Circ-RAPGEF5 promotes intrahepatic cholangiocarcinoma progression by stabilizing SAE1 to facilitate SUMOylation.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with a poor prognosis. The underlying functions and mechanisms of circular RNA and SUMOylation in the development of ICC remain poorly understood. METHODS: Circular RNA hsa_circ_0001681 (termed Circ-RAPGEF5 hereafter) was identified by circular RNA sequencing from 19 pairs of ICC and adjacent tissue samples. The biological function of Circ-RAPGEF5 in tumor proliferation and metastasis was examined by a series of in vitro assays. A preclinical model was used to validate the therapeutic effect of targeting Circ-RAPGEF5. RNA pull-down and dual-luciferase reporter assays were used to access the RNA interactions. Western blot and Co-IP assays were used to detect SUMOylation levels. RESULTS: Circ-RAPGEF5, which is generated from exons 2 to 6 of the host gene RAPGEF5, was upregulated in ICC. In vitro and in vivo assays showed that Circ-RAPGEF5 promoted ICC tumor proliferation and metastasis, and inhibited apoptosis. Additionally, high Circ-RAPGEF5 expression was significantly correlated with a poor prognosis. Further investigation showed that SAE1, a potential target of Circ-RAPGEF5, was also associated with poor oncological outcomes. RNA pull-down and dual-luciferase reporter assays showed an interaction of miR-3185 with Circ-RAPGEF5 and SAE1. Co-IP and western blot assays showed that Circ-RAPGEF5 is capable of regulating SUMOylation. CONCLUSION: Circ-RAPGEF5 promotes ICC tumor progression and SUMOylation by acting as a sponge for miR-3185 to stabilize SAE1. Targeting Circ-RAPGEF5 or SAE1 might be a novel diagnostic and therapeutic strategy in ICC.

The RasGEF MoCdc25 regulates vegetative growth, conidiation and appressorium-mediated infection in the rice blast fungus Magnaporthe oryzae.

Ras guanine nucleotide exchange factors (RasGEFs) can trigger Ras GTPase activities and play important roles in controlling various cellular processes in eukaryotes. Recently, it has been exhibited that RasGEF Cdc25 regulates morphological differentiation and pathogenicity in several plant pathogenic fungi. However, the role of RasGEFs in Magnaporthe oryzae is largely unknown. In this study, we identified and functionally characterized a RasGEF gene MoCDC25 in M. oryzae, which is orthologous to Saccharomyces cerevisiae CDC25. Targeted gene deletion mutants (ΔMocdc25) were completely nonpathogenic and were severely impaired in hyphal growth, conidiation and appressorium formation. The mutants exhibited highly sensitive response to osmotic, cell wall integrity or oxidative stresses. MoCdc25 physically interacts with the MAPK scaffold Mst50 and the putative Cdc42GEF MoScd1 in yeast two-hybrid assays. Moreover, we found that MoCdc25 was involved in regulating the phosphorylation of the MAP kinases (Pmk1, Mps1, and Osm1). In addition, the intracellular cAMP content in hyphae of the ΔMocdc25 mutants was significantly reduced compared to the parent strain Ku80 and the defect of appressorium formation of the mutants could be partially restored by the supplement of exogenous cAMP. Taken together, we conclude that the RasGEF MoCdc25 regulates vegetative growth, conidiation, appressorium formation and pathogenicity via MAPK and cAMP response pathways in M. oryzae.

Genetic variants of SOS2, MAP2K1 and RASGRF2 in the RAS pathway genes predict survival of HBV-related hepatocellular carcinoma patients.

