Regulation of the small GTPase Ran by miR-802 modulates proliferation and metastasis in colorectal cancer cells.

BACKGROUND: The small GTPase Ran is upregulated in multiple cancers and fundamental for cancer cell survival and progression, but its significance and molecular mechanisms in colorectal cancer (CRC) remain elusive. METHODS: Ran expression was detected in CRC cell lines and tumour tissues. In vitro and in vivo functional assays were performed to examine the effects of Ran on cell proliferation and metastasis. The pathways and effectors regulated by Ran were explored by an unbiased screening. Bioinformatics prediction and experimental validation were used to identify the miRNA regulator for Ran. RESULTS: Ran expression was frequently increased in metastatic CRC cells and tissues, especially in metastatic tissues. The upregulation of Ran correlated with poor CRC patient prognosis. Ran silencing reduced proliferation and metastasis of CRC cells both in vitro and in vivo. Ran regulated the expression of EGFR and activation of ERK and AKT signalling pathways. miR-802 was identified as an upstream regulator of Ran and miR-802 overexpression resulted in antiproliferative and antimetastatic activities. CONCLUSION: Our study demonstrates the oncogenic roles and underlying mechanisms of Ran in CRC and the novel miR-802/Ran/EGFR regulatory axis may provide potential biomarkers for the treatment of CRC.

OGDH mediates the inhibition of SIRT5 on cell proliferation and migration of gastric cancer.

SIRT5 has a wide range of functions in different cellular processes such as glycolysis, TCA cycle and antioxidant defense, which mediates lysine desuccinylation, deglutarylation and demalonylation. Recent evidences have implicated that SIRT5 is a potential suppressor of gastric cancer (GC). However, the underlying mechanism of SIRT5 in gastric cancer is still unclear. Here, we show that SIRT5 expression is significantly decreased in human GC tissues. Functional analysis demonstrates that SIRT5 inhibits cell growth in vitro and in vivo, arrests the cell cycle in G1/S transition, and suppresses migration and invasion of GC cells via regulating epithelial-to-mesenchymal transition. Mechanistically, we demonstrate that there is the direct interaction between SIRT5 and 2-oxoglutarate dehydrogenase (OGDH), and desuccinylation of OGDH by SIRT5 inhibits the activity of OGDH complex. Further studies of the relationship between SIRT5 and OGDH show OGDH inhibition by succinyl phosphonate (SP) or siRNA suppresses the increase in cell growth and migration induced by SIRT5 deletion. Moreover, SIRT5 decreases mitochondrial membrane potential (ΔΨm), ATP products and increases the ROS levels and NADP/NADPH ratio in GC cells through the inhibition of OGDH complex activity. Therefore, SIRT5 suppresses GC cell growth and migration through desuccinylating OGDH and inhibiting OGDH complex activity to disturb mitochondrial functions and redox status.

POU2F2-oriented network promotes human gastric cancer metastasis.

BACKGROUND AND AIMS: Aberrant upregulation of POU2F2 expression has been discovered in metastatic gastric cancer (GC). However, the mechanisms underlying the aberrant upregulation and the potential functions of POU2F2 remain uncertain. DESIGN: The role and mechanism of POU2F2 in GC metastasis were investigated in gastric epithelial cells, GC cell lines and an experimental metastasis animal model by gain of function and loss of function. Upstream and downstream targets of POU2F2 were selected by bioinformatics and identified by luciferase reporter assay, electrophoretic mobility shift assay and chromatin immunoprecipitation PCR. The influence of miR-218 on its putative target genes (POU2F2, ROBO1 and IKK-ß) and GC metastasis was further explored via in vitro and in vivo approaches. RESULTS: Increased POU2F2 expression was detected in metastatic GC cell lines and patient samples. POU2F2 was induced by the activation of nuclear factor (NF)-κB and, in turn, regulated ROBO1 transcription, thus functionally contributing to GC metastasis. Finally, miR-218 was found to suppress GC metastasis by simultaneously mediating multiple molecules in the POU2F2-oriented network. CONCLUSIONS: This study demonstrated that NF-κB and the SLIT2/ROBO1 interaction network with POU2F2 as the central part may exert critical effects on tumour metastasis. Blocking the activation of the POU2F2-oriented metastasis network using miR-218 precursors exemplified a promising approach that sheds light on new strategies for GC treatment.

