Direct Binding Site Research Articles

Construction of competitive endogenous RNA network and identification of potential regulatory axis in vascular calcification.

Competitive endogenous RNAs (ceRNA) theory has been proved in numerous biological processes. Nevertheless, there is a lack of research applying the ceRNA theory to the study of vascular calcification (VC) in chronic kidney diseases (CKD). In the present study, a ceRNA network was constructed after conducting transcriptome sequencing of differentially expressed genes, followed by experimental validation to identify a new target for the diagnosis and treatment of vascular calcification. Total RNA was extracted from β-glycerophosphate (β-GP) cultured vascular smooth muscle cells (VSMCs) on Day 7. Illumina HiSeq platform was utilized to build sequencing libraries. GO and KEGG analysis was conducted to identify the function of the differentially expressed genes. Protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. A ceRNA network was established based on TargetScan, miRDB, miRWALK, and miRanda database. Western blot and qRT-PCR were used to explore the expression level of protein and RNA, respectively. The direct binding sites were confirmed by dual-luciferase reporter assay. In total, 647 differentially expressed lncRNAs and 289 differentially expressed mRNAs were identified (|log2FC| ≥ 1, p < .05). The function of differentially expressed mRNAs was mainly enriched in negative regulation of osteoblast differentiation, regulation of RNA metabolic process, and other typical pathways. The ceRNA network was generated with a total of 107 interaction pairs. The lncRNA Prrc2c/miR-145-5p/Smad3 axis was considered a potential regulatory pathway within the ceRNA network. The regulatory relationship and targets of this ceRNA axis were validated via invitro experiments. For the first time, we found that lncRNA Prrc2c was highly expressed and promoted calcification of VSMCs. Luciferase reporter assay showed that lncRNA Prrc2c could bind miR-145-5p at site 1755-1761. Similarly, luciferase reporter assay showed that miR-145-5p inhibited Smad3 expression by binding to its 3'UTR. Our findings provide a comprehensive examination of the ceRNA networks in vascular smooth muscle cells (VSMCs) treated with high phosphorate. Specifically, we have identified the role of lncRNA Prrc2c in promoting VSMC calcification through the miR-145-5p/Smad3 axis.

7615 Genomic Consequences of GRHL2 Overexpression in ER+ Breast Cancer Cells

Abstract Disclosure: K.O. Allen: None. C. Zheng: None. N. Solodin: None. M.S. Ozers: Owner/Co-Owner; Self; Proteovista, LLC. C.L. Warren: None. P.E. Messa: None. D.T. Vogt: None. E.T. Alarid: None. Breast cancer progression is characterized by the presence and prevalence of metastatic disease. At a genomic level, metastasis can be facilitated via rearrangement of the chromatin landscape to expose oncogenic genes for transcription. Grainyhead like protein 2 (GRHL2) is a nuclear transcription factor implicated in epithelial cell differentiation. The GRHL2 motif has been found near activated estrogen receptor (ER) binding sites, and elevated levels of GRHL2 have been linked to worse prognosis in hormone receptor positive breast cancer patients. In its capacity to contribute to a more severe disease state when overexpressed, GRHL2 may function as either a transcription or pioneer factor to aid in the expression of metastasis-associated genes. Our lab previously showed that GRHL2 genomic binding precedes ER binding at co-regulated binding sites. Also, overexpression of GRHL2 in MCF7 cells for 24 hours did not appreciably alter gene expression by RNA-Seq. These data taken together indicate that GRHL2 may be exhibiting pioneer factor activity. However, the specific role of GRHL2 overexpression in hormone positive breast cancer progression remains unknown. In order to examine the mechanism of GRHL2 action, GRHL2 genomic binding was investigated using a tetracycline-inducible MCF7 model that allows controlled overexpression of GRHL2. ChIP studies were performed to determine GRHL2 binding intensity at specific and genome wide GRHL2 binding sites. Specific focus was given to established GRHL2 binding sites near oncogenic genes responsible for epithelial to mesenchymal transition (EMT), dormancy, and proto-oncogenic behavior. A similar analysis examined H3K27 acetylation to validate active gene transcription. An orthogonal technology, Specificity and Affinity for Protein (SNAP) microarrays, which characterizes direct GRHL2 binding to tiled genomic regions identified in ChIP-Seq analyses from multiple labs, was utilized to discriminate direct and indirect GRHL2 binding sites. The requirement for GRHL2 transactivation function in regulation of EMT related genes was further interrogated using transactivation GRHL2 dominant negative mutants. This work aims to distinguish the transcriptional and non-transcriptional activities of GRHL2 in breast cancer cells on a genome wide level and thereby, provide a deeper understanding of how overexpressed GRHL2 contributes to hormone positive breast cancer. Presentation: 6/3/2024