The RAS pathway participates in the cascade of proliferation and cell division process, and the activated RAS pathway can lead to tumorigenesis including hepatocellular carcinoma (HCC). However, few studies have explored the effects of genetic variants in the RAS pathway-related genes on the survival of patients with HBV-related HCC. In the present study, we assessed the associations between 11,658 single-nucleotide polymorphisms (SNPs) in 62 RAS pathway genes and the overall survival (OS) of 866 HBV-related HCC individuals, which were randomly split (1:1) into discovery and validation datasets. As a result, three potentially functional SNPs were identified, based on multivariable cox proportional hazards regression analyses, in SOS Ras/Rho guanine nucleotide exchange factor 2 (SOS2, rs4632055 A > G), Ras protein-specific guanine nucleotide releasing factor 2 (RASGRF2, rs26418A > G) and mitogen-activated protein kinase 1 (MAP2K1,rs57120695 C > T), which were significantly and independently associated with OS of HBV-related HCC patients [adjusted hazards ratios (HRs) of 1.42, 1.32 and 1.50, respectively; 95% confidence intervals (CI), 1.14 to 1.76, 1.15 to 1.53 and 1.15 to 1.97, respectively; P = 0.001, < 0.001 and 0.003, respectively]. Additionally, the joint effects as the unfavorable genotypes of these three SNPs showed a significant association with the poor survival of HCC (trend test P < 0.001). The expression quantitative trait loci (eQTL) analysis further revealed that the rs4632055 G allele and the rs26418 A allele were associated with lower mRNA expression levels of SOS2 and RASGRF2, respectively. Collectively, these potentially functional SNPs of RASGRF2, SOS2 and M2PAK1 may become potential prognostic biomarkers for HBV-related HCC after hepatectomy.

Association between single nucleotide variants and severe chronic pain in older adult patients after lower extremity arthroplasty.

BACKGROUND: Hip or knee osteoarthritis (OA) is one of the main causes of disability worldwide and occurs mostly in the older adults. Total hip or knee arthroplasty is the most effective method to treat OA. However, severe postsurgical pain leading to a poor prognosis. So, investigating the population genetics and genes related to severe chronic pain in older adult patients after lower extremity arthroplasty is helpful to improve the quality of treatment. METHODS: We collected blood samples from elderly patients who underwent lower extremity arthroplasty from September 2020 to February 2021 at the Drum Tower Hospital Affiliated to Nanjing University Medical School. The enrolled patients provided measures of pain intensity using the numerical rating scale on the 90th day after surgery. Patients were divided into the case group (Group A) and the control group (Group B) including 10 patients respectively by the numerical rating scale. DNA was isolated from the blood samples of the two groups for whole-exome sequencing. RESULTS: In total, 661 variants were identified in the 507 gene regions that were significantly different between both groups (P < 0.05), including CASP5, RASGEF1A, CYP4B1, etc. These genes are mainly involved in biological processes, including cell-cell adhesion, ECM-receptor interaction, metabolism, secretion of bioactive substances, ion binding and transport, regulation of DNA methylation, and chromatin assembly. CONCLUSIONS: The current study shows some variants within genes are significantly associated with severe postsurgical chronic pain in older adult patients after lower extremity arthroplasty, indicating a genetic predisposition for chronic postsurgical pain. The study was registered according to ICMJE guidelines. The trial registration number is ChiCTR2000031655 and registration date is April 6th, 2020.

Mutant NPM1 maintains RASGRP3 protein stability via interaction with MID1 to promote acute myeloid leukemia cell proliferation and autophagy.

Acute myeloid leukemia is a heterogeneous hematologic malignancy with high mortality in the world. NPM1 gene mutations are a frequent occurrence in acute myeloid leukemia, leading to abnormal autophagy, while the mechanism of NPM1 mutation-driven acute myeloid leukemia pathogenesis remains to be fully elucidated. GEO microarrays were used to screen for dysregulated autophagy-related genes in NPM1-mutant acute myeloid leukemia and analysis of RASGRP3 expression and prognosis. Next, we explored the potential molecular mechanisms relationship between RASGRP3 and NPM1 through utilizing immunoprecipitation, Western blot, and cycloheximide assay. Further, CCK8, EdU staining, immunofluorescence, and Western blot were performed to explore the effect of RASGRP3 on cell proliferation and apoptosis in NPM1-mutated acute myeloid leukemia. Finally, Western blot was used to study the mechanism of action of RASGRP3. RASGRP3 expression was upregulated in NPM1-mutated acute myeloid leukemia. Mislocalized NPM1-mA in the cytoplasm could bind to E3 ubiquitin-protein ligase MID1 to block degradation of the RASGRP3 protein. RASGRP3 could also activate the EGFR-STAT3 axis to promote proliferation and autophagy in acute myeloid leukemia. In conclusion, our results identified RASGRP3 as a proto-oncogene in NPM1-mutated acute myeloid leukemia. The RASGRP3-EGFR/STAT3 axis may be a promising therapeutic target for this unique leukemic subtype.

