Quantification of urinary podocyte-derived migrasomes for the diagnosis of kidney disease.

Migrasomes represent a recently uncovered category of extracellular microvesicles, spanning a diameter range of 500 to 3000 nm. They are emitted by migrating cells and harbour a diverse array of RNAs and proteins. Migrasomes can be readily identified in bodily fluids like serum and urine, rendering them a valuable non-invasive source for disease diagnosis through liquid biopsy. In this investigation, we introduce a streamlined and effective approach for the capture and quantitative assessment of migrasomes, employing wheat germ agglutinin (WGA)-coated magnetic beads and flow cytometry (referred to as WBFC). Subsequently, we examined the levels of migrasomes in the urine of kidney disease (KD) patients with podocyte injury and healthy volunteers using WBFC. The outcomes unveiled a substantial increase in urinary podocyte-derived migrasome concentrations among individuals with KD with podocyte injury compared to the healthy counterparts. Notably, the urinary podocyte-derived migrasomes were found to express an abundant quantity of phospholipase A2 receptor (PLA2R) proteins. The presence of PLA2R proteins in these migrasomes holds promise for serving as a natural antigen for the quantification of autoantibodies against PLA2R in the serum of patients afflicted by membranous nephropathy. Consequently, our study not only pioneers a novel technique for the isolation and quantification of migrasomes but also underscores the potential of urinary migrasomes as a promising biomarker for the early diagnosis of KD with podocyte injury.

Podocyte SIRPα reduction aggravates lupus nephritis via promoting T cell inflammatory responses.

Signal-regulatory protein alpha (SIRPα) has recently been found to be highly expressed in podocytes and is essential for maintaining podocyte function. However, its immunoregulatory function in podocytes remains elusive. Here, we report that SIRPα controls podocyte antigen presentation in specific T cell activation via inhibiting spleen tyrosine kinase (Syk) phosphorylation. First, podocyte SIRPα under lupus nephritis (LN) conditions is strongly downregulated. Second, podocyte-specific deletion of SIRPα exacerbates renal disease progression in lupus-prone mice, as evidenced by an increase in T cell infiltration. Third, SIRPα deletion or knockdown enhances podocyte antigen presentation, which activates specific T cells, via enhancing Syk phosphorylation. Supporting this, Syk inhibitor GS-9973 prevents podocyte antigen presentation, resulting in a decrease of T cell activation and mitigation of renal disease caused by SIRPα knockdown or deletion. Our findings reveal an immunoregulatory role of SIRPα loss in promoting podocyte antigen presentation to activate specific T cell immune responses in LN.

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.

Blockade of pan-viral propagation by inhibition of host cell PNPT1.

For successful viral propagation within infected cells, the virus needs to overcome the cellular integrated stress response (ISR), triggered during viral infection, which, in turn, inhibits general protein translation. This paper reports a tactic employed by viruses to suppress the ISR by upregulating host cell polyribonucleotide nucleotidyltransferase 1 (PNPT1). The propagation of adenovirus, murine cytomegalovirus and hepatovirus within their respective host cells induces PNPT1 expression. Notably, when PNPT1 is knocked down, the propagation of all three viruses is prevented. Mechanistically, the inhibition of PNPT1 facilitates the relocation of mitochondrial double-stranded RNAs (mt-dsRNAs) to the cytoplasm, where they activate RNA-activated protein kinase (PKR). This activation leads to eukaryotic initiation factor 2α (eIF2α) phosphorylation, resulting in the suppression of translation. Furthermore, by scrutinizing the PNPT1 recognition element and screening 17,728 drugs and bioactive compounds approved by the US Food and Drug Administration, lanatoside C was identified as a potent PNPT1 inhibitor. This compound impedes the propagation of adenovirus, murine cytomegalovirus and hepatovirus, and suppresses production of the severe acute respiratory syndrome coronavirus-2 spike protein. These discoveries shed light on a novel strategy to impede pan-viral propagation by activating the host cell mt-dsRNA-PKR-eIF2α signalling axis.

