination of serum IL-2 and TBIL can be a predictor of the progression of liver failure in patients with primary biliary cholangitis, and it is likely to be related to the expression of GM-CSF and G-CSF.

The Measurement of 25-Hydroxyvitamin-D in Chronic HBV Patients Using LC-MS/MS.

BACKGROUND: Vitamin D deficiency is universal among patients with chronic liver disease. Vitamin D may be involved in the regulation of immune function of chronic hepatitis B and related to disease progression. METHODS: The study was a cross-sectional study. The level of vitamin 25(OH)D was detected in patients with chronic hepatitis B, hepatitis B cirrhosis, hepatitis B cancer, and healthy groups by isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS). At the same time, the clinical data, biochemical indexes, and T lymphocyte subsets were collected to study the relationship between vitamin 25(OH)D deficiency and clinical indexes of hepatitis B patients. RESULTS: The prevalence of vitamin D deficiency (< 20 ng/mL) was higher in patients with liver cancer group (96.97%, 10.59 ± 3.06 ng/mL) and cirrhosis group (93.18%, 11.85 ± 2.66 ng/mL) than in the healthy group (76.92%, 16.38 ± 5.53 ng/mL) and chronic hepatitis B group (77.83%, 15.06 ± 4.91 ng/mL). There were significant differences in vitamin 25(OH)D levels between the cirrhosis groups and the healthy groups, the liver cancer groups and the healthy groups, the hepatitis B cirrhosis groups and the chronic hepatitis B groups, the liver cancer groups and the chronic hepatitis B groups (p < 0.05). There was no significant difference in vitamin 25 (OH)D level between liver cancer group and hepatitis B cirrhosis group, healthy group and chronic hepatitis B group (p > 0.05). Vitamin 25(OH)D level was correlated with age (r = -0.24, p = 0.015), lymphocyte (r = 0.24, p = 0.015), hemoglobin (r = 0.28, p = 0.005), platelet (r = 0.27, p = 0.006), PTA (r = 0.33, p = 0.001), albumin (r = 0.30, p = 0.002), prealbumin (r = 0.39, p = 0.001), cholinesterase (r = 0.29, p = 0.003), CD3+ (r = 0.20, p = 0.04), CD3+ CD8+ (r = 0.20, p = 0.04), CD45+ (r = 0.24, p = 0.017), but none correlated with liver function and HBV-DNA. CONCLUSIONS: Vitamin D deficiency existed in patients with hepatitis B, which was related to the clinical progress of hepatitis B and may be involved in the regulation of immune function in patients with chronic HBV infection.

Isolation and Characterization of the GmMT-II Gene and Its Role in Response to High Temperature and Humidity Stress in Glycine max.

Metallothioneins (MTs) are polypeptide-encoded genes involved in plant growth, development, seed formation, and diverse stress response. High temperature and humidity stress (HTH) reduce seed development and maturity of the field-grown soybean, which also leads to seed pre-harvest deterioration. However, the function of MTs in higher plants is still largely unknown. Herein, we isolated and characterized the soybean metallothionein II gene. The full-length fragment is 255 bp and encodes 85 amino acids and contains the HD domain and the N-terminal non-conservative region. The subcellular location of the GmMT-II-GFP fusion protein was clearly located in the nucleus, cytoplasm, and cell membrane. The highest expression of the GmMT-II gene was observed in seeds both of the soybean Xiangdou No. 3 and Ningzhen No. 1 cultivars, as compared to other plant tissues. Similarly, gene expression was higher 45 days after flowering followed by 30, 40, and 35 days. Furthermore, the GmMT-II transcript levels were significantly higher at 96 and 12 h in the cultivars Xiangdou No. 3 and Ningzhen No. 1 under HTH stress, respectively. In addition, it was found that when the Gm1-MMP protein was deleted, the GmMT-II could bind to the propeptide region of the Gm1-MMP, but not to the signal peptide region or the catalytic region. GmMT-II overexpression in transgenic Arabidopsis increased seed germination and germination rate under HTH conditions, conferring enhanced resistance to HTH stress. GmMT-II overexpressing plants suffered less oxidative damage under HTH stress, as reflected by lower MDA and H2O2 content and ROS production than WT plants. In addition, the activity of antioxidant enzymes namely SOD, CAT, and POD was significantly higher in all transgenic Arabidopsis lines under HTH stress compared wild-tpye plants. Our results suggested that GmMT-II is related to growth and development and confers enhanced HTH stress tolerance in plants by reduction of oxidative molecules through activation of antioxidant activities. These findings will be helpful for us in further understanding of the biological functions of MT-II in plants.