Son of sevenless 1 (SOS1), the RasGEF, interacts with ERα and STAT3 during embryo implantation.

Estrogen accounts for several biological processes in the body; embryo implantation and pregnancy being one of the vital events. This manuscript aims to unearth the nuclear role of Son of sevenless1 (SOS1), its interaction with estrogen receptor alpha (ERα), and signal transducer and activator of transcription 3 (STAT3) in the uterine nucleus during embryo implantation. SOS1, a critical cytoplasmic linker between receptor tyrosine kinase and rat sarcoma virus signaling, translocates into the nucleus via its bipartite nuclear localization signal (NLS) during the 'window of implantation' in pregnant mice. SOS1 associates with chromatin, interacts with histones, and shows intrinsic histone acetyltransferase (HAT) activity specifically acetylating lysine 16 (K16) residue of histone H4. SOS1 is a coactivator of STAT3 and a co-repressor of ERα. SOS1 creates a partial mesenchymal-epithelial transition by acting as a transcriptional modulator. Finally, our phylogenetic tree reveals that the two bipartite NLS surface in reptiles and the second acetyl coenzymeA (CoA) (RDNGPG) important for HAT activity emerges in mammals. Thus, SOS1 has evolved into a moonlighting protein, the special class of multi-tasking proteins, by virtue of its newly identified nuclear functions in addition to its previously known cytoplasmic function.

The prognostic value of RASGEF1A RNA expression and DNA methylation in cytogenetically normal acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a significantly heterogeneous malignancy of the blood. Cytogenetic abnormalities are crucial for the prognosis of AML. However, since more than half of patients with AML are cytogenetically normal AML (CN-AML), predictive prognostic indicators need to be further refined. In recent years, gene abnormalities are considered to be strong prognostic factors of CN-AML, already having clinical significance for treatment. In addition, the relationship of methylation in some genes and AML prognosis predicting has been discovered. RASGEF1A is a guanine nucleotide exchange factors of Ras and widely expressed in brain tissue, bone marrow and 17 other tissues. RASGEF1A has been reported to be associated with a variety of malignant tumors, examples include Hirschsprung disease, renal cell carcinoma, breast cancer, diffuse large B cell lymphoma, intrahepatic cholangiocarcinoma and so on [1, 2]. However, the relationship between the RASGEF1A gene and CN-AML has not been reported. METHODS: By integrating the Cancer Genome Atlas (TCGA) database 75 patients with CN-AML and 240 Gene Expression Omnibus (GEO) database CN-AML samples, we examined the association between RASGEF1A's RNA expression level and DNA methylation of and AML patients' prognosis. Then, we investigated the RASGEF1A RNA expression and DNA methylation's prognostic value in 77 patients with AML after allogeneic hematopoietic stem cell transplantation (Allo-HSCT) as well as 101 AML patients after chemotherapy respectively. We investigated the association between sensitivity to Crenolanib and expression level of RASGED1A in patients by integrating 191 CN-AML patients from BeatAML dadataset. We integrated the expression and methylation of RASGEF1A to predict the CN-AML patients' prognosis and investigated the relationship between prognostic of AML patients with different risk classification and expression levels or methylation levels of RASGEF1A. RESULTS: We found that RASGEF1A gene high expression group predicted poorer event-free survival (EFS) (P< 0.0001) as well as overall survival (OS) (P< 0.0001) in CN-AML samples, and the identical results were found in AML patients receiving chemotherapy (P< 0.0001) and Allo-HSCT (P< 0.0001). RASGEF1A RNA expression level is an CN-AML patients' independent prognostic factor (EFS: HR = 5.5534, 95% CI: 1.2982-23.756, P= 0.0208; OS: HR = 5.3615, 95% CI: 1.1014-26.099, P= 0.0376). The IC50 (half maximal inhibitory concentration) of Crenolanib of CN-AML samples with RASGEF1A high expression level is lower. In addition, patients with high RASGEF1A methylation level had significant favorable prognosis (EPS: P< 0.0001, OS: P< 0.0001). Furthermore, the integrative analysis of expression and methylation of RASGEF1A could classify CN-AML patients into subgroups with different prognosis (EFS: P= 0.034, OS: P= 0.0024). Expression levels or methylation levels of RASGEF1A help to improve risk classification of 2010 European Leukemia Net. CONCLUSION: Higher RASGEF1A RNA expression and lower DNA methylation predicts CN-AML patients' poorer prognosis. The RASGEF1A high expression level from patients with CN-AML have better sensitivity to Crenolanib. The integrative analysis of RASGEF1A RNA expression and DNA methylation can provide a more accurate classification for prognosis. Lower RASGEF1A expression is a favorable prognostic factor for AML patients receiving chemotherapy or Allo-HSCT. 2010 European Leukemia Net's risk classification can be improved by RASGEF1A expression levels or methylation levels.