EBF2 Links KMT2D-Mediated H3K4me1 to Suppress Pancreatic Cancer Progression via Upregulating KLLN.

Mono-methylation of histone H3 on Lys 4 (H3K4me1), which is catalyzed by histone-lysine N-methyltransferase 2D (KMT2D), serves as an important epigenetic regulator in transcriptional control. In this study, the authors identify early B-cell factor 2 (EBF2) as a binding protein of H3K4me1. Combining analyses of RNA-seq and ChIP-seq data, the authors further identify killin (KLLN) as a transcriptional target of KMT2D and EBF2 in pancreatic ductal adenocarcinoma (PDAC) cells. KMT2D-dependent H3K4me1 and EBF2 are predominantly over-lapped proximal to the transcription start site (TSS) of KLLN gene. Comprehensive functional assays show that KMT2D and EBF2 cooperatively inhibit PDAC cells proliferation, migration, and invasion through upregulating KLLN. Such inhibition on PDAC progression is also achieved through increasing H3K4me1 level by GSK-LSD1, a selective inhibitor of lysine-specific demethylase 1 (LSD1). Taken together, these findings reveal a new mechanism underlying PDAC progression and provide potential therapeutic targets for PDAC treatment.

YTHDF1 mitigates acute kidney injury via safeguarding m6A-methylated mRNAs in stress granules of renal tubules.

Acute kidney injury (AKI) presents a daunting challenge with limited therapeutic options. To explore the contribution of N6-methyladenosine (m6A) in AKI development, we have investigated m6A-modified mRNAs within renal tubular cells subjected to injuries induced by diverse stressors. Notably, while the overall level of m6A-modified RNA remains unaltered in renal tubular cells facing stress, a distinct phenomenon emerges-mRNAs bearing m6A methylation exhibit a pronounced tendency to accumulate within stress granules (SGs), structures induced in response to these challenges. Cumulation of m6A-modified mRNA in SGs is orchestrated by YTHDF1, a m6A 'reader' closely associated with SGs. Strikingly, AKI patients and various mouse AKI models showcase elevated levels of renal tubular YTHDF1. Depleting YTHDF1 within renal tubular cells leads to a marked reduction in m6A-modified mRNA accumulation within SGs, accompanied by an escalation in cell apoptosis under stress challenges. The significance of YTHDF1's protective role is further underscored by findings in AKI mouse models triggered by cisplatin or renal ischemia-reperfusion treatments. In particular, renal tubular-specific YTHDF1 knockout mice exhibit heightened AKI severity when contrasted with their wild-type counterparts. Mechanistic insights reveal that YTHDF1 fulfills a crucial function by safeguarding m6A-modified mRNAs that favor cell survival-exemplified by SHPK1-within SGs amid stress-challenged renal tubular cells. Our findings collectively shed light on the pivotal role of YTHDF1 in shielding renal tubules against AKI, through its adeptness in recruiting and preserving m6A-modified mRNAs within stress-induced SGs.

Cold-activated brown fat-derived extracellular vesicle-miR-378a-3p stimulates hepatic gluconeogenesis in male mice.

During cold exposure, activated brown adipose tissue (BAT) takes up a large amount of circulating glucose to fuel non-shivering thermogenesis and defend against hypothermia. However, little is known about the endocrine function of BAT controlling glucose homoeostasis under this thermoregulatory challenge. Here, we show that in male mice, activated BAT-derived extracellular vesicles (BDEVs) reprogram systemic glucose metabolism by promoting hepatic gluconeogenesis during cold stress. Cold exposure facilitates the selective packaging of miR-378a-3p-one of the BAT-enriched miRNAs-into EVs and delivery into the liver. BAT-derived miR-378a-3p enhances gluconeogenesis by targeting p110α. miR-378 KO mice display reduced hepatic gluconeogenesis during cold exposure, while restoration of miR-378a-3p in iBAT induces the expression of gluconeogenic genes in the liver. These findings provide a mechanistic understanding of BDEV-miRNA as stress-induced batokine to coordinate systemic glucose homoeostasis. This miR-378a-3p-mediated interorgan communication highlights a novel endocrine function of BAT in preventing hypoglycemia during cold stress.