Regulation of KLRC and Ceacam gene expression by miR-141 supports cell proliferation and metastasis in cervical cancer cells

IntroductionMicroRNAs (miRNAs) are single RNA molecules that act as global regulators of gene expression in mammalian cells and thus constitute attractive targets in treating cancer. Here we aimed to investigate the possible involvement of miRNA-141 (miR-141) in cervical cancer and to identify its potential targets in cervical cancer cell lines.MethodsThe level of miR-141 in HeLa and C-33A cells has been assessed using the quantitative real-time PCR (qRT-PCR). A new miR-141 construct has been performed in a CMV promoter vector tagged with GFP. Using microarray analysis, we identified the potentially regulated genes by miR-141 in transfected HeLa cells. The protein profile of killer-like receptor C1 (KLRC1), KLRC3, carcinoembryonic antigen‐related cell adhesion molecule 3 (CAM3), and CAM6 was investigated in HeLa cells transfected with either an inhibitor, antagonist miR-141, or miR-141 overexpression vector using immunoblotting and flow cytometry assay. Finally, ELISA assay has been used to monitor the produced cytokines from transfected HeLa cells.ResultsThe expression of miR-141 significantly increased in HeLa and C-33A cells compared to the normal cervical HCK1T cell line. Transfection of HeLa cells with an inhibitor, antagonist miR-141, showed a potent effect on cancer cell viability, unlike the transfection of miR-141 overexpression vector. The microarray data of HeLa cells overexpressed miR-141 provided a hundred of downregulated genes, including KLRC1, KLRC3, CAM3, and CAM6. KLRC1 and KLRC3 expression profiles markedly depleted in HeLa cells transfected with miR-141 overexpression accompanied by decreasing interleukin 8 (IL-8), indicating the role of miR-141 in avoiding programmed cells death in HeLa cells. Likewise, CAM3 and CAM6 expression reduced markedly in miR-141 transduced cells accompanied by an increasing level of transforming growth factor beta (TGF-β), indicating the impact of miR-141 in cancer cell migration. The IntaRNA program and miRWalk were used to check the direct interaction and potential binding sites between miR-141 and identified genes. Based on this, the seeding regions of each potential target was cloned upstream of the luciferase reporter gene in the pGL3 control vector. Interestingly, the luciferase activities of constructed vectors were significantly decreased in HeLa cells pre-transfected with miR-141 overexpression vector, while increasing enormously in cells pre-transfected with miR-141 specific inhibitor.ConclusionTogether, these data uncover an efficient miR-141-based mechanism that supports cervical cancer progression and identifies miR-141 as a credible therapeutic target.

Dehydroevodiamine targeting IKKβ to alleviate acute gastric injury via inhibiting the p65/NLRP3 axis

BackgroundAcute gastric injury, a common and recurring global digestive disorder, significantly impairs patient quality of life and overall health. Dehydroevodiamine (DHE), a bioactive natural product derived from Tetradium ruticarpum (A. Juss.) Hartley, shows potential therapeutic effects on acute gastric injury. This study investigates the underlying mechanisms of DHE's alleviating effects on acute gastric injury. MethodsThe gastric mucosal protective effect of DHE was confirmed through in vivo and in vitro acute gastric injury models. Biotin pulldown MS and molecular dynamics simulations identified DHE's target. CETSA and SPR assays validated DHE's affinity for IKKβ. Protein site mutation validation and MST pinpointed the direct binding sites of DHE on IKKβ. Additionally, the potential mechanism by which DHE ameliorates acute gastric injury was elucidated using WB, IHC, and IF methods, and further confirmed by rescue experiments. ResultsDHE effectively ameliorated IDO-induced gastric injury in GES-1 cells and rat gastric mucosa, both in vitro and in vivo. Biotin pulldown MS identified IKKβ as the target of DHE in alleviating gastric injury. CETSA and SPR assays confirmed DHE's direct binding to IKKβ. Molecular dynamics simulations, protein mutation experiments, and MST results pinpointed GLU-149, GLU-49, and ASP-103 in the ATP-binding pocket as the binding sites of DHE on IKKβ. Notably, DHE was found to competitively bind to IKKβ with ATP. Mechanistically, DHE attenuated IDO-induced gastric injury by inhibiting the IKKβ-p65/NLRP3 signaling pathway. Importantly, exogenous activation of IKKβ reversed the therapeutic effect of DHE, indicating that DHE's efficacy depends on IKKβ. ConclusionDHE attenuated IDO-induced gastric injury by inhibiting the IKKβ-p65/NLRP3 signaling pathway. Notably, DHE is a novel ATP-competitive IKKβ inhibitor that prevents phosphorylation by targeting GLU-149, GLU-49, and ASP-103 in the ATP-binding pocket. This study reveals new targets of action for DHE, providing a new molecular basis for using DHE in treating inflammation-related diseases.

