Comprehensive analysis of zinc and ring finger 3 in prognostic value and pan-cancer immunity.

Zinc and ring finger 3 (ZNRF3) is a negative suppressor of Wnt signal and newly identified as an important regulator in tumorigenesis and development. However, the pan-cancer analysis of ZNRF3 has not been reported. We found that ZNRF3 was significantly decreased in six tumors including CESC, KIRP, KIRC, SKCM, OV, and ACC, but increased in twelve tumors, namely LGG, ESCA, STES, COAD, STAD, LUSC, LIHC, THCA, READ, PAAD, TGCT, and LAML. Clinical outcomes of cancer patients were closely related to ZNRF3 expression in ESCA, GBM, KIRC, LUAD, STAD, UCEC, LGG, and SARC. The highest genetic alteration frequency of ZNRF3 occurred in ACC. Abnormal expression of ZNRF3 could be attributed to the differences of copy number variation (CNV) and DNA methylation as well as ZNRF3-interacting proteins. Besides, ZNRF3 were strongly associated with tumor heterogeneity, tumor stemness, immune score, stromal score and ESTIMATE score in certain cancers. In terms of immune cell infiltration, ZNRF3 was positively correlated to infiltration of cancer-associated fibroblasts in CESC, HNSC, OV, PAAD, PRAD, and THYM, but negatively associated with infiltration of CD8 T cells in HNSC, KIRC, KIRP and THYM. Moreover, ZNRF3 expression was correlated with most immune checkpoint genes in SARC, LUSC, LUAD, PRAD, THCA, UVM, TGCT, and OV, and associated with overwhelming majority of immunoregulatory genes in almost all cancers. Most RNA modification genes were also remarkably related to ZNRF3 level in KIRP, LUAD, LUSC, THYM, UVM, PRAD, and UCEC, indicating that ZNRF3 might have an important effect on cancer epigenetic regulation. Finally, we verified the expression and role of ZNRF3 in clinical specimens and cell lines of renal cancer and liver cancer. This study provides a comprehensive pan-cancer analysis of ZNRF3 and reveals the complexity of its carcinogenic effect.

Exosomal circRNAs in the plasma serve as novel biomarkers for IPF diagnosis and progression prediction.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is a type of chronic interstitial pneumonia, often fatal, with elusive causes and a bleak prognosis. Its treatment options are limited and largely ineffective. Early detection and precise diagnosis are pivotal in managing the disease effectively and enhancing patient survival rates. Recently, the quest for trustworthy biomarkers for IPF has gained momentum. Notably, emerging studies indicate that circular RNAs (circRNAs) found in exosomes may hold significant potential as valuable diagnostic markers. METHODS: In this study, we initially explored the expression profile of circRNAs in exosomes sourced from the blood of IPF patients and healthy volunteers, employing a human circRNA microarray. We then utilized RT-qPCR to corroborate the dysregulated circRNAs identified by the microarray during the training phase. Next, the circRNAs that displayed a significant increase during the training phase were selected for further validation in a larger cohort encompassing 113 IPF patients and 76 healthy volunteers. Ultimately, the expression level and function of hsa_circ_0044226 were substantiated through a series of in vivo and in vitro experiments. RESULTS: Utilizing a human circRNA microarray, we identified 11 dysregulated circRNAs in the exosomes derived from the blood of IPF patients and control volunteers. Subsequent RT-qPCR analysis revealed significant increases in three circRNAs (hsa_circ_0044226, hsa_circ_0004099, hsa_circ_0008898) within the IPF patients. Notably, hsa_circ_0044226 was markedly elevated in patients experiencing acute exacerbation of IPF (AE-IPF) compared to those with stable IPF (S-IPF). Additionally, an upregulation of hsa_circ_0044226 was observed in the blood exosomes derived from a bleomycin-induced IPF mouse model. CONCLUSION: The expression levels of hsa_circ_0044226, hsa_circ_0004099, and hsa_circ_0008898 in plasma exosomes introduce a new paradigm of biomarkers for the diagnosis and progression of IPF.

