Fibroblast Growth Factor Receptor Substrate Research Articles

Hsa_circ_0007765 Promotes Platelet-Derived Growth Factor-BB-Induced Proliferation and Migration of Human Aortic Vascular Smooth Muscle Cells in Atherosclerosis.

Humanaorticvascularsmoothmusclecells (HA-VSMCs) play vital roles in the pathogenesis ofvasculardiseases, including Atherosclerosis (AS). Circular RNAs (circRNAs) have been reported to regulate the biological functions of HA-VSMCs.Therefore, this study aimed to explore the role and mechanism of hsa_circRNA_102353 (circ_0007765) in platelet-derived growth factor-BB (PDGF-BB)-induced HA-VSMCs. Circ_0007765, microRNA-654-3p (miR-654-3p), and Fibroblast Growth Factor Receptor Substrate 2 (FRS2) expression were measured using real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferative ability, invasion, and migration were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), Transwell, and wound healing assays. CyclinD1, MMP2, and FRS2 protein levels were assessed using a Western blot assay. Binding between miR-654-3p and circ_0007765 or FRS2 was predicted by Circinteractome or TargetScan, and verified using dual-luciferase reporter and RNA pull-down assays. PDGF-BB induced HA-VSMC proliferation, invasion, and migration. Circ_0007765 and FRS2 expression levels were increased in PDGF-BB-treated HA-VSMCs, and the miR-654-3p level was reduced. Moreover, circ_0007765 absence hindered PDGF-BB-induced HA-VSMC proliferation, invasion, and migration in vitro. At the molecular level, circ_0007765 increased FRS2 expression by acting as a sponge for miR-654-3p. Our findings revealed that circ_0007765 boosted PDGF-BB-induced HA-VSMC proliferation and migration through elevating FRS2 expression via adsorbing miR-654-3p, providing a feasible therapeutic strategy for AS.

Inhibiting FGFR by toadflax reverses erlotinib resistance in nonsmall cell lung cancer

This study aims to demonstrate the effect of toadflax (bufalin) on erlotinib resistance in nonsmall cell lung cancer (NSCLC) by inhibiting the fibroblast growth factor receptor (FGFR). The microfluidic mobility transferase and caliper mobility-shift assays were employed to detect the FGFR inhibition by bufalin and the binding reversibility. Further, the inhibitory effects of bufalin were determined in HCC827 and HCC827/ER cells in vitro, investigating relative FGFR overexpression by quantitative reverse transcriptase-PCR (RT-qPCR) and FGFR downstream proteins, that is, FGFR substrate 2 (FRS2), extracellular signal-regulated kinase (ERK), and S6 by western blot analysis. Finally, HCC827/ER-inoculated xenograft tumors were constructed to observe the effects of bufalin and bufalin + erlotinib intervention on tumor growth. Bufalin inhibited FGFR by reversibly binding to FGFR1. In addition, the western blot analysis indicated a significant reduction in the expression levels of FGFR, FRS2, ERK, and S6 proteins in HCC827 and HCC827/ER cells, increasing the expression levels of apoptotic caspase-3 and poly-(ADP-ribose) polymerase proteins. Bufalin + erlotinib combination significantly inhibited the apoptosis of HCC827/ER cells and subsequent tumor growth in vivo. In addition, FGFR overexpression significantly reversed the sensitivity of bufalin to HCC827/ER cells, promoting the value-addition of HCC827/ER cells. Further, bufalin + erlotinib significantly reduced the growth of erlotinib-resistant HCC827/ER tumors, induced apoptosis, and inhibited the expression of FGFR and p-ERK proteins. These findings indicated that bufalin could reverse the erlotinib resistance in NSCLC by inhibiting the FGFR expression.

Macrophage-derived FGFR1 drives atherosclerosis through PLCγ-mediated activation of NF-κB inflammatory signalling pathway.

Atherosclerosis (AS) is a leading cause of cardiovascular morbidity and mortality. Atherosclerotic lesions show increased levels of proteins associated with the fibroblast growth factor receptor (FGFR) pathway. However, the functional significance and mechanisms governed by FGFR signalling in AS are not known. In the present study, we investigated fibroblast growth factor receptor 1 (FGFR1) signalling in AS development and progression. Examination of human atherosclerotic lesions and aortas of Apoe-/- mice fed a high-fat diet (HFD) showed increased levels of FGFR1 in macrophages. We deleted myeloid-expressed Fgfr1 in Apoe-/- mice and showed that Fgfr1 deficiency reduces atherosclerotic lesions and lipid accumulations in both male and female mice upon HFD feeding. These protective effects of myeloid Fgfr1 deficiency were also observed when mice with intact FGFR1 were treated with FGFR inhibitor AZD4547. To understand the mechanistic basis of this protection, we harvested macrophages from mice and show that FGFR1 is required for macrophage inflammatory responses and uptake of oxidized LDL. RNA sequencing showed that FGFR1 activity is mediated through phospholipase-C-gamma (PLCγ) and the activation of nuclear factor-κB (NF-κB) but is independent of FGFR substrate 2. Our study provides evidence of a new FGFR1-PLCγ-NF-κB axis in macrophages in inflammatory AS, supporting FGFR1 as a potentially therapeutic target for AS-related diseases.