An examination of mediation by DNA methylation on birthweight differences induced by assisted reproductive technologies.

BACKGROUND: Children born after assisted reproductive technologies (ART) differ in birthweight from those naturally conceived. It has been hypothesized that this might be explained by epigenetic mechanisms. We examined whether cord blood DNA methylation mediated the birthweight difference between 890 newborns conceived by ART (764 by fresh embryo transfer and 126 frozen thawed embryo transfer) and 983 naturally conceived newborns from the Norwegian Mother, Father, and Child Cohort Study (MoBa). DNA methylation was measured by the Illumina Infinium MethylationEPIC array. We conducted mediation analyses to assess whether differentially methylated CpGs mediated the differences in birthweight observed between: (1) fresh embryo transfer and natural conception and (2) frozen and fresh embryo transfer. RESULTS: We observed a difference in birthweight between fresh embryo transfer and naturally conceived offspring of - 120 g. 44% (95% confidence interval [CI] 26% to 81%) of this difference in birthweight between fresh embryo transfer and naturally conceived offspring was explained by differences in methylation levels at four CpGs near LOXL1, CDH20, and DRC1. DNA methylation differences at two CpGs near PTGS1 and RASGRP4 jointly mediated 22% (95% CI 8.1% to 50.3%) of the birthweight differences between fresh and frozen embryo transfer. CONCLUSION: Our findings suggest that DNA methylation is an important mechanism in explaining birthweight differences according to the mode of conception. Further research should examine how gene regulation at these loci influences fetal growth.

Validation of a Chromosome 14 Risk Haplotype for Idiopathic Epilepsy in the Belgian Shepherd Dog Found to Be Associated with an Insertion in the RAPGEF5 Gene.

An idiopathic epilepsy (IE) risk haplotype on canine chromosome (CFA) 14 has been reported to interact with the CFA37 common risk haplotype in the Belgian shepherd (BS). Additional IE cases and control dogs were genotyped for the risk haplotypes to validate these previous findings. In the new cohort, the interaction between the two regions significantly elevated IE risk. When the haplotypes were analyzed individually, particular haplotypes on both CFA14 (ACTG) and 37 (GG) were associated with elevated IE risk, though only the CFA37 AA was significantly associated (p < 0.003) with reduced risk in the new cohort. However, the CFA14 ACTG risk was statistically significant when the new and previous cohort data were combined. The frequency of the ACTG haplotype was four-fold higher in BS dogs than in other breeds. Whole genome sequence analysis revealed that a 3-base pair predicted disruptive insertion in the RAPGEF5 gene, which is adjacent to the CFA14 risk haplotype. RAPGEF5 is involved in the Wnt-ß-catenin signaling pathway that is crucial for normal brain function. Although this risk variant does not fully predict the likelihood of a BS developing IE, the association with a variant in a candidate gene may provide insight into the genetic control of canine IE.