A possible role of GDDR in the development of Helicobacter pylori-associated gastric cancer.

Helicobacter pylori (H. pylori) infection plays an important part in the development of gastric carcinoma. GDDR has been confirmed as a tumor suppressor gene in gastric tumorigenesis. However, the underlying mechanism of GDDR in H. pylori-induced carcinogenesis is not well known. The aim of this study is to investigate the clinicopathological significance and possible molecular mechanism of GDDR in gastric cancer associated with H. pylori. Western blot, real-time quantitative PCR (qRT-PCR), and immunohistochemistry were used to detect the expression level of GDDR with or without H. pylori infection. The function and possible related molecular mechanisms of GDDR were further explored in vitro and in vivo. The variability of GDDR expression appeared in the early stage of gastric carcinogenesis with positive H. pylori infection status. GDDR might inhibit the progression of normal gastric epithelial cells to cancer cells by suppressing NF-kappaB signaling pathway, which in turn could be regulated by H. pylori infection. Our results suggested, for the first time, that the gradual change in GDDR expression might not only be directly related to H. pylori infection but also be an early molecular event in the development of gastric carcinoma.

MiR-19b/20a/92a regulates the self-renewal and proliferation of gastric cancer stem cells.

Human gastric cancers contain a population of gastric cancer stem cells (GCSCs) that can undergo self-renewal and multipotent differentiation. GCSCs can be enriched with EpCAM+/CD44+ gastric cancer cells. However, the underlying mechanisms controlling the balance of GCSC self-renewal and differentiation remain to be explored. Because miRNAs can regulate cancer cell fates, we compared miRNA expression in tumorspheric cancer cells enriched with GCSCs and more differentiated cells. We found that the miR-17-92 cluster members miR-19b, miR-20a and miR-92a were gradually reduced during the differentiation of GCSCs. Therefore, we speculated that miR-17-92 members might regulate the self-renewal ability of GCSCs. By downregulating miR-19b, miR-20a and miR-92a in EpCAM+/CD44+ GCSCs, or overexpressing them in EpCAM-/CD44- non-GCSC populations, we found that miR-19b, miR-20a and miR-92a could sustain the self-renewal function of GCSCs. Furthermore, we found that miR-19b, miR-20a and miR-92a could also promote the proliferation of gastric cancer cells. miR-17-92 targeted the E2F1 and HIPK1 proteins, which suppressed Wnt-ß-catenin signaling. A real-time PCR analysis of miR-19b, miR-20a and miR-92a expression in 97 gastric cancer specimens suggested that miR-92a could be used as an independent prognostic factor in gastric cancer. This study showed that several members of the miR-17-92 cluster, miR-19b, miR-20a and miR-92a, might play important roles in the development of gastric cancer stem cells and that miR-92a has the potential to be used as a predictive prognostic marker in gastric cancer.

c-Myc-mediated epigenetic silencing of MicroRNA-101 contributes to dysregulation of multiple pathways in hepatocellular carcinoma.

UNLABELLED: The MYC oncogene is overexpressed in hepatocellular carcinoma (HCC) and has been associated with widespread microRNA (miRNA) repression; however, the underlying mechanisms are largely unknown. Here, we report that the c-Myc oncogenic transcription factor physically interacts with enhancer of zeste homolog 2 (EZH2), a core enzymatic unit of polycomb repressive complex 2 (PRC2). Furthermore, miR-101, an important tumor-suppressive miRNA in human hepatocarcinomas, is epigenetically repressed by PRC2 complex in a c-Myc-mediated manner. miR-101, in turn, inhibits the expression of two subunits of PRC2 (EZH2 and EED), thus creating a double-negative feedback loop that regulates the process of hepatocarcinogenesis. Restoration of miR-101 expression suppresses multiple malignant phenotypes of HCC cells by coordinate repression of a cohort of oncogenes, including STMN1, JUNB, and CXCR7, and further increases expression of endogenous miR-101 by inhibition of PRC2 activation. In addition, co-overexpression of c-Myc and EZH2 in HCC samples was closely associated with lower expression of miR-101 (P < 0.0001) and poorer prognosis of HCC patients (P < 0.01). CONCLUSIONS: c-Myc collaborates with EZH2-containing PRC2 complex in silencing tumor-suppressive miRNAs during hepatocarcinogenesis and provides promising therapeutic candidates for human HCC.