Circ_0004872 deficiency attenuates ox-LDL-induced vascular smooth muscle cell dysfunction by miR-424-5p-dependent regulation of FRS2.

Atherosclerosis (AS) is a pivotal pathological basis of cardiovascular and cerebrovascular diseases, and circular RNAs (circRNAs) has been disclosed to exert a vital part in the progression of AS. However, the functions of circ_0004872 in the progression of AS is indistinct. In this context, we aimed to elucidate the role of circ_0004872 and the potential mechanism in AS. The level of circ_0004872, miR-424-5p and fibroblast growth factor receptor substrate 2 (FRS2) was detected using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was monitored by Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine (EDU) assays. The invasion and migration capabilities of VSMCs were tested by transwell assays and wound-healing assay, respectively. Western blot was adopted to check the protein levels of CyclinD1, Vimentin and FRS2. Dual-luciferase reporter and RNA immunoprecipitation assay were executed to manifest the interaction between miR-424-5p and circ_0004872 or FRS2. The level of circ_0004872 was increased in the serum samples of AS patients and ox-LDL-exposed VSMCs. Ox-LDL exposure triggered cell proliferation, invasion and migration ability of VSMCs. depletion of circ_0004872 partly weakened ox-LDL-mediated effects in VSMCs. Mechanistically, circ_0004872 functioned as a sponge of miR-424-5p, and miR-424-5p inhibition partly alleviated circ_0004872 deficiency-mediated influences in VSMCs. Additionally, miR-424-5p interacted with FRS2, and miR-424-5p constrained dysfunction in ox-LDL-stimulated VSMCs via reducing FRS2 level. Notably, circ_0004872 functioned as a sponge of miR-424-5p to elevate FRS2 expression. Circ_0004872 accelerated ox-LDL-induced damage via mediating miR-424-5p/FRS2 axis.

Hsa_circ_0032389 Enhances Proliferation and Migration in PDGF-BB-Induced Human Aortic Vascular Smooth Muscle Cells.

Circular RNA (circRNAs) has been confirmed to participate in atherosclerosis (AS) progression. However, the role and mechanism of hsa_circ_0032389 in AS process still need to be further revealed. This study evaluates the role and mechanism of hsa_circ_0032389 in AS process. Platelet-derived growth factor-BB (PDGF-BB) was used to induce human aortic vascular smooth muscle cells (HA-VSMCs). The expression levels of hsa_circ_0032389, microRNA (miR)-513a-5p, and fibroblast growth factor receptor substrate 2 (FRS2) were examined by quantitative real-time PCR. Cell proliferation and migration were analyzed using cell counting kit 8 assay, flow cytometry, EdU assay, transwell assay, and wound healing assay. Protein expression was assessed using western blot analysis. Dual-luciferase reporter and RIP assays were used to confirm RNA interaction. Hsa_circ_0032389 was overexpressed in PDGF-BB-induced HA-VSMCs, and its downregulation inhibited HA-VSMC viability, cell cycle, EdU positive cell rate, migratory cell number, and wound closure rate under PDGF-BB treatment. The luciferase activity of hsa_circ_0032389wt could be reduced by miR-513a-5p mimic, and both hsa_circ_0032389 and miR-513a-5p were enriched in anti-Ago2, confirming that miR-513a-5p could be sponged by hsa_circ_0032389. MiR-513a-5p inhibitor reversed the effect of hsa_circ_0032389 knockdown on PDGF-BB-induced HA-VSMC viability, cell cycle, EdU positive cell rate, migratory cell number, and wound closure rate. Moreover, the luciferase activity of FRS2wt was reduced by miR-513a-5p mimic, and both FRS2 and miR-513a-5p were enriched in anti-Ago2, verifying that FRS2 was targeted by miR-513a-5p. MiR-513a-5p suppressed PDGF-BB-induced HA-VSMC viability, cell cycle, EdU positive cell rate, migratory cell number, and wound closure rate by targeting FRS2. Our results indicated that hsa_circ_0032389 enhanced PDGF-BB-induced HA-VSMC proliferation and migration via regulating miR-513a-5p/FRS2 axis.