Dexmedetomidine directly binds to and inhibits Toll-like receptor 4

BackgroundWhile a number of anesthetics has been shown potentially associated with neurotoxicity in the developing brain, dexmedetomidine, a drug that was rather recently introduced into the perioperative setting, is considered beneficial from neurological wellbeing. However, the underlying mechanism of how dexmedetomidine affects brain health remains to be determined. Based on our recent study, we hypothesized that dexmedetomidine would directly bind to and inhibit Toll-like receptor 4 (TLR4), a critical receptor largely expressed in microglia and responsible for neurological insult. MethodsWe used TLR4 reporter assays to test if dexmedetomidine attenuates TLR4 activation. Furthermore, a direct binding of dexmedetomidine on TLR4 was tested using photoactivatable medetomidine. Lastly, the effect of dexmedetomidine on ketamine (anesthetic)-induced neurotoxicity was tested in rat pups (P7). ResultsWe showed that dexmedetomidine attenuated TLR4 activation using reporter assay (IC50 = 5.8 µg/mL). Photoactivatable dexmedetomidine delineated its direct binding sites on TLR4. We also showed that dexmedetomidine attenuated microglia activation both in vitro and in vivo. DiscussionWe proposed a novel mechanism of dexmedetomidine-mediated neuroprotection.

MicroRNA-141-regulated KLK10 and TNFSF-15 gene expression in hepatoblastoma cells as a novel mechanism in liver carcinogenesis

Liver cancer is one of the most pivotal global health problems, leading hepatocellular carcinoma (HCC) with a significant increase in cases worldwide. The role of non-coding-RNA in cancer proliferation and carcinogenesis has attracted much attention in the last decade; however, microRNAs (miRNAs), as non-coding RNA, are considered master mediators in various cancer progressions. Yet the role of miR-141 as a modulator for specific cellular processes in liver cancer cell proliferation is still unclear. This study identified the role of miR-141 and its potential functions in liver carcinogenesis. The level of miR-141 in HepG2 and HuH7 cells was assessed using quantitative real-time PCR (qRT-PCR) and compared with its expression in normal hepatocytes. A new miR-141 construct has been performed in a CMV promoter vector tagged with GFP. Using microarray analysis, we identified the potentially regulated genes by miR-141 in transfected HepG2 cells. The protein profile of the kallikrein-related peptidase 10 (KLK10) and tumor necrosis factor TNFSF-15 was investigated in HepG2 cells transfected with either an inhibitor, antagonist miR-141, or miR-141 overexpression vector using immunoblotting and flow cytometry assay. Finally, ELISA assay has been used to monitor the produced inflammatory cytokines from transfected HepG2 cells. Our findings showed that the expression of miR-141 significantly increased in HepG2 and HuH7 cells compared to the normal hepatocytes. Transfection of HepG2 cells with an inhibitor, antagonist miR-141, showed a significant reduction of HepG2 cell viability, unlike the transfection of miR-141 overexpression vector. The microarray data of HepG2 cells overexpressed miR-141 provided a hundred downregulated genes, including KLK10 and TNFSF-15. Furthermore, the expression profile of KLK10 and TNFSF-15 markedly depleted in HepG2 cells transfected with miR-141 overexpression accompanied by a decreasing level of interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α), indicating the role of miR-141 in HepG2 cell proliferation and programmed cell death. Interestingly, the experimental rats with liver cancer induced by Diethylnitrosamine injection further confirmed the upregulation of miR-141 level, IL-10, and TNF-α and the disturbance in KLK10 and TNFSF-15 gene expression compared with their expression in normal rats. The in-silico online tools, IntaRNA and miRWalk were used to confirm the direct interaction and potential binding sites between miR-141 and identified genes. Thus, the seeding regions of potential targeted sequences was cloned upstream of luciferase reporter gene in pGL3 control vector. Interestingly, the luciferase activities of constructed vectors were significantly decreased in HepG2 cells pre-transfected with miR-141 overexpression vector, while increasing in cells pre-transfected with miR-141 specific inhibitor. In summary, these data suggest the crucial role of miR-141 in liver cancer development via targeting KLK10 and TNFSF-15 and provide miR-141 as an attractive candidate in liver cancer treatment and protection.