Mechanism study of peptide GMBP1 and its receptor GRP78 in modulating gastric cancer MDR by iTRAQ-based proteomic analysis.

BACKGROUND: Multidrug resistance (MDR) is a major obstacle to the treatment of gastric cancer (GC). Using a phage display approach, we previously obtained the peptide GMBP1, which specifically binds to the surface of MDR gastric cancer cells and is subsequently internalized. Furthermore, GMBP1 was shown to have the potential to reverse the MDR phenotype of gastric cancer cells, and GRP78 was identified as the receptor for this peptide. The present study aimed to investigate the mechanism of peptide GMBP1 and its receptor GRP78 in modulating gastric cancer MDR. METHODS: Fluorescence-activated cell sorting (FACS) and immunofluorescence staining were used to investigate the subcellular location and mechanism of GMBP1 internalization. iTRAQ was used to identify the MDR-associated downstream targets of GMBP1. Differentially expressed proteins were identified in GMBP1-treated compared to untreated SGC7901/ADR and SGC7901/VCR cells. GO and KEGG pathway analyses of the differentially expressed proteins revealed the interconnection of these proteins, the majority of which are involved in MDR. Two differentially expressed proteins were selected and validated by western blotting. RESULTS: GMBP1 and its receptor GRP78 were found to be localized in the cytoplasm of GC cells, and GRP78 can mediate the internalization of GMBP1 into MDR cells through the transferrin-related pathway. In total, 3,752 and 3,749 proteins were affected in GMBP1-treated SGC7901/ADR and SGC7901/VCR cells, respectively, involving 38 and 79 KEGG pathways. Two differentially expressed proteins, CTBP2 and EIF4E, were selected and validated by western blotting. CONCLUSION: This study explored the role and downstream mechanism of GMBP1 in GC MDR, providing insight into the role of endoplasmic reticulum stress protein GRP78 in the MDR of cancer cells.

Loss of vinculin and membrane-bound ß-catenin promotes metastasis and predicts poor prognosis in colorectal cancer.

BACKGROUND: Loss of cell-cell adhesion is important for the development of cancer invasion and metastasis. Vinculin, a key adhesion-related protein, can affect metastasis and prognosis in several tumours. Here, we determined the biological roles of vinculin in the metastasis of colorectal cancer (CRC) and evaluated its clinical significance as a potential disease biomarker. METHODS: The expression level of vinculin in CRC cell lines and tissues was measured using Real-Time PCR and western blotting. Moreover, vinculin function was analysed using Transwell assays and in vivo metastasis assays in gain- and loss-of-function experiments. Furthermore, the impact of vinculin together with membrane-bound ß-catenin on the prognosis of 228 CRC patients was investigated by immunohistochemistry. Additionally, the expression of epithelial-mesenchymal transition (EMT) indicators was verified by immunohistochemistry in CRC tissues obtained from these patients. RESULT: Vinculin expression was found to be significantly downregulated in highly metastatic CRC cell lines and metastatic tissues. Both in vitro and in vivo experiments showed that vinculin suppressed invasion, migration and metastasis in CRC cells and that this suppression could be attenuated by silencing ß-catenin. Moreover, the expression of vinculin and membrane-bound ß-catenin were positively correlated in CRC tissues, and lack of vinculin expression emerged as an independent prognostic factor in patients with CRC. Finally, the loss of vinculin and membrane-bound ß-catenin was associated with node metastasis, organ metastasis and expression of EMT indicators. CONCLUSION: Our results suggest that vinculin may play specific roles in the EMT and metastasis of CRC and that loss of vinculin could be used as a prognostic factor for CRC.

mTORC1 and mTORC2 play different roles in the functional survival of transplanted adipose-derived stromal cells in hind limb ischemic mice via regulating inflammation in vivo.