Expression of fibroblast growth factor receptor substrate 2 (FRS2) in primary retroperitoneal liposarcoma and its clinical implications.

The aim of this study was to investigate the expression level of fibroblast growth factor receptor substrate 2 (FRS2) in tissues of patients with primary retroperitoneal liposarcoma (PRPLS) and its correlation with recurrence and prognosis. The pathological specimens, medical records, and follow-up information of patients with PRPLS who underwent radical surgery for the first time in our hospital from January 2013 to December 2016 were retrospectively analyzed. FRS2 protein expression in tissues was determined by immunohistochemistry staining, and the FRS2 protein positive rates in patients with different clinicopathological features were compared. Factors influencing patients' recurrence and survival were determined using the multivariate Cox stepwise regression model. This research enrolled 87 patients with PRPLS, with the number of cases presenting FRS2 protein positive rate and positive rate in pathological tissues accounting for 62.07% (54/87) and 37.93% (33/87), respectively. The positive expression of FRS2 protein varied markedly among patients with different pathological types, FNCC, LCC grade, number of tumors, positive margin, and recurrence and metastasis (with vs. without) (all p<0.05). The 87 patients were followed up for 3.5-102 months (median, 27.5 months), with a postoperative 5-year overall disease-free survival (DFS) rate of 17.24% [median progression-free survival (PFS): 24.7 months] and a 5-year overall survival (OS) rate of 44.83% (median OS: 47.3 months). Kaplan-Meier survival curves revealed significantly shorter PFS and OS in patients with positive FRS2 protein expression vs. those with negative FRS2 protein expression (χ2=6.396, 5.032, p<0.05). According to the univariate analysis, the 5-year overall DFS rate varied significantly among patients with different pathological types, Fédération Nationale des Centres De Lutte Contre le Cancer (FNCLCC) grades, number of tumors, positive margin, and FRS2 protein expression (all p<0.05). Pathological type, FNCLCC grading, tumor number, recurrence and metastasis, positive margin, and FRS2 protein expression were significantly correlated with the 5-year OS rate of patients (all p<0.05). Furthermore, pathological type, FNCLCC grading, multiple tumors, positive margin, and FRS2 protein expression were identified by multivariate Cox regression analysis to be independent factors that affected patients' 5-year DFS and OS rates (all p<0.05), and that relapsed and metastasized patients had a 4.586-fold risk of death than those without recurrence and metastasis. FRS2 shows a high positive rate in the tissues of PRPLS patients and is significantly related to the prognostic recurrence and survival of patients, with potential value in judging the prognosis of patients.

Mutation S249C of FGFR3b Promotes Bladder Cancer through Downstream Signaling Proteins FRS2 and FRS3: A Computational Approach

Background: Bladder cancer is the 9th most prevalent malignancy worldwide. Fibroblast Growth Factor Receptor 3b (FGFR3b), involved in cell proliferation, differentiation, and migration, is a mutations hotspot for bladder cancer with the most prevalent aberration being S249C. Objective: Impact of S249C of FGFR3b on bladder tumorigenesis via immediate downstream adapter proteins, Fibroblast Growth Factor Receptor Substrate (FRS2 and FRS3) is analyzed computationally. Methods: Wildtype FGFR3b monomer was modeled using I-TASSER and Phyre2. Whereas, S249C mutation was introduced via DynaMut. Wildtype FGFR3b homodimer and mutant heterodimer were structured and docked with downstream proteins using HADDOCK. PDBSum was used to study the amino acid residues involved in intermolecular and intramolecular interactions. Results: Parameters of molecular flexibility and interatomic interactions predicted S249C heterodimer mutation of FGFR3b to be stable. Furthermore, docking with FRS2 protein revealed greater stability and higher binding affinity for S249C heterodimer mutant compared to wildtype homodimer. However, FRS3 docking showed a negligible decline in binding affinity for the S249C mutation but based on Van der Waal’s energy and insights into the interacting residues, it was revealed that these interactions might be stronger and for longer duration in comparison to the wildtype homodimer. Conclusion: S249C heterodimer mutation of FGFR3b is predicted to be stable with a tumorigenic potential where FRS2 and FRS3 might be among the key players of altered downstream signaling. Further investigations are required for a detailed picture.

Hsa_circ_0093741 competes with FRS2 for miR-562 binding sites to promote nephroblastoma progression.