HIF-1α drives resistance to ferroptosis in solid tumors by promoting lactate production and activating SLC1A1.

Solid tumors have developed robust ferroptosis resistance. The mechanism underlying ferroptosis resistance regulation in solid tumors, however, remains elusive. Here, we report that the hypoxic tumor microenvironment potently promotes ferroptosis resistance in solid tumors in a hypoxia-inducible factor 1α (HIF-1α)-dependent manner. In combination with HIF-2α, which promotes tumor ferroptosis under hypoxia, HIF-1α is the main driver of hypoxia-induced ferroptosis resistance. Mechanistically, HIF-1α-induced lactate contributes to ferroptosis resistance in a pH-dependent manner that is parallel to the classical SLC7A11 and FSP1 systems. In addition, HIF-1α also enhances transcription of SLC1A1, an important glutamate transporter, and promotes cystine uptake to promote ferroptosis resistance. In support of the role of hypoxia in ferroptosis resistance, silencing HIF-1α sensitizes mouse solid tumors to ferroptosis inducers. In conclusion, our results reveal a mechanism by which hypoxia drives ferroptosis resistance and identify the combination of hypoxia alleviation and ferroptosis induction as a promising therapeutic strategy for solid tumors.

CD8 T Cell-Derived Exosomal miR-186-5p Elicits Renal Inflammation via Activating Tubular TLR7/8 Signal Axis.

T cells play an important role in the development of focal segmental glomerulosclerosis (FSGS). The mechanism underlying such T cell-based kidney disease, however, remains elusive. Here the authors report that activated CD8 T cells elicit renal inflammation and tissue injury via releasing miR-186-5p-enriched exosomes. Continuing the cohort study identifying the correlation of plasma level of miR-186-5p with proteinuria in FSGS patients, it is demonstrated that circulating miR-186-5p is mainly derived from activated CD8 T cell exosomes. Renal miR-186-5p, which is markedly increased in FSGS patients and mice with adriamycin-induced renal injury, is mainly delivered by CD8 T cell exosomes. Depleting miR-186-5p strongly attenuates adriamycin-induced mouse renal injury. Supporting the function of exosomal miR-186-5p as a key circulating pathogenic factor, intravenous injection of miR-186-5p or miR-186-5p-containing T cell exosomes results in mouse renal inflammation and tissue injury. Tracing the injected T cell exosomes shows their preferential distribution in mouse renal tubules, not glomerulus. Mechanistically, miR-186-5p directly activates renal tubular TLR7/8 signal and initiates tubular cell apoptosis. Mutating the TLR7-binding sequence on miR-186-5p or deleting mouse TLR7 largely abolishes renal tubular injuries induced by miR-186-5p or adriamycin. These findings reveal a causative role of exosomal miR-186-5p in T cell-mediated renal dysfunction.

A Novel Pathway of Functional microRNA Uptake and Mitochondria Delivery.

Extracellular microRNAs (miRNAs) play a critical role in horizontal gene regulation. Uptake of extracellular miRNAs by recipient cells and their intracellular transport, however, remains elusive. Here RNA phase separation is shown as a novel pathway of miRNA uptake. In the presence of serum, synthetic miRNAs rapidly self-assembly into ≈110 nm discrete nanoparticles, which enable miRNAs' entry into different cells. Depleting serum cationic proteins prevents the formation of such nanoparticles and thus blocks miRNA uptake. Different from lipofectamine-mediated miRNA transfection in which majority of miRNAs are accumulated in lysosomes of transfected cells, nanoparticles-mediated miRNA uptake predominantly delivers miRNAs into mitochondria in a polyribonucleotide nucleotidyltransferase 1(PNPT1)-dependent manner. Functional assays further show that the internalized miR-21 via miRNA phase separation enhances mitochondrial translation of cytochrome b (CYB), leading to increase in adenosine triphosphate (ATP) and reactive oxygen species (ROS) reduction in HEK293T cells. The findings thus reveal a previously unrecognized mechanism for uptake and delivery functional extracellular miRNAs into mitochondria.