Transcriptomics-proteomics Integration reveals alternative polyadenylation driving inflammation-related protein translation in patients with diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is a complex disease involving the upregulation of many inflammation-related proteins. Alternative polyadenylation (APA), a crucial post-transcriptional regulatory mechanism, has been proven to play vital roles in many inflammatory diseases. However, it is largely unknown whether and how APA exerts function in DN. METHODS: We performed transcriptomics and proteomics analysis of glomeruli samples isolated from 50 biopsy-proven DN patients and 25 control subjects. DaPars and QAPA algorithms were adopted to identify APA events from RNA-seq data. The qRT-PCR analysis was conducted to verify 3'UTR length alteration. Short and long 3'UTRs isoforms were also overexpressed in podocytes under hyperglycemia condition for examining protein expression. RESULTS: We detected transcriptome-wide 3'UTR APA events in DN, and found that APA-mediated 3'UTR lengthening of genes (APA genes) increased their expression at protein but not mRNA level. Increased protein level of 3'UTR lengthening gene was validated in podocytes under hyperglycemia condition. Pathway enrichment analysis showed that APA genes were enriched in inflammation-related biological processes including endoplasmic reticulum stress pathways, NF-κB signaling and autophagy. Further bioinformatics analysis demonstrated that 3'UTR APA of genes probably altered the binding sites for RNA-binding proteins, thus enhancing protein translation. CONCLUSION: This study revealed for the first time that 3'UTR lengthening of APA genes contributed to the progression of DN by elevating the translation of corresponding proteins, providing new insight and a rich resource for investigating DN mechanisms.

Pyruvate kinase M2 mediates fibroblast proliferation to promote tubular epithelial cell survival in acute kidney injury.

Acute kidney injury (AKI) is a devastating condition with high morbidity and mortality rates. The pathological features of AKI are tubular injury, infiltration of inflammatory cells, and impaired vascular integrity. Pyruvate kinase is the final rate-limiting enzyme in the glycolysis pathway. We previously showed that pyruvate kinase M2 (PKM2) plays an important role in regulating the glycolytic reprogramming of fibroblasts in renal interstitial fibrosis. The present study aimed to determine the role of PKM2 in fibroblast activation during the pathogenesis of AKI. We found increased numbers of S100A4 positive cells expressing PKM2 in renal tissues from mice with AKI induced via folic acid or ischemia/reperfusion (I/R). The loss of PKM2 in fibroblasts impaired fibroblast proliferation and promoted tubular epithelial cell death including apoptosis, necroptosis, and ferroptosis. Mechanistically, fibroblasts produced less hepatocyte growth factor (HGF) in response to a loss of PKM2. Moreover, in two AKI mouse models, fibroblast-specific deletion of PKM2 blocked HGF signal activation and aggravated AKI after it was induced in mice via ischemia or folic acid. Fibroblast proliferation mediated by PKM2 elicits pro-survival signals that repress tubular cell death and may help to prevent AKI progression. Fibroblast activation mediated by PKM2 in AKI suggests that targeting PKM2 expression could be a novel strategy for treating AKI.

3'-Terminal 2'-O-methylation of lung cancer miR-21-5p enhances its stability and association with Argonaute 2.

Methylation of miRNAs at the 2'-hydroxyl group on the ribose at 3'-end (2'-O-methylation, 2'Ome) is critical for miRNA function in plants and Drosophila. Whether this methylation phenomenon exists for mammalian miRNA remains unknown. Through LC-MS/MS analysis, we discover that majority of miR-21-5p isolated from human non-small cell lung cancer (NSCLC) tissue possesses 3'-terminal 2'Ome. Predominant 3'-terminal 2'Ome of miR-21-5p in cancer tissue is confirmed by qRT-PCR and northern blot after oxidation/ß-elimination procedure. Cancerous and the paired non-cancerous lung tissue miRNAs display different pattern of 3'-terminal 2'Ome. We further identify HENMT1 as the methyltransferase responsible for 3'-terminal 2'Ome of mammalian miRNAs. Compared to non-methylated miR-21-5p, methylated miR-21-5p is more resistant to digestion by 3'→5' exoribonuclease polyribonucleotide nucleotidyltransferase 1 (PNPT1) and has higher affinity to Argonaute-2, which may contribute to its higher stability and stronger inhibition on programmed cell death protein 4 (PDCD4) translation, respectively. Our findings reveal HENMT1-mediated 3'-terminal 2'Ome of mammalian miRNAs and highlight its role in enhancing miRNA's stability and function.