Circular RNA (circRNA) has been shown to play an essential role in cancer progression, including nephroblastoma. Hsa_circ_0093741 was discovered to be highly expressed in nephroblastoma. However, its function and mechanism in nephroblastoma development are still vague. The expression levels of hsa_circ_0093741, miR-562 and FRS2 (Fibroblast Growth Factor Receptor Substrate 2) were detected using western blotting and quantitative real-time polymerase chain reaction. Functional experiments were performed by using cell counting kit-8, colony formation, 5-ethynyl-2'-deoxyuridine (EdU), transwell, scratch assays in vitro and animal experiments in vivo. The interaction analysis was conducted using dual-luciferase reporter assay and RIP assay. Hsa_circ_0093741 was highly expressed in nephroblastoma tissues and cells. Functionally, hsa_circ_0093741 silencing significantly suppressed the growth, invasion, and migration of nephroblastoma cells in vitro. MiR-562 was decreased in nephroblastoma, and was validated to be a target of hsa_circ_0093741. Inhibition of miR-562 reversed the anticancer functions of hsa_circ_0093741 silencing on nephroblastoma cells. FRS2 expression was increased in nephroblastoma and served as a target of miR-562, moreover, FRS2 overexpression attenuated the inhibitory functions of miR-562 on the nephroblastoma cell malignant phenotypes mentioned above. Pre-clinically, lentivirus-mediated hsa_circ_0093741 silencing also impeded nephroblastoma tumor growth and metastasis in vivo. Knockdown of hsa_circ_0093741 suppresses nephroblastoma cell growth, migration and invasion by regulating the miR-562/FRS2 axis, suggesting the potential involvement of hsa_circ_0093741 in nephroblastoma progression.

MiR-338-3p acts as a tumor suppressor in lung squamous cell carcinoma by targeting FGFR2/FRS2

BackgroundLung cancer refers to the occurrence of malignant tumors in the lung, and squamous cell carcinoma is one of the most common pathological types of non-small cell lung cancer. Studies have shown that microRNAs (miRNAs) play an important role in the occurrence, development, early diagnosis, and treatment of lung cancer. This study aimed to explore the role and possible mechanism of MicroRNA-338-3p (miR-338-3p) in lung squamous cell carcinoma (LUSC). MethodIn this study, we compared 238 LUSC patients with relatively high miR-338-3p expression levels with 238 miR-338-3p expression levels in The Cancer Genome Atlas (TCGA)-LUSC dataset using first-line gene set enrichment analysis (GSEA). Second, the mRNA expression of miR-338-3p, FGFR2, and fibroblast growth factor receptor substrate 2 (FRS2) in 30 lung cancers and adjacent lung tissues was detected using quantitative real-time polymerase chain reaction (qRT-PCR). Finally, in vitro experiments were conducted, whereby the expression levels of miR-338-3p in lung cancer cells (H1703, SKMES1, H2170, H520) and normal lung epithelial cells (16HBE) were detected using qRT-PCR. miR-338-3p was overexpressed in lung cancer cells (H1703), and the cell proliferation (cell counting kit-8 [CCK8] assay), colony formation, cell apoptosis, cell cycle (BD-FACSVerse assay, Becton Dickinson, Bedford, MA, USA), cell invasion, and migration (Transwell assay, Thermo Fischer Corporation, Waltham, MA, USA) were detected. ResultsWe found that the expression of miR-338-3p was significantly reduced in LUSC tissues (p ​< ​0.001) and cancer cell lines (P < 0.01), and miR-338-3p was significantly negatively correlated with the expression of FGFR2 (P < 0.001) and FRS2 (P < 0.01). Furthermore, overexpression of miR-338-3p inhibited proliferation (P < 0.001), migration, and invasion (P < 0.001) of LUSC cell lines and increased apoptosis in the G1 phase (P < 0.001) and cell cycle arrest (P < 0.05). ConclusionsOur study demonstrates that miR-338-3p inhibits tumor cell proliferation and migration by targeting FGFR2 and FRS2 in LUSC. We believe that miR-338-3p may be a promising target for the treatment of LUSC.

Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action

Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase that plays a major role in developmental processes and metabolism. The dysregulation of FGFR1 through genetic aberrations leads to skeletal and metabolic diseases as well as cancer. For this reason, FGFR1 is a promising therapeutic target, yet a very challenging one due to potential on-target toxicity. More puzzling is that both agonistic and antagonistic FGFR1 antibodies are reported to exhibit similar toxicity profiles invivo, namely weight loss. In this study, we aimed to assess and compare the mechanism of action of these molecules to better understand this apparent contradiction. By systematically comparing the binding of these antibodies and the activation or the inhibition of the major FGFR1 signaling events, we demonstrated that the molecules displayed similar properties and can behave either as an agonist or antagonist depending on the presence or the absence of the endogenous ligand. We further demonstrated that these findings translated in xenografts mice models. In addition, using time-resolved FRET and mass spectrometry analysis, we showed a functionally distinct FGFR1 active conformation in the presence of an antibody that preferentially activates the FGFR substrate 2 (FRS2)-dependent signaling pathway, demonstrating that modulating the geometry of a FGFR1 dimer can effectively change the signaling outputs and ultimately the activity of the molecule in preclinical studies. Altogether, our results highlighted how bivalent antibodies can exhibit both agonistic and antagonistic activities and have implications for targeting other receptor tyrosine kinases with antibodies.