Construction of a nomogram for predicting HNSCC distant metastasis and identification of EIF5A as a hub gene

Patients with distant metastasis of head and neck squamous cell carcinoma (HNSCC) often have a poor prognosis. However, early diagnosis of distant metastasis is challenging in clinical practice, and distant metastasis is often only detected in the late stages of tumor metastasis through imaging techniques. In this study, we utilized data from HNSCC patients collected from the TCGA database. Patients were divided into distant metastasis and nonmetastasis groups based on the tumor–node–metastasis (TNM) stage. We analyzed the differentially expressed genes between the two groups (DM/non-M DEGs) and their associated lncRNAs and generated a predictive model based on 23 lncRNAs that were significantly associated with the occurrence of distant metastasis in HNSCC patients. On this basis, we built a nomogram to predict the distant metastasis of HNSCC patients. Moreover, through WGCNA and Cytoscape software analysis of DM/non-M DEGs, we identified the gene most closely related to HNSCC distant metastasis: EIF5A. Our findings were validated using GEO data; EIF5A expression was significantly increased in the tumor tissues of HNSCC patients with distant metastasis. We then predicted miRNAs that can directly bind to EIF5A via the TargetScan and miRWalk websites, intersected them with differentially expressed miRNAs in the two groups from the TCGA cohort, and identified the only overlapping miRNA, miR-424; we predicted the direct binding site of EIF5A and miR-424 via the miRWalk website. Immunohistochemistry further revealed high expression of EIF5A in the primary tumor tissue of HNSCC patients with distant metastasis. These results provide a new perspective for the early diagnosis of distant metastasis in HNSCC patients and the study of the mechanisms underlying HNSCC distant metastasis.

Greasy Cations Bind to Neutral Macromolecules in Aqueous Solution.

Ions influence the solution properties of macromolecules. Although much is known about anions, cationic effects are considered mostly in terms of weak interactions or exclusion from neutral interfaces. Herein, we have systematically studied the effect of quaternary tetraalkylammonium cations (NH4+, NMe4+, NEt4+, NPr4+, NBu4+) on the phase transition of poly(N-isopropylacrylamide) (PNIPAM) in aqueous solution. Solubility measurements were coupled to 1H NMR and ATR-FTIR spectroscopic measurements. The solubility and NMR measurements revealed a direct binding between the greasiest cations and the isopropyl group of the macromolecule, evidenced from the nonlinear, Langmuir-type chemical shift response only at the isopropyl NMR signals with increasing salt concentrations. The ATR-FTIR measurements focusing on the amide oxygen showed that it is not the main direct-binding site. Additionally, the salting-out effects of the greasier cations correlate with their hydration entropies. These results demonstrate that the most weakly hydrated cations can bind to macromolecules as strongly as the weakly hydrated Hofmeister anions.

BCR-ABL kinase domain mutations in CML patients, experience from a tertiary care center in North India