MiR-155 inhibits TP53INP1 expression leading to enhanced glycolysis of psoriatic mesenchymal stem cells.

BACKGROUND: Psoriasis is a systemic disease with multiple associated comorbidities, including metabolic syndrome. Studies suggest that chronic inflammation is a central link between psoriasis and metabolic abnormalities. MiR-155 is a well-known microRNA that plays an important regulatory role in inflammation. Studies from our group and others have demonstrated an upregulation of miR-155 in psoriasis. OBJECTIVES: Here, we investigated whether miR-155 regulates glycolysis of psoriasis and the underlying mechanisms. METHODS: Human dermal-derived mesenchymal stem cells (MSC) were treated with miR-155 mimic or inhibitor, followed by assessments of cells proliferation, metabolism and inflammatory response. Target gene prediction and GO/Pathway analysis were used to screen the putative targets involved in the pathways of metabolism, and verified by dual-luciferase reporter assay. To determine whether TP53INP1/p53 signaling pathway is involved in miR-155-mediated regulation of glycolysis, changes in glycolysis were assessed in psoriatic MSC (PM) with either overexpression or knockdown of TP53INP1, or activation/inhibition of p53 signaling pathway. RESULTS: Our results showed that miR-155 promoted proliferation, migration, inflammatory response and metabolite levels of MSC, while inhibiting apoptosis. In comparison to the MSC from normal subjects, the glycolysis levels were increased in PM. GO and KEGG analyses indicated that TP53INP1 was miR-155 target gene with negative regulation of cellular metabolic process. Moreover, miR-155 promoted glycolysis of PM by negative regulation of TP53INP1/p53 signaling pathway. CONCLUSIONS: MiR-155 could promote glycolysis via targeting of TP53INP1 in PM. These findings suggest a pathogenic role of miR-155 in metabolic abnormalities in psoriasis.

Microbiota in mesenteric adipose tissue from Crohn's disease promote colitis in mice.

BACKGROUND: Mesenteric adipose tissue (mAT) hyperplasia, known as creeping fat is a pathologic characteristic of Crohn's disease (CD). The reserve of creeping fat in surgery is associated with poor prognosis of CD patients, but the mechanism remains unknown. METHODS: Mesenteric microbiome, metabolome, and host transcriptome were characterized using a cohort of 48 patients with CD and 16 non-CD controls. Multidimensional data including 16S ribosomal RNA gene sequencing (16S rRNA), host RNA sequencing, and metabolome were integrated to reveal network interaction. Mesenteric resident bacteria were isolated from mAT and functionally investigated both in the dextran sulfate sodium (DSS) model and in the Il10 gene-deficient (Il10-/-) mouse colitis model to validate their pro-inflammatory roles. RESULTS: Mesenteric microbiota contributed to aberrant metabolites production and transcripts in mATs from patients with CD. The presence of mAT resident microbiota was associated with the development of CD. Achromobacter pulmonis (A. pulmonis) isolated from CD mAT could translocate to mAT and exacerbate both DSS-induced and Il10 gene-deficient (Il10-/-) spontaneous colitis in mice. The levels of A. pulmonis in both mAT and mucous layer from CD patients were higher compared to those from the non-CD group. CONCLUSIONS: This study suggests that the mesenteric microbiota from patients with CD sculpt a detrimental microenvironment and promote intestinal inflammation. Video abstract.

Tumor cell-derived SPON2 promotes M2-polarized tumor-associated macrophage infiltration and cancer progression by activating PYK2 in CRC.