The SMARCA4R1157W mutation facilitates chromatin remodeling and confers PRMT1/SMARCA4 inhibitors sensitivity in colorectal cancer.

Genomic studies have demonstrated a high frequency of genetic alterations in components of the SWI/SNF complex including the core subunit SMARCA4. However, the mechanisms of tumorigenesis driven by SMARCA4 mutations, particularly in colorectal cancer (CRC), remain largely unknown. In this study, we identified a specific, hotspot mutation in SMARCA4 (c. 3721C>T) which results in a conversion from arginine to tryptophan at residue 1157 (R1157W) in human CRC tissues associated with higher-grade tumors and controls CRC progression. Mechanistically, we found that the SMARCA4R1157W mutation facilitated its recruitment to PRMT1-mediated H4R3me2a (asymmetric dimethylation of Arg 3 in histone H4) and enhanced the ATPase activity of SWI/SNF complex to remodel chromatin in CRC cells. We further showed that the SMARCA4R1157W mutant reinforced the transcriptional expression of EGFR and TNS4 to promote the proliferation of CRC cells and patient-derived tumor organoids. Importantly, we demonstrated that SMARCA4R1157W CRC cells and mutant cell-derived xenografts were more sensitive to the combined inhibition of PRMT1 and SMARCA4 which act synergistically to suppress cell proliferation. Together, our findings show that SMARCA4-R1157W is a critical activating mutation, which accelerates CRC progression through facilitating chromatin recruitment and remodeling. Our results suggest a potential precision therapeutic strategy for the treatment of CRC patients carrying the SMARCA4R1157W mutation.

Comparative Analyses of Human Exosome Proteomes.

Exosomes are responsible for cell-to-cell communication and serves as a valuable drug delivery vehicle. However, exosome heterogeneity, non-standardized isolation methods and proteomics/bioinformatics approaches limit its clinical applications. To better understand exosome heterogeneity, biological function and molecular mechanism of its biogenesis, secretion and uptake, techniques in proteomics or bioinformatics were applied to investigate human embryonic kidney cell (293T cell line)-derived exosome proteome and enable an integrative comparison of exosomal proteins and protein-protein interaction (PPI) networks of eleven exosome proteomes extracted from diverse human samples, including 293T (two datasets), dermal fibroblast, mesenchymal stem cell, thymic epithelial primary cell, breast cancer cell line (MDA-MB-231), patient neuroblastoma cell, plasma, saliva, serum and urine. Mapping of exosome biogenesis/secretion/uptake-related proteins onto exosome proteomes highlights exosomal origin-specific routes of exosome biogenesis/secretion/uptake and exosome-dependent intercellular communication. The finding provides insight into comparative exosome proteomes and its biogenesis, secretion and uptake, and potentially contributes to clinical applications.

Polynucleotide phosphorylase protects against renal tubular injury via blocking mt-dsRNA-PKR-eIF2α axis.

Renal tubular atrophy is a hallmark of chronic kidney disease. The cause of tubular atrophy, however, remains elusive. Here we report that reduction of renal tubular cell polynucleotide phosphorylase (PNPT1) causes renal tubular translation arrest and atrophy. Analysis of tubular atrophic tissues from renal dysfunction patients and male mice with ischemia-reperfusion injuries (IRI) or unilateral ureteral obstruction (UUO) treatment shows that renal tubular PNPT1 is markedly downregulated under atrophic conditions. PNPT1 reduction leads to leakage of mitochondrial double-stranded RNA (mt-dsRNA) into the cytoplasm where it activates protein kinase R (PKR), followed by phosphorylation of eukaryotic initiation factor 2α (eIF2α) and protein translational termination. Increasing renal PNPT1 expression or inhibiting PKR activity largely rescues IRI- or UUO-induced mouse renal tubular injury. Moreover, tubular-specific PNPT1-knockout mice display Fanconi syndrome-like phenotypes with impaired reabsorption and significant renal tubular injury. Our results reveal that PNPT1 protects renal tubules by blocking the mt-dsRNA-PKR-eIF2α axis.