Poor cell survival severely limits the beneficial effects of stem cell therapy for peripheral arterial disease (PAD). This study was designed to investigate the role of mammalian target of rapamycin (mTOR) in the survival and therapeutic function of transplanted murine adipose-derived stromal cells (mADSCs) in a murine PAD model. mADSCs (1.0 × 10(7)) were isolated from dual-reporter firefly luciferase and enhanced green fluorescent protein-positive transgenic mice, intramuscularly implanted into the hind limb of C57BL/6 mice after femoral artery ligation/excision, and monitored using noninvasive bioluminescence imaging (BLI). Although engrafted mADSCs produced antiapoptotic/proangiogenic effects in vivo by modulating the inflammatory and angiogenic cytokine response involving the mTOR pathway, longitudinal BLI revealed progressive death of post-transplant mADSCs within ~4 weeks in the ischemic hind limb. Selectively targeting mTOR complex-1 (mTORC1) using low-dose rapamycin treatment with mADSCs attenuated proinflammatory cytokines (interleukin [IL]-1ß and tumor necrosis factor-alpha [TNF-α]) expression and neutrophil/macrophage infiltration, which overtly promoted mADSCs viability and antiapoptotic/proangiogenic efficacy in vivo. However, targeting dual mTORC1/mTORC2 using PP242 or high-dose rapamycin caused IL-1ß/TNF-α upregulation and anti-inflammatory IL-10, IL-6, and vascular endothelial growth factor/vascular endothelial growth factor receptor 2 downregulation, undermining the survival and antiapoptotic/proangiogenic action of mADSCs in vivo. Furthermore, low-dose rapamycin abrogated TNF-α secretion by mADSCs and rescued the cells from hypoxia/reoxygenation-induced death in vitro, while PP242 or high-dose rapamycin exerted proinflammatory effects and promoted cell death. In conclusion, mTORC1 and mTORC2 may differentially regulate inflammation and affect transplanted mADSCs' functional survival in ischemic hind limb. These findings uncover that mTOR may evolve into a promising candidate for mechanism-driven approaches to facilitate the translation of cell-based PAD therapy.

Single-cell analysis reveals the COL11A1+ fibroblasts are cancer-specific fibroblasts that promote tumor progression.

Background: Cancer-associated fibroblasts (CAFs) promote tumor progression through extracellular matrix (ECM) remodeling and extensive communication with other cells in tumor microenvironment. However, most CAF-targeting strategies failed in clinical trials due to the heterogeneity of CAFs. Hence, we aimed to identify the cluster of tumor-promoting CAFs, elucidate their function and determine their specific membrane markers to ensure precise targeting. Methods: We integrated multiple single-cell RNA sequencing (scRNA-seq) datasets across different tumors and adjacent normal tissues to identify the tumor-promoting CAF cluster. We analyzed the origin of these CAFs by pseudotime analysis, and tried to elucidate the function of these CAFs by gene regulatory network analysis and cell-cell communication analysis. We also performed cell-type deconvolution analysis to examine the association between the proportion of these CAFs and patients' prognosis in TCGA cancer cohorts, and validated that through IHC staining in clinical tumor tissues. In addition, we analyzed the membrane molecules in different fibroblast clusters, trying to identify the membrane molecules that were specifically expressed on these CAFs. Results: We found that COL11A1+ fibroblasts specifically exist in tumor tissues but not in normal tissues and named them cancer-specific fibroblasts (CSFs). We revealed that these CSFs were transformed from normal fibroblasts. CSFs represented a more activated CAF cluster and may promote tumor progression through the regulation on ECM remodeling and antitumor immune responses. High CSF proportion was associated with poor prognosis in bladder cancer (BCa) and lung adenocarcinoma (LUAD), and IHC staining of COL11A1 confirmed their specific expression in tumor stroma in clinical BCa samples. We also identified that CSFs specifically express the membrane molecules LRRC15, ITGA11, SPHK1 and FAP, which could distinguish CSFs from other fibroblasts. Conclusion: We identified that CSFs is a tumor specific cluster of fibroblasts, which are in active state, may promote tumor progression through the regulation on ECM remodeling and antitumor immune responses. Membrane molecules LRRC15, ITGA11, SPHK1 and FAP could be used as therapeutic targets for CSF-targeting cancer treatment.