Injured liver-released miRNA-122 elicits acute pulmonary inflammation via activating alveolar macrophage TLR7 signaling pathway.

Hepatic injury is often accompanied by pulmonary inflammation and tissue damage, but the underlying mechanism is not fully elucidated. Here we identify hepatic miR-122 as a mediator of pulmonary inflammation induced by various liver injuries. Analyses of acute and chronic liver injury mouse models confirm that liver dysfunction can cause pulmonary inflammation and tissue damage. Injured livers release large amounts of miR-122 in an exosome-independent manner into the circulation compared with normal livers. Circulating miR-122 is then preferentially transported to mouse lungs and taken up by alveolar macrophages, in which it binds Toll-like receptor 7 (TLR7) and activates inflammatory responses. Depleting miR-122 in mouse liver or plasma largely abolishes liver injury-induced pulmonary inflammation and tissue damage. Furthermore, alveolar macrophage activation by miR-122 is blocked by mutating the TLR7-binding GU-rich sequence on miR-122 or knocking out macrophage TLR7. Our findings reveal a causative role of hepatic miR-122 in liver injury-induced pulmonary dysfunction.

High-throughput sequencing provides insights into oral microbiota dysbiosis in association with inflammatory bowel disease.

Although the prevalence of inflammatory bowel disease (IBD) has been increasing worldwide, the etiology remains elusive. Investigating oral microbiota dysbiosis is essential to understanding IBD pathogenesis. Our study evaluated variations in salivary microbiota and identified potential associations with IBD. The saliva microbiota of 22 IBD patients and 8 healthy controls (HCs) was determined using 16S ribosomal RNA (rRNA) gene sequencing and analyzed using QIIME2. A distinct saliva microbiota dysbiosis in IBD, characterized by alterations in microbiota biodiversity and composition, was identified. Saccharibacteria (TM7), Absconditabacteria (SR1), Leptotrichia, Prevotella, Bulleidia, and Atopobium, some of which are oral biofilm-forming bacteria, were significantly increased. Moreover, levels of inflammatory cytokines associated with IBD were elevated and positively correlated with TM7 and SR1. Functional variations include down-regulation of genetic information processing, while up-regulation of carbohydrate metabolism and protein processing in the endoplasmic reticulum in IBD. Our data implicate salivary microbiota dysbiosis involving in IBD pathogenesis.

Podocytes present antigen to activate specific T cell immune responses in inflammatory renal disease.

Infiltration of activated T cells into renal tissue plays an essential role in inflammatory nephropathy. However, the mechanism enabling the renal recruitment and activation of T cells remains elusive. Here we report that inflammatory cytokine-promoted antigen presentation by podocytes is a key for recruiting and activating specific T cells. Our results showed that diabetes-associated inflammatory cytokines IFNγ and IL-17 all upregulated expression of MHC-I, MHC-II, CD80 and CD86 on the podocyte surface. Both IFNγ and IL-17 stimulated the uptake and processing of ovalbumin (OVA) by mouse podocytes, resulting in presentation of OVA antigen peptide on the cell surface. OVA antigen presentation by podocytes was also validated using human podocytes. Furthermore, OVA antigen-presenting mouse podocytes were able to activate OT-I mouse T cell proliferation and inflammatory cytokine secretion, which in turn caused podocyte injury and apoptosis. Finally, OT-I mice subjected to direct renal injection of OVA plus IFNγ/IL-17 but not OVA alone exhibited OVA antigen presentation by podocytes and developed nephropathy in 4 weeks. In conclusion, antigen presentation by podocytes under inflammatory conditions plays an important role in activating T cell immune responses and facilitating immune-mediated glomerular disease development. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tubule-derived lactate is required for fibroblast activation in acute kidney injury.