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.

Mesenchymal stem cell-derived exosome-educated macrophages alleviate systemic lupus erythematosus by promoting efferocytosis and recruitment of IL-17+ regulatory T cell.

BACKGROUND: Anti-inflammatory polarized macrophages are reported to alleviate systemic lupus erythematosus (SLE). Our previous studies have demonstrated that exosomes from adipose-derived stem cells promote the anti-inflammatory polarization of macrophages. However, the possible therapeutic effect of exosomes from stem cells on SLE remains unexplored. METHODS: Exosomes were isolated from the conditioned medium of bone marrow-derived mesenchymal stem cells using ultrafiltration and size-exclusion chromatography and were identified by nanoparticle tracking analysis and immunoblotting of exosomal-specific markers. Macrophages were collected from the MRL/lpr mouse kidney. The phenotype of macrophages was identified by immunoblotting for intracellular markers-inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1), and flow cytometry for macrophage markers F4/80, CD86, CD206, B7H4, and CD138. Pristane-induced murine lupus nephritis models were employed for in vivo study. RESULTS: When macrophages from the kidney of the MRL/lpr mice were treated with exosomes from bone marrow-derived mesenchymal stem cells (BM-MSCs), the upregulation of CD206, B7H4, CD138, Arg-1, CCL20, and anti-inflammatory cytokines was observed, which suggested that the macrophages were polarized to a specific anti-inflammatory phenotype. These anti-inflammatory macrophages produced low levels of reactive oxygen species (ROS) but had a high efferocytosis activity and promoted regulatory T (Treg) cell recruitment. Moreover, exosome injection stimulated the anti-inflammatory polarization of macrophages and increased the production of IL-17+ Treg cells in a pristane-induced murine lupus nephritis model. We observed that exosomes from BMMSCs depleted of microRNA-16 (miR-16) and microRNA-21 (miR-21) failed to downregulate PDCD4 and PTEN in macrophages, respectively, and attenuated exosome-induced anti-inflammatory polarization. CONCLUSION: Our findings provide evidence that exosomes from BMMSCs promote the anti-inflammatory polarization of macrophages. These macrophages alleviate SLE nephritis in lupus mice by consuming apoptotic debris and inducing the recruitment of Treg cells. We identify that exosomal delivery of miR-16 and miR-21 is a significant contributor to the polarization of macrophages.

A novel method to purify small RNAs from human tissues for methylation analysis by LC-MS/MS.

Methylation modification of small RNAs, including miRNA, piRNA, and tsRNA, is critical for small RNA biogenesis and biological function. Methylation of individual small RNA can be defined by liquid chromatography-coupled with mass spectrometry (LC-MS/MS). However, LC-MS/MS analysis requires a high purity of individual small RNA. Due to the difficulty of purifying specific small RNA from tissues or cells, the progress in characterizing small RNA methylation by LC-MS/MS is limited. Here, we report a novel method that can efficiently purify small RNA from human tissues for LC-MS/MS analysis. This method includes two steps: 1) pull down the target small RNA by incubating total small RNAs (18-24 nt) extracted from human tissues with a biotinylated antisense oligonucleotide of the target small RNA, followed by capturing the binding duplex of biotinylated antisense and small RNA via streptavidin magnetic beads, and 2) protect the target small RNA by pairing it with a single-strand DNA, which sequence is complementary to the target small RNA, to form a DNA/RNA hybrid double-strand, followed by sequential digestion with exonuclease I, nuclease S1, and DNase I, respectively. Furthermore, employing a mixture of four pairs of synthetic methylated and non-methylated small RNAs, we further refined this two-step method by optimizing the nuclease S1 treatment condition. With this method, we successfully purified miR-21-5p, miR-26-5p, piR-020485, and tsRNA from human lung and sperm tissue samples and analyzed their 2'-O-methylation modification at the 3'-end by LC-MS/MS.

Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis.