TRIM44 Promotes Endometrial Carcinoma Progression by Activating the FRS2 Signalling Pathway.

The Tripartite Motif Containing 44 (TRIM44) is highly expressed in a variety of tumours. However, the TRIM44's role in endometrial carcinoma (EC) progression remains unknown. To investigate the TRIM44's role in the development and metastasis of EC, we detected TRIM44 expression in EC cell lines and surgical specimens from patients with EC using immunohistochemistry, real-time reverse transcription-polymerase chain reaction, and western blotting analysis. The biological functions of TRIM44 by loss-of-function analysis in RL95-2 and Ishikawa cells were studied. The effect of TRIM44 on the progression of EC in terms of cell proliferation, apoptosis, and invasion was examined and revealed its underlying mechanism in vitro using EC cell lines and in vivo using mouse xenograft models. The TRIM44's expression was positively correlated with EC progression and poor prognosis. The TRIM44 knockdown reduced the EC cell proliferation and invasion while promoting cell apoptosis. Mechanism experiments showed that the TRIM44 interacts with Fibroblast Growth Factor Receptor Substrate 2 (FRS2) and negatively regulates the expression of Bone Morphogenetic Protein 4(BMP4), β-catenin, and Transforming Growth Factor Beta Receptor 1(TGF-βR1). Moreover, the effect of TRIM44 overexpression on EC cell proliferation, invasion, and apoptosis is reversed by the FRS2 knockdown. Our study may provide a new perspective on targeting the TRIM44/FRS2 signaling pathway in treating EC, which deserves further investigation.

Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) represent a major component of the tumor microenvironment and interplay with cancer cells by secreting cytokines, growth factors and extracellular matrix proteins. When estrogen receptor-negative breast cancer MDA-MB-231 cells were treated with the CAF-conditioned medium (CAF-CM), Akt and STAT3 involved in cell proliferation and survival were activated through phosphorylation. CAFs secrete fibroblast growth factor 2 (FGF2), thereby stimulating breast cancer cell progression. Akt activation induced by CAF-CM in MDA-MB-231 cells was abolished when FGF2-neutralizing antibody was added. Treatment of MDA-MB-231 cells directly with FGF2 enhanced the phosphorylation of Akt and the FGF receptor (FGFR) substrate, FRS2α. These events were abrogated by siRNA-mediated silencing of FGFR1. In a xenograft mouse model, co-injection of MDA-MB-231 cells with activated fibroblasts expressing FGF2 dramatically enhanced activation of Akt. Stable knockdown of FGFR1 blunted Akt phosphorylation in xenograft tumors. MDA-MB-231 cells co-cultured with CAFs or directly stimulated with FGF2 exhibited enhanced nuclear localization of FGFR1. Notably, FGF2 stimulation produced reactive oxygen species (ROS) accumulation in MDA-MB-231 cells, and FGF2-induced nuclear accumulation of FGFR1 was abrogated by the ROS scavenging agent, N-acetylcysteine.

Expression of FRS2 in atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma: an immunohistochemical analysis of 182 cases with genetic data