Acute kidney injury (AKI) is a highly prevalent medical syndrome associated with high mortality and morbidity. Several types of cells, including epithelial cells, vascular endothelial cells, pericytes, and macrophages, participate in the development of AKI. Recently, renal fibroblasts were found to play an important role in the regulation of tubular injury, repair, and recovery after AKI. However, the mechanisms underlying fibroblast activation and proliferation during the progression of AKI remain unclear. In the present study, we found many activated myofibroblasts located in the renal interstitium with an abundance of extracellular matrix deposition following folic acid-induced AKI. The proliferative pattern of tubular epithelial cells and interstitial cells following acute injury was different, indicating that the proliferation of fibroblasts followed the proliferation of tubular epithelial cells. Furthermore, we observed that proliferative tubular epithelial cells preferred aerobic glycolysis as the dominating metabolic pathway in the progression of AKI. Lactate generated from injured tubules was taken up by interstitial fibroblasts in the later stages of AKI, which induced fibroblast activation and proliferation in vitro. Early inhibition of lactate production in tubules by glycolytic inhibitors suppressed fibroblast activation after folic acid-induced injury. Collectively, these results support the important role of fibroblasts in the development of AKI and suggest that lactate produced by glycolysis in tubular epithelial cells is a novel regulator of fibroblast activation and proliferation.

Tuberous sclerosis 1 (Tsc1) mediated mTORC1 activation promotes glycolysis in tubular epithelial cells in kidney fibrosis.

Energy reprogramming to glycolysis is closely associated with the development of chronic kidney disease. As an important negative regulatory factor of the mammalian target of rapamycin complex 1 (mTORC1) signal, tuberous sclerosis complex 1 (Tsc1) is also a key regulatory point of glycolysis. Here, we investigated whether Tsc1 could mediate the progression of kidney interstitial fibrosis by regulating glycolysis in proximal tubular epithelial cells. We induced mTORC1 signal activation in tubular epithelial cells in kidneys with fibrosis via unilateral ureteral occlusion. This resulted in increased tubular epithelial cell proliferation and glycolytic enzyme upregulation. Prior incubation with rapamycin inhibited mTORC1 activation and abolished the enhanced glycolysis and tubular epithelial cell proliferation. Furthermore, knockdown of Tsc1 expression promoted glycolysis in the rat kidney epithelial cell line NRK-52E. Specific deletion of Tsc1 in the proximal tubules of mice resulted in enlarged kidneys characterized by a high proportion of proliferative tubular epithelial cells, dilated tubules with cyst formation, and a large area of interstitial fibrosis in conjunction with elevated glycolysis. Treatment of the mice with the glycolysis inhibitor 2-deoxyglucose notably ameliorated tubular epithelial cell proliferation, cystogenesis, and kidney fibrosis. Thus, our findings suggest that Tsc1-associated mTORC1 signaling mediates the progression of kidney interstitial fibrosis by regulating glycolysis in proximal tubular epithelial cells.

The feedback loop between miR-21, PDCD4 and AP-1 functions as a driving force for renal fibrogenesis.

Renal fibrosis is a final common pathway of chronic kidney disease. Sustained activation of fibroblasts is considered to play a key role in perpetuating renal fibrosis but the driving force in the perpetuation stage is only partially understood. To date, some investigations have specifically identified overexpression of microRNA 21 (miR-21) in the progression of kidney fibrosis. Nevertheless, the precise role of miR-21 in fibroblast activation remains largely unknown. In this study, we found that miR-21 was significantly upregulated in activated fibroblasts and that it maintained itself at constant high levels by employing an auto-regulatory loop between miR-21, PDCD4 and AP-1. Persistently upregulated miR-21 suppressed protein expression of Smad7 and, eventually, enhanced the TGF-ß1/Smad pathway to promote fibroblast activation. More importantly, we found miR-21 sequestration with miR-21 antagomir or AP-1 inhibitors attenuated unilateral ureteral obstruction (UUO)-induced renal fibrosis. miR-21-knockout mice also suffered far less interstitial fibrosis in response to kidney injury. Altogether, these data suggest that miR-21 is a main driving force of fibroblast activation and keeps its high expression level by employing a double negative autoregulatory loop. Targeting this aberrantly activated feedback loop may provide new therapeutic strategy in treating fibrotic kidneys.

Direct quantification of 3' terminal 2'-O-methylation of small RNAs by RT-qPCR.