Self-Assembly of Small Organic Molecules into Luminophores for Cancer Theranostic Applications.

Self-assembled biomaterials have been widely explored for real-time fluorescence imaging, imaging-guided surgery, and targeted therapy for tumors, etc. In particular, small molecule-based self-assembly has been established as a reliable strategy for cancer theranostics due to the merits of small-sized molecules, multiple functions, and ease of synthesis and modification. In this review, we first briefly introduce the supramolecular chemistry of small organic molecules in cancer theranostics. Then, we summarize and discuss advanced small molecule-based self-assembly for cancer theranostics based on three types, including peptides, amphiphilic molecules, and aggregation-induced emission luminogens. Finally, we conclude with a perspective on future developments of small molecule-based self-assembled biomaterials integrating diagnosis and therapy for biomedical applications. These applications highlight the opportunities arising from the rational design of small organic molecules with self-assembly properties for precision medicine.

MiR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.

Background: Gastric cancer (GC) is one of the most malignant and lethal cancers worldwide. Multiple microRNAs (miRNAs) have been identified as key regulators in the progression of GC. However, the underlying pathogenesis that miRNAs govern GC malignancy remains uncertain. Here, we identified a novel miR-585-5p as a key regulator in GC development. Methods: The expression of miR-585-5p in the context of GC tissue was detected by in situ hybridization for GC tissue microarray and assessed by H-scoring. The gain- and loss-of-function analyses comprised of Cell Counting Kit-8 assay and Transwell invasion and migration assay. The expression of downstream microphthalmia-associated transcription factor (MITF), cyclic AMP-responsive element-binding protein 1 (CREB1) and mitogen-activated protein kinase 1 (MAPK1) were examined by Immunohistochemistry, quantitative real-time PCR and western blot. The direct regulation between miR-585-5p and MITF/CREB1/MAPK1 were predicted by bioinformatic analysis and screened by luciferase reporter assay. The direct transcriptional activation of CREB1 on MITF was verified by luciferase reporter assay, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSAs). The interaction between MAPK1 and MITF was confirmed by co-immunoprecipitation (Co-IP) and immunofluorescent double-labelled staining. Results: MiR-585-5p is progressively downregulated in GC tissues and low miR-585-5p levels were strongly associated with poor clinical outcomes. Further gain- and loss-of-function analyses showed that miR-585-5p possesses strong anti-proliferative and anti-metastatic capacities in GC. Follow-up studies indicated that miR-585-5p targets the downstream molecules CREB1 and MAPK1 to regulate the transcriptional and post-translational regulation of MITF, respectively, thus controlling its expression and cancer-promoting activity. MiR-585-5p directly and negatively regulates MITF together with CREB1 and MAPK1. According to bioinformatic analysis, promotor reporter gene assays, ChIP and EMSAs, CREB1 binds to the promotor region to enhance transcriptional expression of MITF. Co-IP and immunofluorescent double-labelled staining confirmed interaction between MAPK1 and MITF. Protein immunoprecipitation revealed that MAPK1 enhances MITF activity via phosphorylation (Ser73). MiR-585-5p can not only inhibit MITF expression directly, but also hinder MITF expression and pro-cancerous activity in a CREB1-/MAPK1-dependent manner indirectly. Conclusions: In conclusion, this study uncovered miR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.

Targeting the miR-34a/LRPPRC/MDR1 axis collapse the chemoresistance in P53 inactive colorectal cancer.

P53 mutation is an important cause of chemoresistance in colorectal cancer (CRC). The investigation and identification of the downstream targets and underlying molecular mechanism of chemoresistance induced by P53 abnormalities are therefore of great clinical significance. In this study, we demonstrated and reported for the first time that leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) is a key functional downstream factor and therapeutic target for P53 mutation-induced chemoresistance. Due to its RNA binding function, LRPPRC specifically bound to the mRNA of multidrug resistance 1 (MDR1), increasing MDR1 mRNA stability and protein expression. In normal cells, P53 induced by chemotherapy inhibited the expression of LRPPRC via miR-34a and in turn reduced the expression of MDR1. However, chemotherapy-induced P53/miR-34a/LRPPRC/MDR1 signalling pathway activation was lost when P53 was mutated. Additionally, the accumulated LRPPRC and MDR1 promoted drug resistance. Most importantly, gossypol-acetic acid (GAA) was recently reported by our team as the first specific inhibitor of LRPPRC. In CRC cells with P53 mutation, GAA effectively induced degradation of the LRPPRC protein and reduced chemoresistance. Both in vivo and in vitro experiments revealed that combination chemotherapy with GAA and 5-fluorouracil (5FU) yielded improved treatment outcomes. In this study, we reported a novel mechanism and target related to P53-induced drug resistance and provided corresponding interventional strategies for the precision treatment of CRC.