Identification of immune-related and autophagy-related genes for the prediction of survival in bladder cancer.

BACKGROUND: Bladder cancer has the characteristics of high morbidity and mortality, and the prevalence of bladder cancer has been increasing in recent years. Immune and autophagy related genes play important roles in cancer, but there are few studies on their effects on the prognosis of bladder cancer patients. METHODS: Using gene expression data from the TCGA-BLCA database, we clustered bladder cancer samples into 6 immune-related and autophagy-related molecular subtypes with different prognostic outcomes based on 2208 immune-related and autophagy-related genes. Six subtypes were divided into two groups which had significantly different prognosis. Differential expression analysis was used to explore genes closely related to the progression of bladder cancer. Then we used Cox stepwise regression to define a combination of gene expression levels and immune infiltration indexes to construct the risk model. Finally, we built a Nomogram which consist of risk score and several other prognosis-related clinical indicators. RESULTS: The risk model suggested that high expression of C5AR2, CSF3R, FBXW10, FCAR, GHR, OLR1, PGLYRP3, RASGRP4, S100A12 was associated with poor prognosis, while high expression level of CD96, IL10, MEFV pointed to a better prognosis. Validation by internal and external dataset suggested that our risk model had a high ability to discriminate between the outcomes of patients with bladder cancer. The immunohistochemical results basically confirmed our results. The C-Index value and Calibration curves verified the robustness of Nomogram. CONCLUSIONS: Our study constructed a model that included a risk score for patients with bladder cancer, which provided a lot of helps to predict the prognosis of patients with bladder cancer.

Ras guanine nucleotide-releasing protein-4 promotes renal inflammatory injury in type 2 diabetes mellitus.

INTRODUCTION: Ras guanine nucleotide-releasing protein-4 (RasGRP4) is an activator of Ras protein, which plays significant roles in both the inflammatory response and immune activation. This study determined the role of RasGRP4 in diabetic kidney disease (DKD) progression. METHODS: CRISPR/Cas9 technology was used to establish RasGRP4 knockout (KO) mice. Diabetes was induced by a high-fat diet combined with five consecutive daily intraperitoneal injections of streptozotocin (60 mg/kg) in C57BL/6J wild-type (WT) mice and RasGRP4 KO mice. Hematoxylin and eosin, periodic acid-Schiff, and Masson's trichrome staining were used to observe the histology of pathological injury. Immunohistochemical staining was used to analyze inflammatory cell infiltration. Quantitative PCR and Western blotting were used to detect the expression of inflammatory mediators and the activation of signaling pathways in renal tissues. In vitro cell co-culture experiments were performed to explore the interactions between peripheral blood mononuclear cells (PBMCs) and glomerular endothelial cells (GEnCs). RESULTS: RasGRP4 KO mice developed less severe diabetic kidney injury compared to WT mice, exhibiting lower proteinuria, reduced CD3+ T lymphocyte and F4/80+ macrophage infiltration, less inflammatory mediator expression including interleukin 6, tumor necrosis alpha, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, and lower expression levels of critical signal transduction molecules in the NLR family pyrin domain-containing 3 inflammasome and mitogen-activated protein kinase (MAPK)/nuclear factor kappa B (NF-κB) signaling pathways in the diabetic kidney. In vitro experiments showed that the adhesion function of PBMCs of RasGRP4 KO mice was reduced compared to that of WT mice. Moreover, the expression of adhesion molecules and critical signal transduction molecules in the NLRP3 inflammasome and MAPK/NF-κB signaling pathways in GEnCs was stimulated by the supernatant of PBMCs, which were derived from RasGRP4 KO mice treated with high glucose and were also significantly reduced compared to those derived from WT mice. CONCLUSION: RasGRP4 promotes the inflammatory injury mediated by PBMCs in diabetes, probably by regulating the interaction between PBMCs and GEnCs and further activating the NLRP3 inflammasome and MAPK/NF-κB signaling pathways.