BackgroundChronic Myeloid Leukemia is characterized by the presence of the Philadelphia Chromosome (Ph) which contains the BCR::ABL1 fusion gene that occurs due to a reciprocal translocation between chromosomes 9 and 22. This accounts for up to 15 % of all adult leukemias [1]. Most patients treated with first line tyrosine kinase inhibitor (TKI) imatinib achieve durable response but may undergo relapse at some stage [2]. The most important mechanism that may confer imatinib resistance is point mutation within BCR::ABL kinase domain. Other generation ABL tyrosine kinase inhibitors such as dasatinib, nilotinib, bosutinib and ponatinib help to overcome imatinib resistance [3]. Sensitivity of the patient to each of the above TKIs depends upon the individual candidate mutation present. Thus, it is important to perform mutation analysis for effective therapeutic management of CML patients once they show imatinib resistance. We used direct sequencing to identify the different types of mutations responsible for resistance of imatinib treatment from north India. MethodsIn this study, the patient resistance for the imatinib were analyzed for BCR::ABL kinase domain mutation by direct sequencing and the detected mutations along with their percentage prevalence were reported. Results329 patients with CML-CP were analyzed for BCR::ABL kinase domain mutation. Total 66 (20.06 %) patients out of 329 had mutation in at least one of the domains of BCR::ABL conferring resistance to different generations of TKI. Mutations in BCR::ABL kinase domain was observed in different domain of BCR::ABL. ATP binding P-Loop (42.42 %), Direct binding site (36.36 %), C-Loop (10.60 %), A-Loop (6.06 %), SH2 contact (3.03 %), SH3 contact (1.51 %). ConclusionTotal 20.06 % patients (66/329) show mutation in at least one of the structural motifs of BCR-ABL kinase domain, which further confer the resistance to a particular generation of TKI.

Inhibitory effect of theaflavin-3,3'-digallate caninvolve its binding to the "stem" domain ofα-hemolysin of Staphylococcus aureus.

Infections caused by Staphylococcus aureus are currently a worldwide threat affecting millions of individuals. The pathogenicity of S. aureus is associated with numerous virulence factors, including cell surface proteins, polysaccharides, and secreted toxins. The pore-forming α-hemolysin, known as α-toxin, is produced by nearly all virulent strains of S. aureus and is implicated in several diseases including skin and soft tissue infections, atopic dermatitis, and pneumonia. There are currently no vaccines available for the prevention of S. aureus infections and the efficacy of available antibiotics has been fading. In this study we examined the mode of antihemolytic activity of theaflavin-3,3'-digallate against α-hemolysin of methicillin-resistant S. aureus by molecular docking using AutoDock Vina asthemolecular docking tool. The theaflavin-3,3'-digallate docked the molecular sequence of the Hla(PDB ID:7ahl). The scores of the top 10 binding modes obtained were between -9.0 and -8.5 kcalmol-1, and the best binding mode was -9.0 kcal mol-1. Direct binding sites of theaflavin-3,3'-digallate to the "stem" domain of Hla were revealed which primarily targeted of the residues Met113, Thr117, Asn139. The disclosure of this potential binding mode warrants further clinical evaluation of theaflavin-3,3'-digallate as an anti-hemolytic compound in order to practically validate our results.

Molecular basis of RanGTP-activated release of Histones H2A-H2B from Importin-9

Imp9 is the primary importin for shuttling H2A-H2B from the cytoplasm to the nucleus. It employs an unusual mechanism where the binding of RanGTP is insufficient to release H2A-H2B. The resulting stable RanGTP·Imp9·H2A-H2B complex gains nucleosome assembly activity with H2A-H2B able to be deposited into an assembling nucleosome invitro. Using hydrogen-deuterium exchange coupled with mass spectrometry (HDX), we show that Imp9 stabilizes H2A-H2B beyond the direct-binding site, like other histone chaperones. HDX also shows that binding of RanGTP releases H2A-H2B contacts at Imp9 HEAT repeats 4-5, but not 18-19. DNA- and histone-binding surfaces of H2A-H2B are exposed in the ternary complex, facilitating nucleosome assembly. We also reveal that RanGTP has a weaker affinity for Imp9 when H2A-H2B is bound. Imp9 thus provides a connection between the nuclear import of H2A-H2B and its deposition into chromatin.

GAP-43 closely interacts with BDNF in hippocampal neurons and is associated with Alzheimer's disease progression.