Modification of nucleotides significantly increases the diversity of functional nucleic acids. As one of the most common modifications of RNAs, methylation of the 2'-hydroxyl-group of ribonucleotides (2'-O-methylation) has been found in various RNAs in eukaryotes. However, due to the lack of an efficient method for quantifying small RNA 3' terminal 2'-O-methylation, it is difficult to monitor the dynamic change of 3' terminal 2'-O-methylation during various biological processes. Capitalizing on the finding that 3' terminal RNA 2'-O-methylation can inhibit the activity of poly(A) polymerase, an enzyme that can add the poly(A)-tail to RNA, we develop a method by which the 2'-O-methylation level of small RNAs, such as microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), can be directly quantified based on the poly(A)-tailed RT-qPCR technique. With this method, we successfully determine the 2'-O-methylation level of miRNAs in Arabidopsis thaliana and mouse lung tissue, piRNA in human seminal plasma, and monitor the alteration of miRNA 2'-O-methylation in Drosophila Schneider 2 cells after knockdown of Drosophila methyltransferase protein Hua enhancer 1 (DmHen-1).

UCP2-dependent improvement of mitochondrial dynamics protects against acute kidney injury.

Acute kidney injury (AKI) is a public health concern, with high morbidity and mortality rates in hospitalized patients and because survivors have an increased risk of progression to chronic kidney disease. Mitochondrial damage is the critical driver of AKI-associated dysfunction and loss of tubular epithelial cells; however, the pathways that mediate these events are poorly defined. Here, in murine ischemia/reperfusion (I/R)-induced AKI, we determined that mitochondrial damage is associated with the level of renal uncoupling protein 2 (UCP2). In hypoxia-damaged proximal tubular cells, a disruption of mitochondrial dynamics demonstrated by mitochondrial fragmentation and disturbance between fusion and fission was clearly indicated. Ucp2-deficient mice (knockout mice) with I/R injury experienced more severe AKI and mitochondrial fragmentation than wild-type mice. Moreover, genetic or pharmacological treatment increased UCP2 expression, improved renal function, reduced tubular injury and limited mitochondrial fission. In cultured proximal tubular epithelial cells, hypoxia-induced mitochondrial fission was exacerbated in cells with UCP2 deletion, whereas an increase in UCP2 ameliorated the hypoxia-induced disturbance of the balance between mitochondrial fusion and fission. Furthermore, results following modulation of UCP2 suggested it has a role in preserving mitochondrial integrity by preventing loss of membrane potential and reducing subsequent mitophagy. Taken together, our results indicate that UCP2 is protective against AKI and suggest that enhancing UCP2 to improve mitochondrial dynamics has potential as a strategy for improving outcomes of renal injury. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Nuclear miR-122 directly regulates the biogenesis of cell survival oncomiR miR-21 at the posttranscriptional level.

Hepatic miR-122 can serve as a pro-apoptotic factor to suppress tumorigenesis. The underlying mechanism, however, remains incompletely understood. Here we present the first evidence that miR-122 promotes hepatocellular carcinoma cell apoptosis through directly silencing the biogenesis of cell survival oncomiR miR-21 at posttranscriptional level. We find that miR-122 is strongly expressed in primary liver cell nucleus but its nuclear localization is markedly decreased in transformed cells particularly in chemoresistant tumor cells. MiRNA profiling and RT-qPCR confirm an inverse correlation between miR-122 and miR-21 in hepatocellular carcinoma tissues/cells, and increasing or decreasing nuclear level of miR-122 respectively reduces or increases miR-21 expression. Mechanistically, nuclear miR-122 suppresses miR-21 maturation via binding to a 19-nt UG-containing recognition element in the basal region of pri-miR-21 and preventing the Drosha-DGCR8 microprocessor's conversion of pri-miR-21 into pre-miR-21. Furthermore, both in vitro and in vivo studies demonstrate that nuclear miR-122 participates in the regulation of HCC cell apoptosis through modulating the miR-21-targeted programmed cell death 4 (PDCD4) signal pathway.

Plant microRNAs in larval food regulate honeybee caste development.

The major environmental determinants of honeybee caste development come from larval nutrients: royal jelly stimulates the differentiation of larvae into queens, whereas beebread leads to worker bee fate. However, these determinants are not fully characterized. Here we report that plant RNAs, particularly miRNAs, which are more enriched in beebread than in royal jelly, delay development and decrease body and ovary size in honeybees, thereby preventing larval differentiation into queens and inducing development into worker bees. Mechanistic studies reveal that amTOR, a stimulatory gene in caste differentiation, is the direct target of miR162a. Interestingly, the same effect also exists in non-social Drosophila. When such plant RNAs and miRNAs are fed to Drosophila larvae, they cause extended developmental times and reductions in body weight and length, ovary size and fecundity. This study identifies an uncharacterized function of plant miRNAs that fine-tunes honeybee caste development, offering hints for understanding cross-kingdom interaction and co-evolution.