BackgroundThe fibroblast growth factor receptor substrate 2 (FRS2) gene is located close to MDM2 and CDK4 within the 12q13-15 chromosomal region. FRS2 gene was recently found to be consistently amplified in atypical lipomatous tumor (ALT)/well-differentiated liposarcoma (WDL) and dedifferentiated liposarcoma (DDL), suggesting the detection of FRS2 amplification could be a diagnostic tool for ALT/WDL/DDLs. However, the expression of FRS2 protein and diagnostic value of FRS2 immunohistochemistry (IHC) has not been evaluated in a large cohort of ALT/WDL/DDLs.MethodsA SNOMED search of hospital surgical pathology files from January 2007 to July 2020 identified 182 ALT/WDL/DDLs with available materials. FRS2 fluorescence in situ hybridization (FISH) and IHC were performed on 182 ALT/WDL/DDLs and 64 control samples. The expression of FRS2 was also compared with that of classic immunomarkers (MDM2 and CDK4) of this tumor entity.ResultsThis study included 91 ALT/WDLs and 91 DDLs. The FISH results showed 172 of 182 (94.5%) cases were FRS2-amplified, and 10 cases were FRS2-nonamplified. Immunostaining results showed 171 (94.0%) ALT/WDL/DDLs were positive for FRS2 and 11 cases (6.0%) were FRS2-immunonegative. In 172 FRS2-amplified cases, 166 (96.5%) were FRS2-immunopositive, and 6 (3.5%) were negative. Among 10 FRS2-nonamplified ALT/WDL/DDL cases, 5 cases were FRS2-immunonegative, and 5 tumors displayed 1+ staining for this marker. In 64 control cases, none of them exhibited FRS2 amplification. Forty-seven (73.5%) control cases were negative for FRS2 immunostaining, while 17 cases (26.5%) were FRS2-immunopositive. Fifteen of these false positive samples (15/17, 88.2%) showed 1+ positivity and only 2 cases (2/17, 11.8%) displayed 2+ positivity. In ALT/WDL/DDLs, the sensitivity of FRS2 immunostaining was slightly lower than MDM2 (FRS2 vs. MDM2: 94.0% vs 100.0%) and CDK4 (FRS2 vs. CDK4: 94.0% vs 97.0%). However, the specificity of FRS2 (73.5%) was slightly higher than that of MDM2 (67.8%) and CDK4 (64.4%).ConclusionThis study indicated that FRS2 IHC had relatively good consistency with FRS2 FISH, suggesting that FRS2 immunostaining could be utilized as an additional screening tool for the diagnosis of ALT/WDL/DDL. It must be emphasized that MDM2/CDK4/FRS2 especially MDM2 FISH remains the gold standard and the most recommended method to diagnose this entity.

Circ_0002984 Enhances Growth, Invasion, and Migration in PDGF-bb-Induced Vascular Smooth Muscle Cells Through miR-379-5p/FRS2 Axis.

The accumulation of vascular smooth muscle cells (VSMCs) is considered to play important roles in atherosclerosis (AS) development and progression. Circ_0002984 was found to be increased in oxidized low-density lipoprotein (ox-LDL) human VSMCs (HVSMCs). However, the function and mechanism of circ_0002984 in VSMC dysfunction remain unknown. In this study, the expression of circ_0002984, microRNA (miR)-379-5p, and fibroblast growth factor receptor substrate 2 (FRS2) was detected using quantitative real-time polymerase chain reaction and western blot. Cell proliferation, cell cycle, migration, and invasion were detected using Cell Counting Kit-8, flow cytometry, and transwell assays. The binding interaction between miR-379-5p and circ_0002984 or FRS2 was confirmed by the dual-luciferase reporter assay. Collectively, this study found that circ_0002984 was elevated in platelet-derived growth factor type bb (PDGF-bb)-induced HVSMCs. Circ_0002984 knockdown abrogated PDGF-bb-induced proliferation, migration, and invasion in HVSMCs. Mechanistically, circ_0002984 was confirmed to target miR-379-5p, and miR-379-5p upregulation reversed the protective effects of circ_0002984 knockdown on PDGF-bb-induced HVSMCs. Besides, when FRS2 was a target of miR-379-5p, miR-379-5p restoration abolished PDGF-bb-evoked HVSMC dysfunction, which was attenuated by the overexpression of FRS2. Moreover, circ_0002984 could regulate FRS2 expression through sponging miR-379-5p in HVSMCs. Collectively, these results demonstrated that circ_0002984 promoted PDGF-bb-induced VSMC proliferation, migration, and invasion through the regulation of miR-379-5p/FRS2 axis, suggesting a new insight into the pathogenesis of AS and the potential application of circ_0002984 in AS treatment.

Circular RNA circUBR4 regulates ox-LDL-induced proliferation and migration of vascular smooth muscle cells through miR-185-5p/FRS2 axis.

Circular RNAs (circRNAs) have been reported to play vital roles in atherosclerosis. However, the precise roles of circUBR4 in atherosclerosis remain unclear. The purpose of this study is to investigate the regulatory roles of circUBR4 in atherosclerosis. The expression levels of circUBR4, miR-185-5p, and Fibroblast growth factor receptor substrate 2 (FRS2) were analyzed by real-time quantitative polymerase chain reaction (RT-qPCR) assay. Human vascular smooth muscle cells (VSMCs) were treated with oxidized low-density lipoprotein (ox-LDL) to mimic atherosclerosis condition in vitro. Cell proliferation was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT), colony-forming, and 5-ethynyl-2'-deoxyuridine (EdU) assays. Wound healing and transwell assays were used to assess cell migration. The interaction relationship between miR-185-5p and circUBR4 or FRS2 was confirmed by dual-luciferase reporter and RNA pull-down assays. CircUBR4 was overexpressed in atherosclerosis patients and VSMCs treated with ox-LDL, and the knockdown of circUBR4 abolished ox-LDL-induced enhanced effects on the proliferation and migration of VSMCs. MiR-185-5p, interacted with FRS2, was a target of circUBR4 in VSMCs. The silencing of miR-185-5p reversed the effects caused by circUBR4 knockdown on ox-LDL-induced VSMCs. In addition, overexpression of miR-185-5p suppressed the proliferation and migration of VSMCs by targeting FRS2. CircUBR4 contributed to ox-LDL-induced VSMC proliferation and migration through up-regulating FRS2 via miR-185-5p.