QKI degradation in macrophage by RNF6 protects mice from MRSA infection via enhancing PI3K p110ß dependent autophagy.

BACKGROUND: Sepsis is a fatal condition commonly caused by Methicillin-resistant Staphylococcus aureus (MRSA) with a high death rate. Macrophages can protect the host from various microbial pathogens by recognizing and eliminating them. Earlier we found that Quaking (QKI), an RNA binding protein (RBP), was involved in differentiation and polarization of macrophages. However, the role of QKI in sepsis caused by pathogenic microbes, specifically MRSA, is unclear. This study aimed to investigate the role of QKI in regulation of host-pathogen interaction in MRSA-induced sepsis and explored the underlying mechanisms. METHODS: Transmission electron microscope and immunofluorescence were used to observe the autophagy level in macrophages. Real-time PCR and western blot were used to analyzed the expression of mRNA and protein respectively. The potential protein interaction was analyzed by iTRAQ mass spectrometry and Immunoprecipitation. RNA fluorescence in situ hybridization, dual-luciferase reporter assay and RNA immunoprecipitation were used to explore the mechanism of QKI regulating mRNA of PI3K-p110ß. RESULTS: The mRNA level of QKI was aberrantly decreased in monocytes and PBMCs of septic patients with the increasing level of plasma procalcitonin (PCT). Then the mice with myeloid specific knockout of QKI was challenged with MRSA or Cecal Ligation and Puncture (CLP). Mice in these two models displayed higher survival rates and lower bacterial loads. Mechanistically, QKI deletion promoted phagocytosis and autophagic degradation of MRSA via activating p110ß (a member of Class IA phosphoinositide 3-kinases) mediated autophagic response. QKI expression in macrophages led to the sequestration of p110ß in mRNA processing (P) bodies and translational repression. Upon infection, the direct interaction of RNF6, a RING-type E3 ligase, mediated QKI ubiquitination degradation and facilitated PI3K-p110ß related autophagic removal of pathogen. The administration of nanoparticles with QKI specific siRNA significantly protected mice from MRSA infection. CONCLUSIONS: This study disclosed the novel function of QKI in the P body mRNA regulation during infection. QKI degradation in macrophage by RNF6 protects mice from MRSA infection via enhancing PI3K-p110ß dependent autophagy. It suggested that QKI may serve as a potential theranostic marker in MRSA-induced sepsis.

Notch-mediated lactate metabolism regulates MDSC development through the Hes1/MCT2/c-Jun axis.

Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) play critical roles in tumorigenesis. However, the mechanisms underlying MDSC and TAM development and function remain unclear. In this study, we find that myeloid-specific activation of Notch/RBP-J signaling downregulates lactate transporter MCT2 transcription via its downstream molecule Hes1, leading to reduced intracellular lactate levels, blunted granulocytic MDSC (G-MDSC) differentiation, and enhanced TAM maturation. We identify c-Jun as a novel intracellular sensor of lactate in myeloid cells using liquid-chromatography-mass spectrometry (LC-MS) followed by CRISPR-Cas9-mediated gene disruption. Meanwhile, lactate interacts with c-Jun to protect from FBW7 ubiquitin-ligase-mediated degradation. Activation of Notch signaling and blockade of lactate import repress tumor progression by remodeling myeloid development. Consistently, the relationship between the Notch-MCT2/lactate-c-Jun axis in myeloid cells and tumorigenesis is also confirmed in clinical lung cancer biopsies. Taken together, our current study shows that lactate metabolism regulated by activated Notch signaling might participate in MDSC differentiation and TAM maturation.