SIRPα deficiency accelerates the pathologic process in models of Parkinson disease.

Microglia-mediated neuroinflammation is a crucial pathophysiological contributor to several aging-related neurodegenerative disorders, including Parkinson's disease (PD). During the process of aging or stress, microglia undergoes several transcriptional and morphological changes that contribute to aberrant immunological responses, which is known as priming. Key molecules involved in the process, however, are not clearly defined. In the present study, we have demonstrated that level of microglial signal regulatory protein α (SIRPα) decreased during aging or inflammatory challenge. Functional studies suggested that downregulation of SIRPα released the brake of inflammatory response in microglia, revealing an inhibitory effect of SIRPα in microglial activation. Furthermore, we assessed the impact of SIRPα downregulation in PD pathogenesis using both cell culture and animal models. Our results showed that SIRPα deficiency resulted in abnormal inflammatory response and phagocytic activity of microglia, which in turn, further accelerated degeneration of dopaminergic neurons in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine or lipopolysaccharides mice models. These results collectively demonstrate that dysregulation of SIRPα signaling in microglia during aging plays a critical role in the pathogenesis of age-related neurological disorders such as PD.

Arginase-1 is neither constitutively expressed in nor required for myeloid-derived suppressor cell-mediated inhibition of T-cell proliferation.

Although previous reports suggest that tumor-induced myeloid-derived suppressor cells (MDSC) inhibit T cells by L-arginine depletion through arginase-1 activity, we herein show that arginase-1 is neither inherently expressed in MDSC nor required for MDSC-mediated inhibition. Employing Percoll density gradients, large expansions of MDSC in the bone marrow of tumor-bearing mice were isolated and demonstrated potent inhibition in T-cell proliferation activated by TCR-ligation, Concanavalin A, PMA plus ionomycin, or IL-2. Despite demonstrating characteristic immunosuppressive capacity, these MDSC exhibit no arginase-1 expression and/or exert their inhibitory effects independent of arginase-1 activity. However, arginase-1 expression in MDSC can be induced by exposure to TCR-activated T cells or their culture medium, but not T cells activated by other means or growing tumor cells. Further investigation reveals multiple cytokines secreted by TCR-activated T cells as orchestrating two signaling-relay axes, IL-6-to-IL-4 and GM-CSF/IL-4-to-IL-10, leading to arginase-1 expression in MDSC. Specifically, IL-6 signaling increases IL-4R, enabling IL-4 to induce arginase-1 expression; similarly, GM-CSF in concert with IL-4 induces IL-10R, allowing IL-10-mediated induction. Surprisingly, our study indicates that induction of arginase-1 expression is not conducive to the critical MDSC-mediated inhibition toward T cells, which is rather dependent on direct cell contacts undiminished by PD-L1 blockade or SIRPα deficiency.

Tumor conditions induce bone marrow expansion of granulocytic, but not monocytic, immunosuppressive leukocytes with increased CXCR2 expression in mice.

Myeloid-derived suppressor cells (MDSCs) promote tumor growth through, in part, inhibiting T-cell immunity. However, mechanisms underlying MDSC expansion and guidance of MDSCs toward the tumor microenvironment remain unclear. Employing Percoll density gradients, we separate bone marrow (BM) leukocytes from tumor-bearing mice into four density-increasing bands with myeloid leukocytes enriched in bands III and IV. Band III comprises monocytes and low-density granulocytes, both confirmed to be M-MDSCs and G-MDSCs, respectively, by displaying potent inhibition of T-cell proliferation. However, monocytes act as M-MDSCs not only under tumor conditions but also the healthy condition. In contrast, band IV contains non-inhibitory, mature granulocytes. Only band III G-MDSCs display significant expansion in mice bearing B16 melanoma, Lewis lung carcinoma, or MC38 colon carcinoma. The expanded G-MDSCs also show increased CXCR2 expression, which guides egress out of BM, and produce arginase-1 and ROS upon encountering antigen-activated T cells. Adoptive transfer assays demonstrate that both G-MDSCs and mature granulocytes infiltrate tumors, but only the former displays sustention and accumulation. Intratumoral administrations of granulocytes further demonstrate that G-MDSCs promote tumor growth, whereas mature granulocytes exert minimal effects, or execute powerful anti-tumor effects providing the presence of PMN activation mechanisms in the tumor microenvironment.