Kaempferol alleviates human endothelial cell injury through circNOL12/miR-6873-3p/FRS2 axis

BackgroundAtherosclerosis, inflammatory disease, is a major reason for cardiovascular diseases and stroke. Kaempferol (Kae) has been well-documented to have pharmacological activities in the previous studies. However, the detailed mechanisms by which Kae regulates inflammation, oxidative stress, and apoptosis in Human Umbilical Vein Endothelial Cells (HUVECs) remain unknown. Methods and resultsThe real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure expression levels of circNOL12, nucleolar protein 12 (NOL12), miR-6873-3p, and Fibroblast growth factor receptor substrate 2 (FRS2) in HUVECs treated with either oxidized low-density lipoprotein (ox-LDL) alone or in combination with Kae. The cells viability was assessed by 3-(4, 5-dimethylthiazol-2-yl)−2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay. The inflammation and oxidative stress were assessed by checking inflammatory factors, Reactive Oxygen Species (ROS), Superoxide Dismutase (SOD), and Malondialdehyde (MDA) levels in ox-LDL-induced HUVECs. The apoptotic cells were quantified by flow cytometry assay. The western blot assay was used for measuring protein expression. The interaction relationship between miR-6873-3p and circNOL12 or FRS2 was analyzed by dual-luciferase reporter and RNA pull-down assays. Treatment with Kae could inhibit ox-LDL-induced the upregulation of circNOL12 in HUVECs. Importantly, Kae weakened ox-LDL-induced inflammation, oxidative stress, and apoptosis in HUVECs, which was abolished by overexpression of circNOL12. What’s more, miR-6873-3p was a target of circNOL12 in HUVECs, and the upregulation of miR-6873-3p overturned circNOL12 overexpression-induced effects on HUVECs treated with ox-LDL and Kae. FRS2 was negatively regulated by miR-6873-3p in HUVECs. ConclusionKae alleviated ox-LDL-induced inflammation, oxidative stress, and apoptosis in HUVECs by regulating circNOL12/miR-6873-3p/FRS2 axis.

Enriched Environment Promotes Adult Hippocampal Neurogenesis through FGFRs.

The addition of new neurons to existing neural circuits in the adult brain remains of great interest to neurobiology because of its therapeutic implications. The premier model for studying this process has been the hippocampal dentate gyrus in mice, where new neurons are added to mature circuits during adulthood. Notably, external factors such as an enriched environment (EE) and exercise markedly increase hippocampal neurogenesis. Here, we demonstrate that EE acts by increasing fibroblast growth factor receptor (FGFR) function autonomously within neurogenic cells to expand their numbers in adult male and female mice. FGFRs activated by EE signal through their mediators, FGFR substrate (FRS), to induce stem cell proliferation, and through FRS and phospholipase Cγ to increase the number of adult-born neurons, providing a mechanism for how EE promotes adult neurogenesis.SIGNIFICANCE STATEMENT How the environment we live in affects cognition remains poorly understood. In the current study, we explore the mechanism underlying the effects of an enriched environment on the production of new neurons in the adult hippocampal dentate gyrus, a brain area integral in forming new memories. A mechanism is provided for how neural precursor cells in the adult mammalian dentate gyrus respond to an enriched environment to increase their neurogenic output. Namely, an enriched environment acts on stem and progenitor cells by activating fibroblast growth factor receptor signaling through phospholipase Cγ and FGF receptor substrate proteins to expand the pool of precursor cells.

Long non-coding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) regulates fibroblast growth factor receptor substrate 2 (FRS2) by targeting microRNA (miR)-29-3p in hypertrophic scar fibroblasts

ABSTRACT Long non-coding RNAs (lncRNAs) play crucial roles in human diseases. However, the detailed role of lncRNAs in hypertrophic scar fibroblasts (HSFs) is inadequately understood. This study aimed to investigate the potential role of lncRNA nuclear enriched abundant transcript 1 (NEAT1) in hypertrophic scarring. Expression of lncRNAs, miRNAs, and genes were detected by polymerase chain reaction; protein expression was evaluated using western blotting. Cellular function was determined using the CCK-8 assay. The interaction between microRNA (miR)-29-3p and NEAT1 or fibroblast growth factor receptor substrate 2 (FRS2) was verified by luciferase and RNA pull-down assays. The results showed that NEAT1 was overexpressed in the hypertrophic dermis and in HSFs. However, knockdown of NEAT1 suppressed the proliferation and extracellular matrix (ECM) production of HSFs. Moreover, NEAT1 functioned as a competing endogenous RNA to upregulate FRS2 by sponging miR-29-3p. Downregulation of miR-29-3p or overexpression of FRS2 antagonized the effects of NEAT1 knockdown and promoted HSF proliferation and ECM release. In conclusion, NEAT1 knockdown protected against hypertrophic scarring by modulating the miR-29-3p/FRS2 axis, which is a viable target in scar treatment.