Mutated RAS-associating proteins and ERK activation in relapse/refractory diffuse large B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is successfully treated with combination immuno-chemotherapy, but relapse with resistant disease occurs in ~ 40% of patients. However, little is known regarding relapsed/refractory DLBCL (rrDLBCL) genetics and alternative therapies. Based on findings from other tumors, we hypothesized that RAS-MEK-ERK signaling would be upregulated in resistant tumors, potentially correlating with mutations in RAS, RAF, or associated proteins. We analyzed mutations and phospho-ERK levels in tumor samples from rrDLBCL patients. Unlike other tumor types, rrDLBCL is not mutated in any Ras or Raf family members, despite having increased expression of p-ERK. In paired biopsies comparing diagnostic and relapsed specimens, 33% of tumors gained p-ERK expression, suggesting a role in promoting survival. We did find mutations in several Ras-associating proteins, including GEFs, GAPs, and downstream effectors that could account for increased ERK activation. We further investigated mutations in one such protein, RASGRP4. In silico modeling indicated an increased interaction between H-Ras and mutant RASGRP4. In cell lines, mutant RASGRP4 increased basal p-ERK expression and lead to a growth advantage in colony forming assays when challenged with doxorubicin. Relapsed/refractory DLBCL is often associated with increased survival signals downstream of ERK, potentially corresponding with mutations in protein controlling RAS/MEK/ERK signaling.

Regulation of Leukaemia Associated Rho GEF (LARG/ARHGEF12).

The Ras homologous (Rho) protein family of GTPases (RhoA, RhoB and RhoC) are the members of the Ras superfamily and regulate cellular processes such as cell migration, proliferation, polarization, adhesion, gene transcription and cytoskeletal structure. Rho GTPases function as molecular switches that cycle between GTP-bound (active state) and GDP-bound (inactive state) forms. Leukaemia-associated RhoGEF (LARG) is a guanine nucleotide exchange factor (GEF) that activates RhoA subfamily GTPases by promoting the exchange of GDP for GTP. LARG is selective for RhoA subfamily GTPases and is an essential regulator of cell migration and invasion. Here, we describe the mechanisms by which LARG is regulated to facilitate the understanding of how LARG mediates functions like cell motility and to provide insight for better therapeutic targeting of these functions.

Natural selection at the RASGEF1C (GGC) repeat in human and divergent genotypes in late-onset neurocognitive disorder.

Expression dysregulation of the neuron-specific gene, RASGEF1C (RasGEF Domain Family Member 1C), occurs in late-onset neurocognitive disorders (NCDs), such as Alzheimer's disease. This gene contains a (GGC)13, spanning its core promoter and 5' untranslated region (RASGEF1C-201 ENST00000361132.9). Here we sequenced the (GGC)-repeat in a sample of human subjects (N = 269), consisting of late-onset NCDs (N = 115) and controls (N = 154). We also studied the status of this STR across various primate and non-primate species based on Ensembl 103. The 6-repeat allele was the predominant allele in the controls (frequency = 0.85) and NCD patients (frequency = 0.78). The NCD genotype compartment consisted of an excess of genotypes that lacked the 6-repeat (divergent genotypes) (Mid-P exact = 0.004). A number of those genotypes were not detected in the control group (Mid-P exact = 0.007). The RASGEF1C (GGC)-repeat expanded beyond 2-repeats specifically in primates, and was at maximum length in human. We conclude that there is natural selection for the 6-repeat allele of the RASGEF1C (GGC)-repeat in human, and significant divergence from that allele in late-onset NCDs. STR alleles that are predominantly abundant and genotypes that deviate from those alleles are underappreciated features, which may have deep evolutionary and pathological consequences.

Circ_RASGEF1B Promotes LPS-Induced Apoptosis and Inflammatory Response by Targeting MicroRNA-146a-5p/Pdk1 Axis in Septic Acute Kidney Injury Cell Model.