Cd47-Sirpα interaction and IL-10 constrain inflammation-induced macrophage phagocytosis of healthy self-cells.

Rapid clearance of adoptively transferred Cd47-null (Cd47(-/-)) cells in congeneic WT mice suggests a critical self-recognition mechanism, in which CD47 is the ubiquitous marker of self, and its interaction with macrophage signal regulatory protein α (SIRPα) triggers inhibitory signaling through SIRPα cytoplasmic immunoreceptor tyrosine-based inhibition motifs and tyrosine phosphatase SHP-1/2. However, instead of displaying self-destruction phenotypes, Cd47(-/-) mice manifest no, or only mild, macrophage phagocytosis toward self-cells except under the nonobese diabetic background. Studying our recently established Sirpα-KO (Sirpα(-/-)) mice, as well as Cd47(-/-) mice, we reveal additional activation and inhibitory mechanisms besides the CD47-SIRPα axis dominantly controlling macrophage behavior. Sirpα(-/-) mice and Cd47(-/-) mice, although being normally healthy, develop severe anemia and splenomegaly under chronic colitis, peritonitis, cytokine treatments, and CFA-/LPS-induced inflammation, owing to splenic macrophages phagocytizing self-red blood cells. Ex vivo phagocytosis assays confirmed general inactivity of macrophages from Sirpα(-/-) or Cd47(-/-) mice toward healthy self-cells, whereas they aggressively attack toward bacteria, zymosan, apoptotic, and immune complex-bound cells; however, treating these macrophages with IL-17, LPS, IL-6, IL-1ß, and TNFα, but not IFNγ, dramatically initiates potent phagocytosis toward self-cells, for which only the Cd47-Sirpα interaction restrains. Even for macrophages from WT mice, phagocytosis toward Cd47(-/-) cells does not occur without phagocytic activation. Mechanistic studies suggest a PKC-Syk-mediated signaling pathway, to which IL-10 conversely inhibits, is required for activating macrophage self-targeting, followed by phagocytosis independent of calreticulin Moreover, we identified spleen red pulp to be one specific tissue that provides stimuli constantly activating macrophage phagocytosis albeit lacking in Cd47(-/-) or Sirpα(-/-) mice.

Decreased miR-200a-3p is a key regulator of renal carcinoma growth and migration by directly targeting CBL.

Although emerging evidence has revealed that microRNAs (miRNAs) dysregulation contribute to carcinogenesis, the mechanism underlying their roles in renal cell carcinoma (RCC) is unclear. The purpose of the current study was to analyze the association of miR-200a-3p expression with RCC and to understand potential novel target genes, functions and mechanisms of miR-200a-3p in RCC. MiR-200a-3p expression levels were first measured by quantitative real-time polymerase chain reaction and in situ hybridization in pairs of RCC tissue samples. Next, the potential miR-200a-3p target gene was analyzed using a combination of computer-aided algorithms, luciferase reporter assays and Western blot analysis. Finally, the biological roles of miR-200a-3p in RCC tumorigenesis were investigated both in vitro by 5-ethynyl-20-deoxyuridine, apoptosis assay and transwell assay, as well as in vivo using a xenograft mouse model. Our results demonstrated that miR-200a-3p was remarkably downregulated in RCC tissues compared with normal adjacent tissue, and CBL is a direct target of miR-200a-3p. An inverse correlation between miR-200a-3p and CBL was observed in RCC tissue samples. Mechanistic investigations revealed that ectopic expression of miR-200a-3p in RCC cell lines suppressed cell proliferation and migration and enforced cell apoptosis by directly inhibiting CBL in vitro and in vivo, whereas silencing miR-200a-3p resulted in the opposite effects. Additionally, overexpressing CBL abolished the effects induced by miR-200a-3p overexpression. Taken together, our results show that the miR-200a-3p/CBL regulation axis is a novel mechanism underlying RCC pathogenesis and may serve as a candidate biomarker and therapeutic target in RCC.