Growth-associated protein 43 (GAP-43) is known as a neuronal plasticity protein because it is widely expressed at high levels in neuronal growth cones during axonal regeneration. GAP-43 expressed in mature adult neurons is functionally important for the neuronal communication of synapses in learning and memory. Brain-derived neurotrophic factor (BDNF) is closely related to neurodegeneration and synaptic plasticity during the aging process. However, the molecular mechanisms regulating neurodegeneration and synaptic plasticity underlying the pathogenesis and progression of Alzheimer's disease (AD) still remain incompletely understood. Remarkably, the expressions of GAP-43 and BDNF perfectly match in various neurons in the Human Brain Atlas database. Moreover, GAP-43 and BDNF are highly expressed in a healthy adults' hippocampus brain region and are inversely correlated with the amyloid beta (Aβ), which is the pathological peptide of amyloid plaques found in the brains of patients with AD. These data led us to investigate the impact of the direct molecular interaction between GAP-43 and BDNF in hippocampal neuron fate. In this study, we show that GAP-43 and BDNF are inversely associated with pathological molecules for AD (Tau and Aβ). In addition, we define the three-dimensional protein structure for GAP-43 and BDNF, including the predictive direct binding sites via analysis using ClusPro 2.0, and demonstrate that the deprivation of GAP-43 and BDNF triggers hippocampal neuronal death and memory dysfunction, employing the GAP-43 or BDNF knock-down cellular models and 5XFAD mice. These results show that GAP-43 and BDNF are direct binding partners in hippocampal neurons and that their molecular signaling might be potential therapeutic targets for AD.

WTAP-Mediated m6A RNA Methylation Regulates the Differentiation of Bone Marrow Mesenchymal Stem Cells via the miR-29b-3p/HDAC4 Axis.

N6-methyladenosine (m6A) methylation, a well-known modification with new epigenetic functions, has been reported to participate in the progression of osteoporosis (OP), providing novel insights into the pathogenesis of OP. However, as the key component of m6A methylation, Wilms tumor 1-associated protein (WTAP) has not been studied in OP. Here we explored the biological role and underlying mechanism of WTAP in OP and the differentiation of bone marrow mesenchymal stem cells (BMMSCs). We demonstrated that WTAP was expressed at low levels in bone specimens from patients with OP and OVX mice. Functionally, WTAP promoted osteogenic differentiation and inhibited adipogenic differentiation of BMMSCs in vitro and in vivo. In addition, microRNA-29b-3p (miR-29b-3p) was identified as a downstream target of WTAP. M6A modifications regulated by WTAP led to increased miR-29b-3p expression. WTAP interacted with the microprocessor protein DGCR8 and accelerated the maturation of pri-miR-29b-3p in an m6A-dependent manner. Target prediction and dual-luciferase reporter assays identified the direct binding sites of miR-29b-3p with histone deacetylase 4 (HDAC4). WTAP-mediated m6A modification promoted osteogenic differentiation and inhibited adipogenic differentiation of BMMSCs through the miR-29b-3p/HDAC4 axis. Furthermore, WTAP-mediated m6A methylation negatively regulates osteoclast differentiation. Collectively, our study first identified a critical role of WTAP-mediated m6A methylation in BMMSC differentiation and highlighted WTAP as a potential therapeutic target for OP treatment.

A Comprehensive Reassessment of the Direct DNA Binding Sites of PML-Rara within the Genomes of Primary Hematopoietic Progenitor Cells, and Their Effects on Chromatin Structure and Gene Expression

A Comprehensive Reassessment of the Direct DNA Binding Sites of PML-Rara within the Genomes of Primary Hematopoietic Progenitor Cells, and Their Effects on Chromatin Structure and Gene Expression

High-Throughput Analysis Reveals miRNA Upregulating α-2,6-Sialic Acid through Direct miRNA-mRNA Interactions.

Chemical biology has revealed the importance of sialic acids as a major signal in physiology and disease. The terminal modification α-2,6-sialic acid is controlled by the enzymes ST6GAL1 and ST6GAL2. Dysregulation of this glycan impacts immunological recognition and cancer development. microRNAs (miRNA, miR), noncoding RNAs that downregulate protein expression, are important regulators of glycosylation. Using our recently developed high-throughput fluorescence assay (miRFluR), we comprehensively mapped the miRNA regulatory landscape of α-2,6-sialyltransferases ST6GAL1 and ST6GAL2. We found, contrary to expectations, the majority of miRNAs upregulate ST6GAL1 and α-2,6-sialylation in a variety of cancer cells. In contrast, miRNAs that regulate ST6GAL2 were predominantly downregulatory. Mutational analysis identified direct binding sites in the 3'-untranslated region (UTR) responsible for upregulation, confirming it is a direct effect. The miRNA binding proteins AGO2 and FXR1 were required for upregulation. Our results upend common assumptions surrounding miRNA, arguing that upregulation by these noncoding RNA is common. Indeed, for some proteins, upregulation may be the dominant function of miRNA. Our work also suggests that upregulatory miRNAs enhance overexpression of ST6GAL1 and α-2,6-sialylation, providing another potential pathway to explain the dysregulation observed in cancer and other disease states.