Expression of T-cell immunoglobulin- and mucin-domain-containing molecules-1 and -3 (Tim-1 and Tim-3) in Helicobacter pylori infection.

BACKGROUND: Th immune response plays an important role in Helicobacter pylori (H. pylori) infection. Tim-1 and Tim-3 are expressed on terminally differentiated Th2 and Th1 cells, respectively, and participate in the regulation of Th immune response. Until now, the role of Tim in H. pylori infection remains unclear. MATERIALS AND METHODS: (1) Lymphocytes isolated from the spleen of BALB/c mice were co-cultured with different concentrations of viable H. pylori. Alternatively, mice were challenged by viable H. pylori to set up the H. pylori infection model. (2) The expression of Tim-1 and Tim-3 on mRNA level in lymphocytes or spleen of mice was determined by RT-PCR. The percentage of Tim-3-positive cells was determined by flow cytometric analysis. The production of cytokine in supernatants was measured by standard sandwich cytokine ELISA. RESULTS: (1) Co-culture: At 12 hours, there was markedly decreased production of Tim-1 and increased production of Tim-3 in lymphocytes co-cultured with H. pylori compared with normal control. The change of Th2 cytokine had the similar tendency as that of Tim-1 expression; alternatively, the change of Th1 cytokine had the similar tendency as that of Tim-3 expression. (2) INFECTION: Tim-1 expression was declined in infected mice compared with control group; in the contrast, Tim-3 expression was increased. Furthermore, the expression of Tim-1 and Tim-3 mRNA in spleen was significantly positively correlated with the level of Th2 and Th1 cytokine in gastric homogenized supernatant, respectively. CONCLUSION: H. pylori could inhibit the differentiation of T lymphocytes toward Th2 cells, promote the Th1 cell differentiation, and induce Th1-biased immune response. The expression of Tim-1 and Tim-3 could reflect Th2 and Th1 immune response, respectively, which provide evidence for the prevention and treatment of H. pylori infection and correlation diseases through regulation of Tim-1 and Tim-3.

Microsurgical Skills of Establishing Permanent Jugular Vein Cannulation in Rats for Serial Blood Sampling of Orally Administered Drug.

Blood sampling in small laboratory animals is necessary for pharmaceutical lead optimization but can cause great harm and stress to experimental animals, which could potentially affect results. The jugular vein cannulation (JVC) in rats is a widely used model for repeated blood collection but requires adequate training of surgery skills and animal care. This article details the microsurgical procedures for establishing and maintaining a permanent JVC rat model with specific focus on the placement and sealing of the jugular cannula. The importance of monitoring physiological (e.g., body weight, food, and water intake) and hematological parameters, was highlighted with results presented for 6 days post-surgery during the rat's recovery. The drug-plasma concentration-time profile of orally administered natural phenol ellagic acid was determined in the JVC rat model.

Chaperone-mediated autophagy controls the turnover of E3 ubiquitin ligase MARCHF5 and regulates mitochondrial dynamics.

As a highly dynamic organelle, mitochondria undergo constant fission and fusion to change their morphology and function, coping with various stress conditions. Loss of the balance between fission and fusion leads to impaired mitochondria function, which plays a critical role in the pathogenesis of Parkinson disease (PD). Yet the mechanisms behind mitochondria dynamics regulation remain to be fully illustrated. Chaperone-mediated autophagy (CMA) is a lysosome-dependent process that selectively degrades proteins to maintain cellular proteostasis. In this study, we demonstrated that MARCHF5, an E3 ubiquitin ligase required for mitochondria fission, is a CMA substrate. MARCHF5 interacted with key CMA regulators and was degraded by lysosomes. Severe oxidative stress compromised CMA activity and stabilized MARCHF5, which facilitated DNM1L translocation and led to excessive fission. Increase of CMA activity promoted MARCHF5 turnover, attenuated DNM1L translocation, and reduced mitochondria fragmentation, which alleviated mitochondrial dysfunction under oxidative stress. Furthermore, we showed that conditional expression of LAMP2A, the key CMA regulator, in dopaminergic (DA) neurons helped maintain mitochondria morphology and protected DA neuronal viability in a rodent PD model. Our work uncovers a critical role of CMA in maintaining proper mitochondria dynamics, and loss of this regulatory control may occur in PD and underlie its pathogenic process.Abbreviations: CMA: chaperone-mediated autophagy; DA: dopaminergic; DNM1L: dynamin 1 like; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; HSPA8: heat shock protein family A (Hsp70) member 8; LAMP2A: lysosomal associated membrane protein 2A; MARCHF5: membrane-associated ring-CH-type finger 5; MMP: mitochondria membrane potential; OCR: oxygen consumption rate; 6-OHDA: 6-hydroxydopamine; PD: Parkinson disease; SNc: substantia nigra pars compacta; TEM: transmission electron microscopy; TH: tyrosine hydroxylase; TMRE: tetramethylrhodamine ethyl ester perchlorate; WT: wild type.