BACKGROUND: Tumor-associated macrophages (TAMs) are key regulators of the complex interplay between cancer and the immune microenvironment. Tumor cell-derived spondin 2 (SPON2) is an extracellular matrix glycoprotein that has complicated roles in recruitment of macrophages and neutrophils during inflammation. Overexpression of SPON2 has been shown to promote tumor cell migration in colorectal cancer (CRC). However, the mechanism by which SPON2 regulates the accumulation of TAMs in the tumor microenvironment (TME) of CRC is unknown. METHODS: Immunohistochemistry was used to examine SPON2 expression in clinical CRC tissues. In vitro migration assays, transendothelial migration assays (iTEM), and cell adhesion assays were used to investigate the effects of SPON2 on monocyte/macrophage migration. Subcutaneous tumor formation and orthotopic implantation assays were performed in C57 BL/6 mice to confirm the effects of SPON2 on TAM infiltration in tumors. RESULTS: SPON2 expression is positively correlated with M2-TAM infiltration in clinical CRC tumors and poor prognosis of CRC patients. In addition, SPON2 promotes cytoskeletal remodeling and transendothelial migration of monocytes by activating integrin ß1/PYK2 axis. SPON2 may indirectly induce M2-polarization through upregulating cytokines including IL10, CCL2 and CSF1 expression in tumor cells. Blocking M2 polarization and Macrophage depletion inhibited the SPON2-induced tumors growth and invasion. Furthermore, blocking the SPON2/integrin ß1/PYK2 axis impairs the transendothelial migration of monocytes and cancer-promoting functions of TAMs in vivo. CONCLUSIONS: Our findings demonstrate that SPON2-driven M2-TAM infiltration plays an important role during CRC tumor growth and metastasis. SPON2 may be a valuable biomarker guiding the use of macrophage-targeting strategies and a potential therapeutic target in advanced CRC.

Preconditioning With Intermittent Hypobaric Hypoxia Attenuates Stroke Damage and Modulates Endocytosis in Residual Neurons.

Background: Moderate hypobaric hypoxia induces cerebral ischemic tolerance. We investigated the optimal method for applying hypobaric hypoxia preconditioning at 5,000 m to ischemic brain tissue and combined it with proteomics to determine the mechanisms underlying this effect. Methods: Male SD rats were randomly grouped as S (sham, n = 20), M (middle cerebral artery occlusion [MCAO], n = 28), H2M (intermittent hypobaric hypoxia preconditioned MCAO group, 2 h/day, 10 days, n = 20), H6M (intermittent hypobaric hypoxia preconditioned MCAO group, 6 h/day, 10 days, n = 28), and HpM (persistent hypobaric hypoxia preconditioned MCAO group, 10 days, n = 28). The permanent MCAO model was established based on the Zea Longa method. Infarction was assessed with the modified neurological severity score (mNSS) and 2,3,5-triphenyl tetrazolium chloride staining. The total protein expression of the neuron-specific nuclear protein (NeuN), cysteinyl aspartate specific proteinase 3 (caspase-3), cleaved-caspase-3, and interleukin 6 (IL-6) was determined using western blotting. We assessed the peri-infarct cortex's ultrastructural changes. A label-free proteomic study and western blot verification were performed on the most effective preconditioned group. Results: The H6M group showed a lower infarct volume (p = 0.0005), lower mNSS score (p = 0.0009) than the M group. The H2M showed a lower level of IL-6 (p = 0.0213) than the M group. The caspase-3 level decreased in the H2M (p = 0.0002), H6M (p = 0.0025), and HpM groups (p = 0.0054) compared with that in the M group. Cleaved-caspase-3 expression decreased in the H2M (p = 0.0011), H6M (p < 0.0001), and HpM groups (p < 0.0001) compared with that in the M group. The neurons' ultrastructure and the blood-brain barrier in the peri-infarct tissue improved in the H2M and H6M groups. Immunofluorescence revealed increased NeuN-positive cells in the peri-infarct tissue in the H6M group (p = 0.0003, H6M vs. M). Protein expression of Chmp1a, Arpc5, and Hspa2 factors related to endocytosis were upregulated in the H6M compared with those of the M group (p < 0.05 for all) on western blot verification of label-free proteomics. Conclusions: Intermittent hypobaric hypoxia preconditioning exerts a neuroprotective effect in a rat stroke model. Persistent hypobaric hypoxia stimulation exhibited no significant neuroprotective effect. Intermittent hypoxic preconditioning for 6 h/day for 10 days upregulates key proteins in clathrin-dependent endocytosis of neurons in the cortex.

Circular RNA Hsa_circ_0004018 Inhibits Wnt/ß-Catenin Signaling Pathway by Targeting microRNA-626/DKK3 in Hepatocellular Carcinoma.