Tumor cells can evade attack by phagocytes by upregulating the self-marker CD47. The mechanisms underlying tumor CD47 upregulation, however, remain unclear. Here, we report that human lung adenocarcinoma CD47 is upregulated by interferon-γ (IFN-γ), the level in the tumor microenvironment of which is markedly increased after tumor metastasis and chemotherapy. The IFN-γ receptor is expressed in various human lung adenocarcinoma tissues regardless of the CD47 protein expression, and lung adenocarcinoma CD47 expression is significantly enhanced following tumor metastasis or chemotherapy treatment. In line with this, CD47 expression in various lung cancer cells is markedly increased by IFN-γ treatment. Mechanistically, IFN-γ promotes CD47 expression by activating interferon regulatory factor-1 (IRF-1), which binds to an IRF-1-binding domain within the CD47 promoter region and increases CD47 transcription. Functionally, IFN-γ-enhanced CD47 expression facilitates human lung cancer cell invasion both in vitro and in vivo, whereas IFN-γ-induced CD47 upregulation and cancer metastasis are blocked by mutating the IRF-1-binding site within the CD47 promoter. Our results reveal IFN-γ-enhanced CD47 expression as a novel mechanism promoting human lung adenocarcinoma progression.

Myeloid-Specific Pyruvate-Kinase-Type-M2-Deficient Mice Are Resistant to Acute Lung Injury.

Infiltration of polymorphonuclear neutrophils (PMNs) plays a central role in acute lung injury (ALI). The mechanisms governing PMN inflammatory responses, however, remain incompletely understood. Based on our recent study showing a non-metabolic role of pyruvate kinase type M2 (PKM2) in controlling PMN degranulation of secondary and tertiary granules and consequent chemotaxis, here we tested a hypothesis that Pkm2-deficient mice may resist ALI due to impaired PMN inflammatory responses. We found that PMN aerobic glycolysis controlled the degranulation of secondary and tertiary granules induced by fMLP and PMA. Compared to WT PMNs, Pkm2-deficient (Pkm2-/-) PMNs displayed significantly less capacity for fMLP- or PMA-induced degranulation of secondary and tertiary granules, ROS production, and transfilter migration. In line with this, myeloid-specific Pkm2-/- mice exhibited impaired zymosan-induced PMN infiltration in the peritoneal cavity. Employing an LPS-induced ALI mouse model, LPS-treated Pkm2-/- mice displayed significantly less infiltration of inflammatory PMNs in the alveolar space and a strong resistance to LPS-induced ALI. Our results thus reveal that PKM2 is required for PMN inflammatory responses and deletion of PKM2 in PMN leads to an impaired PMN function but protection against LPS-induced ALI.

Accurate quantification of 3'-terminal 2'-O-methylated small RNAs by utilizing oxidative deep sequencing and stem-loop RT-qPCR.

The continuing discoveries of novel classes of RNA modifications in various organisms have raised the need for improving sensitive, convenient, and reliable methods for quantifying RNA modifications. In particular, a subset of small RNAs, including microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), are modified at their 3'-terminal nucleotides via 2'-O-methylation. However, quantifying the levels of these small RNAs is difficult because 2'-O-methylation at the RNA 3'-terminus inhibits the activity of polyadenylate polymerase and T4 RNA ligase. These two enzymes are indispensable for RNA labeling or ligation in conventional miRNA quantification assays. In this study, we profiled 3'-terminal 2'-O-methyl plant miRNAs in the livers of rice-fed mice by oxidative deep sequencing and detected increasing amounts of plant miRNAs with prolonged oxidation treatment. We further compared the efficiency of stem-loop and poly(A)-tailed RT-qPCR in quantifying plant miRNAs in animal tissues and identified stem-loop RT-qPCR as the only suitable approach. Likewise, stem-loop RT-qPCR was superior to poly(A)-tailed RT-qPCR in quantifying 3'-terminal 2'-O-methyl piRNAs in human seminal plasma. In summary, this study established a standard procedure for quantifying the levels of 3'-terminal 2'-O-methyl miRNAs in plants and piRNAs. Accurate measurement of the 3'-terminal 2'-O-methylation of small RNAs has profound implications for understanding their pathophysiologic roles in biological systems.