FGFR Regulation of Dendrite Elaboration in Adult-born Granule Cells Depends on Intracellular Mediator and Proximity to the Soma

Fibroblast Growth Factor Receptors (FGFRs) play crucial roles in promoting dendrite growth and branching during development. In mice, three FGFR genes, Fgfr1, Fgfr2, and Fgfr3, remain expressed in the adult neurogenic niche of the hippocampal dentate gyrus. However, the function of FGFRs in the dendritic maturation of adult-born neurons remains largely unexplored. Here, using conditional alleles of Fgfr1, Fgfr2, and Fgfr3 as well as Fgfr1 alleles lacking binding sites for Phospholipase-Cγ (PLCγ) and FGF Receptor Substrate (FRS) proteins, we test the requirement for FGFRs in dendritogenesis of adult-born granule cells. We find that deleting all three receptors results in a small decrease in proximal dendrite elaboration. In contrast, specifically mutating Tyr766 in FGFR1 (a PLCγ binding site) in an Fgfr2;Fgfr3 deficient background results in a dramatic increase of overall dendrite elaboration, while blocking FGFR1-FRS signaling causes proximal dendrite deficits and, to a lesser extent than Tyr766 mutants, increases distal dendrite elaboration. These findings reveal unexpectedly complex roles for FGFRs and their intracellular mediators in regulating proximal and distal dendrite elaboration, with the most notable role in suppressing distal elaboration through the PLCγbinding site.

Abstract 992: IL-6/STAT3-mediated inflammatory signaling results in dysregulated fibroblast growth factor receptor activation and tumor cell signaling in human pancreatic adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy characterized by delayed diagnosis and the lowest five-year survival amongst all cancer sites. PDAC development and progression are attributed to chronic inflammation and associated tissue repair mechanisms which contribute to high density tumor formation and chemoresistance. PDAC-associated inflammation is, in part, owed to high levels of interleukin 6 (IL-6) and its activated pathways including JAK/STAT3. In Addition, Fibroblast growth factor (FGF) signaling pathways are known to be dysregulated in cancer and have been implicated in various malignancies including PDAC. As such, we sought to explore the association between IL-6-induced inflammatory signaling and FGF signaling activation. To observe this effect as it pertains to carcinogenesis and cancer progression we used four human cell lines ranging from healthy pancreatic ductal epithelial (HPDE) cells to various grade (G) PDAC cells. Cells were treated with IL-6 at a concentration of 100 ng/mL for 24 hours. The results showed increased phosphorylation of STAT3 in BxPC-3 (G2) and PANC-1 (G3) cells with no change noted in HPDE and SW1990 (G2, metastatic) cells. IL-6 stimulation likewise resulted in markedly increased phosphorylation of the fibroblast growth factor receptor substrate 2-alpha (FRS2-α) and its downstream component Erk1/2 in BxPC3 cells, indicating activation of FGF signaling through the MAPK/Erk pathway in our low grade PDAC cell model. In conclusion, the results demonstrate a potential link between IL-6 induced inflammatory signaling and dysregulation of FGF signaling in human PDAC. Activated STAT3 signaling seems to be involved in this process. Existing cancer therapies aimed at controlling growth factor signaling broadly target vascular endothelial or epithelial growth factor receptors (VEGF/EGFR) with marginal efficacy and adverse side effects in PDAC. Identifying the pivotal link between inflammatory signaling and dysregulation of fibroblast growth factor signaling will allow for the development of more precisely targeted and effective cancer therapy in pancreatic cancer. This may minimize clinically observed exaggerated fibrotic events within the tumor and may hence result in higher vulnerability for established chemotoxic therapies in the tumor cells. Citation Format: Karol Nawalaniec, Yuxiang Wang, Romana Moench, Minghua Cao, Yueming Luo, Li-Li Hsiao, Ana Maria Waaga-Gasser. IL-6/STAT3-mediated inflammatory signaling results in dysregulated fibroblast growth factor receptor activation and tumor cell signaling in human pancreatic adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 992.