Bmp4 Synexpression Gene, Sizzled, Transcription Is Collectively Modulated by Smad1 and Ventx1.1/Ventx2.1 in Early Xenopus Embryos.

Sizzled (Szl) is a secreted frizzled protein, having a sequence homology with the extracellular cysteine-rich domain (CRD) of the Wnt receptor, 'Frizzled'. Contrary to the other secreted frizzled like proteins (Sfrps), szl belongs to the bone morphogenetic protein 4 (Bmp4) synexpression group and is tightly coexpressed with Bmp4. What is not known is how the szl transcription achieves its Bmp4 synexpression pattern. To address the molecular details of szl transcription control, we cloned a promoter of size 1566 base pairs for szl (bps) from the Xenopus laevis genomic DNA. Luciferase and eGFP reporter gene results of this szl promoter (-1566 bp) in its activation and repression patterns by Bmp4/Smad1 and a dominant negative Bmp4 receptor (DNBR) were similar to those of the endogenous szl expression. Reporter gene assays and site-directed mutagenesis of the szl promoter mapped an active Bmp4/Smad1 response element (BRE) and a cis-acting element, which competitively share a direct binding site for Ventx1.1 and Ventx2.1 (a Ventx response element, VRE). Smad1 and ventx2.1 alone increased szl promoter activity; in addition, the binding of each protein component was enhanced with their coexpression. Interestingly, Ventx1.1 repressed this reporter gene activity; however, Ventx1.1 and Ventx2.1 together positively regulated the szl promoter activity. From our analysis, Ventx2.1 binding was enhanced by Ventx1.1, but Ventx1.1 inhibitory binding was inhibited by co-injection of Ventx2.1 for the VRE site. The inhibitory Ventx1.1 co-injection decreased Smad1 binding on the szl promoter. In a triple combination of overexpressed Smad1/Ventx1.1/Ventx2.1, the reduced binding of Smad1 from Ventx1.1 was recovered to that of the Smad1/Ventx2 combination. Collectively, this study provides evidence of Bmp4/Smad1 signaling for a primary immediate early response and its two oppositely behaving target transcription factors, Ventx1.1 and Ventx2.1, for a secondary response, as they together upregulate the szl promoter's activity to achieve szl expression in a Bmp4 synexpression manner.

METTL3 contributes to slow transit constipation by regulating miR-30b-5p/PIK3R2/Akt/mTOR signaling cascade through DGCR8.

N6-methyladenosine (m6A) is the most prevalent methylation modification of eukaryotic RNA, and methyltransferase-like 3 (METTL3) plays a vital role in multiple cell functions. This study aimed to investigate the role of m6A methylase METTL3 in slow transit constipation (STC). The expression of METTL3 and DGCR8 was measured in STC tissues and glutamic acid-induced interstitial cells of Cajal (ICCs). The effects of METTL3, miR-30b-5p, and DGCR8 on the biological characteristics of ICCs were investigated on the basis of loss-of-function analyses. Luciferase reporter assay was used to identify the direct binding sites of miR-30b-5p with PIK3R2. The results showed that the METTL3, DGCR8, miR-30b-5p, and the methylation level of m6A were significantly increased in STC tissues and glutamic acid-induced ICCs. Silencing of METTL3 and miR-30b-5p inhibited apoptosis, autophagy, and pyroptosis of glutamic acid-induced ICCs. Moreover, overexpression of miR-30b-5p reversed the cytoprotection of METTL3 knockdown in glutamic acid-induced ICCs. Besides, DGCR8 knockdown could facilitate cell growth and decrease apoptotic glutamic acid-induced ICCs. Mechanically, we illustrated that METTL3 in glutamic acid-induced ICCs significantly accelerated the maturation of pri-miR-30b-5p by m6A methylation modification, resulting in the reduction of PIK3R2, which results in the inhibition of PI3K/Akt/mTOR pathway and ultimately leads to the cell death of STC. Collectively, these data demonstrated that METTL3 promoted the apoptosis, autophagy, and pyroptosis of glutamic acid-induced ICCs by interacting with the DGCR8 and successively modulating the miR-30b-5p/PIK3R2 axis in an m6A-dependent manner, and METTL3 may be a potential therapeutic target for STC.