BACKGROUND: We intended to investigate the function of circular RNA RasGEF domain family member 1B (circ_RASGEF1B) in lipopolysaccharide (LPS)-induced septic acute kidney injury (AKI) cell model and its associated mechanism. METHODS: TCMK-1 cells were exposed to 10 µg/mL LPS for 24 h to establish a septic AKI cell model. Mice were intraperitoneally injected with 10 mg/kg LPS to establish a septic AKI mice model. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were used to measure RNA and protein expression, respectively. Cell viability and apoptosis were assessed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry. Cell inflammatory response was analyzed using enzyme-linked immunosorbent assay. Dual-luciferase reporter assay was conducted to confirm the predicted target relationship between microRNA-146a-5p (miR-146a-5p) and circ_RASGEF1B or pyruvate dehydrogenase kinase 1 (Pdk1). RESULTS: The circ_RASGEF1B level was upregulated in LPS-induced TCMK-1 cells and septic AKI mice models. LPS exposure reduced cell viability and promoted cell apoptosis and inflammatory response partly by upregulating circ_RASGEF1B. Circ_RASGEF1B bound to miR-146a-5p and miR-146a-5p interference partly overturned circ_RASGEF1B silencing-mediated effects in LPS-induced TCMK-1 cells. Pdk1 was a target of miR-146a-5p, and Pdk1 accumulation partly counteracted miR-146a-5p-induced influences in TCMK-1 cells upon LPS stimulation. CONCLUSION: Circ_RASGEF1B promoted LPS-induced apoptosis and inflammatory response in renal tubular epithelial cells partly by upregulating Pdk1 via acting as miR-146a-5p sponge.

Identification of biomarkers associated with metabolic cardiovascular disease using mRNA-SNP-miRNA regulatory network analysis.

BACKGROUND: CVD is the leading cause of death in T2DM patients. However, few biomarkers have been identified to detect and diagnose CVD in the early stage of T2DM. The aim of our study was to identify the important mRNAs, micro (mi)RNAs and SNPs (single nucleotide polymorphisms) that are associated with metabolic cardiovascular disease. MATERIALS AND METHODS: Expression profiles and GWAS data were obtained from Gene Expression Omnibus (GEO) database. MiRNA-sequencing was conducted by Illumina HiSeq 2000 platform in T2DM patients and T2DM with CVD patients. EQTL analysis and gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted. MRNA-miRNA co-expression network and mRNA-SNP-miRNA interaction network were established and visualized by Cytoscape 3.7.2. RESULTS: In our study, we identified 56 genes and 16 miRNAs that were significantly differentially expressed. KEGG analyses results indicated that B cell receptor signaling pathway and hematopoietic cell lineage were included in the biological functions of differentially expressed genes. MRNA-miRNA co-expression network and mRNA-SNP-miRNA interaction network illustrated that let-7i-5p, RASGRP3, KRT1 and CEP41 may be potential biomarkers for the early detection and diagnosis of CVD in T2DM patients. CONCLUSION: Our results suggested that downregulated let-7i-5p, and upregulated RASGRP3, KRT1 and CEP41 may play crucial roles in molecular mechanisms underlying the initiation and development of CVD in T2DM patients.

RASGRF2 gene fusions identified in a variety of melanocytic lesions with distinct morphological features.

The WHO classification identifies nine classes of melanocytic proliferations according to location, UV exposure, histological, and genetic features. Only a minority of lesions remain unclassified. We describe five cases that harbored either an ERBIN-RASGRF2 or an ATP2B4-RASGRF2 in-frame fusion transcript. These lesions were collected from different studies, unified only by the lack of identifiable known mutations, with a highly variable phenotype. One case was a large abdominal congenital nevus, three were slowly growing pigmented nodules, and the last was an ulcerated nodule arising on the site of a preexisting small nevus, known since childhood. The latter was diagnosed as a 4 mm thick melanoma with loss of BAP1 expression. The four other cases were compound, melanocytic proliferations with an unusual deep pattern of small dense nests of bland melanocytes encased in a fibrous background. The RASGRF2 fusion was confirmed by a break-apart FISH technique. Array CGH performed in three cases found non-recurrent secondary copy number alterations. Follow-up was uneventful. In silico analysis identified a single RASGRF2 fusion in the TCGA pan-cancer database, whereas RASGRF2 variants were stochastically distributed in all cancer subtypes.