An MD2-perturbing peptide has therapeutic effects in rodent and rhesus monkey models of stroke.

Studies have failed to translate more than 1000 experimental treatments from bench to bedside, leaving stroke as the second leading cause of death in the world. Thrombolysis within 4.5 hours is the recommended therapy for stroke and cannot be performed until neuroimaging is used to distinguish ischemic stroke from hemorrhagic stroke. Therefore, finding a common and critical therapeutic target for both ischemic and hemorrhagic stroke is appealing. Here, we report that the expression of myeloid differentiation protein 2 (MD2), which is traditionally regarded to be expressed only in microglia in the normal brain, was markedly increased in cortical neurons after stroke. We synthesized a small peptide, Trans-trans-activating (Tat)-cold-inducible RNA binding protein (Tat-CIRP), which perturbed the function of MD2 and strongly protected neurons against excitotoxic injury in vitro. In addition, systemic administration of Tat-CIRP or genetic deletion of MD2 induced robust neuroprotection against ischemic and hemorrhagic stroke in mice. Tat-CIRP reduced the brain infarct volume and preserved neurological function in rhesus monkeys 30 days after ischemic stroke. Tat-CIRP efficiently crossed the blood-brain barrier and showed a wide therapeutic index for stroke because no toxicity was detected when high doses were administered to the mice. Furthermore, we demonstrated that MD2 elicited neuronal apoptosis and necroptosis via a TLR4-independent, Sam68-related cascade. In summary, Tat-CIRP provides robust neuroprotection against stroke in rodents and gyrencephalic nonhuman primates. Further efforts should be made to translate these findings to treat both ischemic and hemorrhagic stroke in patients.

SOD3 Is Secreted by Adipocytes and Mitigates High-Fat Diet-Induced Obesity, Inflammation, and Insulin Resistance.

Aims: To study the expression and regulatory role of SOD3 in adipocytes and adipose tissue. Results: SOD3 expression was determined in various tissues of adult C57BL/6J mice, human adipose tissue and epididymal adipose tissue, subcutaneous adipose tissue and brown adipose tissue of high-fat diet (HFD)-induced obese mice. SOD3 expression and release were evaluated in adipocytes differentiated from primary human preadipocytes and murine bone marrow-derived mesenchymal stem cells (BM-MSCs). The regulatory role for SOD3 was determined by SOD3 lentivirus knockdown in human adipocytes and global sod3 knockout (KO) mice. SOD3 was expressed at high levels in white adipose tissue, and adipocytes were the main cells expressing SOD3 in adipose tissue. SOD3 expression was significantly elevated in adipose tissue of HFD-fed mice. Moreover, SOD3 expression and release were markedly increased in differentiated human adipocytes and adipocytes differentiated from mouse BM-MSCs compared with undifferentiated cells. In addition, SOD3 silencing in human adipocytes increased expression of genes involved in lipid metabolic pathways such as PPARγ and SREBP1c and promoted the accumulation of triglycerides. Finally, global sod3 KO mice were more obese and insulin resistant with enlarged adipose tissue and increased triglyceride accumulation. Innovation: Our data showed that SOD3 is secreted from adipocytes and regulates lipid metabolism in adipose tissue. This important discovery may open up new avenues of research for the cytoprotective role of SOD3 in obesity and its associated metabolic disorders. Conclusion: SOD3 is a protective factor secreted by adipocytes in response to HFD-induced obesity and regulates adipose tissue lipid metabolism.