Tanshinone IIA improves cardiac function via regulating miR-499-5p dependent angiogenesis in myocardial ischemic mice

Background/aimsMyocardial ischemia-reperfusion injury leads to aggravated cardiac remodeling and heart failure. After myocardial infarction (MI), angiogenesis plays a vital role in the repair and regeneration of tissue. The purpose of the current study was to determine the effect of Tanshinone IIA (Tan IIA) on angiogenesis and elucidate its related mechanism. MethodsThe C57BL/6 mice MI model was established to evaluate the therapeutic effect of Tan IIA in vivo. MicroRNA (miRNA) microarray and bioinformatics analysis were performed to determine the differential expressions of miRNAs after Tan IIA administration. Cell proliferation, migration, and angiogenesis capacity were detected by EdU, Transwell, and Tube formation assay in vitro, respectively. The relationship between miR-499-5p (miR-499) and paired phosphate and tension homolog deleted on chromosome ten (PTEN) was confirmed by using a Dual-luciferase reporter assay. ResultsOur results showed that Tan IIA administration improved cardiac function after MI by activating angiogenesis. Further miRNA microarray and bioinformatics analysis revealed that miR-499 was significantly down-regulated, while PTEN was remarkably upregulated after Tan IIA administration post-MI. In addition, we found that miR-499 knock-down effectively promotes cell proliferation, migration, and tube formation ability of HUVECs. Dual-luciferase reporter assay demonstrated that PTEN contains a direct binding site for miR-499-5p. ConclusionTan IIA improves cardiac function post-MI by inducing angiogenesis. In terms of the mechanism, Tan IIA promotes therapeutic angiogenesis by regulating miR-499-5p/PTEN signaling pathway.

MET Exon 14 Skipping Mutations: Essential Considerations for Current Management of Non–Small-Cell Lung Cancer

MET Exon 14 Skipping Mutations: Essential Considerations for Current Management of Non–Small-Cell Lung Cancer

Bile acids directly bind and allosterically regulate the epithelial sodium channel

Bile acids are critical for the digestion and absorption of dietary fats and have important roles as signaling molecules. Bile acids are normally restricted to the enterohepatic circulation, but can be elevated in the blood and urine of patients with advanced liver diseases. The epithelial sodium channel (ENaC) mediates Na+ transport in the biliary epithelium, where it regulates bile viscosity, and in the aldosterone‐sensitive distal nephron, where it regulates extracellular fluid volume. We previously showed that bile acid regulation of ENaC depends on the position and stereochemistry of specific functional groups, but not on their chemico‐physical properties. To determine whether bile acids regulate ENaC in ex vivo native tissues, we performed patch clamp experiments on isolated split open renal tubules from mice, using recording pipettes backfilled with taurocholic acid (t‐CA) or taurohyodeoxycholic acid (t‐HDCA). Consistent with in vitro experiments, t‐CA increased ENaC open probability (PO) by 2‐fold while t‐HDCA decreased ENaC PO by nearly two‐thirds. To determine whether bile acids regulate ENaC through direct binding, we performed photoaffinity labeling experiments. We employed a bile acid derivative (p‐DCA) with diazirine and terminal alkyne groups that facilitate uv‐crosslinking and affinity purification, respectively. Using cultured Fisher Rat Thyroid cells transfected with ENaC, we found that the deoxycholic acid derivative (p‐DCA) crosslinked to the α, β, and γ subunits. To determine whether crosslinking was specific, we performed experiments in the presence of competing bile acids. Crosslinking to the β subunit was significantly reduced in the presence of deoxycholic acid (DCA). Crosslinking to the γ subunit was significantly reduced by taurocholic acid (t‐CA). These data suggest direct binding and multiple sites. To determine the effect of subunit composition on regulation, we measured the effect of t‐CA on Li+ currents in Xenopusoocytes expressing different functional ENaC subunit combinations. Channels composed of α subunits alone were strongly activated by t‐CA, followed by αβγ‐ and αβ‐ channels, suggesting that the α subunit is sufficient for ENaC sensitivity to t‐CA. Our data are consistent with bile acids directly binding all three ENaC subunits at multiple sites. Bile acid moieties provide for selective preferences for each of the sites, resulting in distinct effects for each bile acid.