Critical requirement of SOS1 RAS-GEF function for mitochondrial dynamics, metabolism, and redox homeostasis.

SOS1 ablation causes specific defective phenotypes in MEFs including increased levels of intracellular ROS. We showed that the mitochondria-targeted antioxidant MitoTEMPO restores normal endogenous ROS levels, suggesting predominant involvement of mitochondria in generation of this defective SOS1-dependent phenotype. The absence of SOS1 caused specific alterations of mitochondrial shape, mass, and dynamics accompanied by higher percentage of dysfunctional mitochondria and lower rates of electron transport in comparison to WT or SOS2-KO counterparts. SOS1-deficient MEFs also exhibited specific alterations of respiratory complexes and their assembly into mitochondrial supercomplexes and consistently reduced rates of respiration, glycolysis, and ATP production, together with distinctive patterns of substrate preference for oxidative energy metabolism and dependence on glucose for survival. RASless cells showed defective respiratory/metabolic phenotypes reminiscent of those of SOS1-deficient MEFs, suggesting that the mitochondrial defects of these cells are mechanistically linked to the absence of SOS1-GEF activity on cellular RAS targets. Our observations provide a direct mechanistic link between SOS1 and control of cellular oxidative stress and suggest that SOS1-mediated RAS activation is required for correct mitochondrial dynamics and function.

Ras GEF Mouse Models for the Analysis of Ras Biology and Signaling.

Animal models have become in recent years a crucial tool to understand the physiological and pathological roles of many cellular proteins. They allow analysis of the functional consequences of [1] complete or partial (time- or organ-limited) removal of specific proteins (knockout animals), [2] the exchange of a wild-type allele for a mutant or truncated version found in human illnesses (knock-in), or [3] the effect of overexpression of a given protein in the whole body or in specific organs (transgenic mice). In this regard, the study of phenotypes in Ras GEF animal models has allowed researchers to find specific functions for otherwise very similar proteins, uncovering their role in physiological contexts such as memory formation, lymphopoiesis, photoreception, or body homeostasis. In addition, mouse models have been used to unveil the functional role of Ras GEFs under pathological conditions, including Noonan syndrome, skin tumorigenesis, inflammatory diseases, diabetes, or ischemia among others. In the following sections, we will describe the methodological approaches employed for Ras GEF animal model analyses, as well as the main discoveries made.

Impaired contextual fear conditioning in RasGRF2 mutant mice is likely Ras-ERK-dependent.

Ras/Raf/MEK/ERK (Ras-ERK) signaling has been shown to play an important role in fear acquisition. However, little information is known regarding the mechanisms that contribute to the regulation of this pathway in terms of the learning of conditioned fears. Ras Guanine Nucleotide Releasing Factor 2 (RasGRF2) is one of two guanine nucleotide exchange factors (GEF) that regulates the Ras-ERK signaling pathway in a Ca2+-dependent manner via control of the cycling of Ras isoforms between an inactive and active state. Here we sought to determine the role of RasGRF2 on contextual fear conditioning in RasGRF2 knockout (KO) and their wild type (WT) counterparts. Male KO and WT mice underwent a single session of contextual fear conditioning (12 min, 4 unsignaled shocks), followed by either daily 12-min retention trials or the molecular analysis of Ras activation and pERK1/2 activity. KO mice showed an impaired acquisition of contextual fear, as demonstrated by reduced freezing during fear conditioning and 24-hr retention tests relative to WT mice. Ras analysis following fear conditioning demonstrated a reduction in Ras activation in the hippocampus as well as a reduction in pERK1/2 in the CA1 region of the hippocampus in KO mice, suggesting that the decrease in fear conditioning in KO mice is at least in part due to the impairment of Ras-ERK signaling in the hippocampus during learning. These data indicate a role for RasGRF2 in contextual fear conditioning in mice that may be Ras-ERK-dependent.