BACKGROUND AND AIM: Dysexpression of circular RNAs has been identified in multiple types of cancer. Hsa_circ_0004018 was reported to be significantly downregulated in hepatocellular carcinoma (HCC) and to display HCC-stage-specific expression features. However, the role of hsa_circ_0004018 in HCC progression remains unclear. METHODS: The expression of hsa_circ_0004018 or microRNA-626 (miR-626) was detected in tumor tissues and paired non-tumor tissues from HCC patients, as well as in one normal human liver cell line and 5 HCC cell lines by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, dye exclusion assay, clonogenic assay, scratch migration assay and transwell assay were used to measure cell proliferation and migration capacity, respectively. Luciferase report assay and RNA pull down assay were performed to explore the regulatory effect of certain molecules on the expression of target genes. RESULTS: We found that the expression of hsa_circ_0004018 was lower in tumor tissues than in their paired non-tumor tissues from 28 out of 41 HCC patients. The difference in the expression between tumor tissues and non-tumor tissues was statistically significant (p<0.001). Further analysis revealed that such lower expression in tumor tissues was much more common in bigger tumor size group (≥5cm) compared with the smaller tumor size group (<5cm) (85% vs 42%, p=0.0007). Similarly, hsa_circ_0004018 was downregulated in HCC cell lines. Additionally, a negative correlation between hsa_circ_0004018 and miR-626 expression was noticed in HCC tissues. Moreover, we observed that hsa_circ_0004018 interacted with miR-626/DKK3 and contributed to HCC cell proliferation and migration through inhibiting Wnt/ß-catenin signaling pathway in vitro. Furthermore, hsa_circ_0004018 blocked xenograft tumor growth in vivo through inhibiting Wnt/ß-catenin signaling pathway by targeting miR-626/DKK3. CONCLUSION: We revealed that hsa_circ_0004018/miR-626/DKK3 regulatory axis may be a possible novel therapeutic target for HCC.

Single-cell transcriptomic analysis defines the interplay between tumor cells, viral infection, and the microenvironment in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignancy with a complex tumor ecosystem. How the interplay between tumor cells, EBV, and the microenvironment contributes to NPC progression and immune evasion remains unclear. Here we performed single-cell RNA sequencing on ~104,000 cells from 19 EBV+ NPCs and 7 nonmalignant nasopharyngeal biopsies, simultaneously profiling the transcriptomes of malignant cells, EBV, stromal and immune cells. Overall, we identified global upregulation of interferon responses in the multicellular ecosystem of NPC. Notably, an epithelial-immune dual feature of malignant cells was discovered and associated with poor prognosis. Functional experiments revealed that tumor cells with this dual feature exhibited a higher capacity for tumorigenesis. Further characterization of the cellular components of the tumor microenvironment (TME) and their interactions with tumor cells revealed that the dual feature of tumor cells was positively correlated with the expression of co-inhibitory receptors on CD8+ tumor-infiltrating T cells. In addition, tumor cells with the dual feature were found to repress IFN-γ production by T cells, demonstrating their capacity for immune suppression. Our results provide new insights into the multicellular ecosystem of NPC and offer important clinical implications.

Psoriasis-associated angiogenesis is mediated by EDIL3.

The dermal mesenchymal stem cells (DMSCs) from psoriasis display higher expression level of epidermal growth factor-like repeats and discoidin I-like domains 3 (EDIL3), while EDIL3 can bind integrins, including αvß3 and αvß5, to regulate angiogenesis. To assess the role of EDIL3 derived from DMSCs of psoriasis (P-DMSCs) in angiogenesis, in vitro, EDIL3 of DMSCs from psoriasis was silenced by interfering EDIL3. Then the efficacy of silencing EDIL3 was tested by fluorescent flag, qRT-PCR and western blotting. And, in vitro, the relationship of EDIL3 in DMSCs with the angiogenesis of HUVECs were investigated through co-culture system. In vivo, EDIL3 recombinant protein was injected into IMQ cream-induced psoriasis-like skin lesions of mouse and EDIL3-associated tube formation were determined using Image J software. Our results showed the capacity of the adhesion, migration and tube formation of HUVECs in all psoriatic DMSCs groups were significantly higher compared with the control and si-EDIL3 groups (all P<0.05) in vitro. Moreover, under stimulated by EDIL3 recombinant protein, EDIL3-associated tube formation was dramatically elevated in vivo (P<0.01). In this study, EDIL3 could promote the adhesion, migration and tube formation of ECs and participant in the angiogenesis pathogenesis of psoriasis through affecting biological function on ECs both in vitro and in vivo. The results suggest a potential role of the critical pro-angiogenic factor EDIL3 in psoriasis therapy.