Potential Binding Sites Research Articles

MicroRNA‑15a promotes prostate cancer cell ferroptosis by inhibiting GPX4 expression

Ferroptosis is a novel form of regulated cell death characterized by accumulated lipid reactive oxygen species (ROS) and inactivation of glutathione peroxidase 4 (GPX4). The present study aimed to investigate the role of microRNA (miRNA/miR)-15a in ferroptosis of prostate cancer cells. Bioinformatics analysis was performed to predict the potential interaction between miR-15a and the 3'-untranslated region (UTR) of GPX4 mRNA. The prostate cancer cell line, LNCAP was transfected with miR-15a mimics or small interfering (si)-GPX4. Reverse transcription-quantitative PCR and western blot analyses were performed to detect the mRNA and protein expression levels of GPX4, respectively. Biotin-RNA pull-down and dual-luciferase reporter assays were performed to verify the interaction between miR-15a and GPX4 mRNA. The Cell Counting Kit-8 assay was performed to assess cell proliferation, while lactate dehydrogenase (LDH) and intracellular ferrous iron levels were detected via ELISA. Lipid ROS and mitochondrial membrane potential (MMP) were assessed via flow cytometry and staining with C11-BIODIPY probes or JC-1. Furthermore, lipid peroxidation was identified by measuring malondialdehyde (MDA) levels. The results demonstrated that transfection with miR-15a mimics decreased GPX4 protein expression. Bioinformatics analysis revealed potential binding sites between miR-15a and the 3'-UTR region of GPX4, and RNA pull-down and the dual-luciferase reporter assays further confirmed the interaction between miR-15a and GPX4 mRNA. Both transfection with miR-15a mimics and si-GPX4 suppressed cell proliferation, elevated LDH release, accumulated intracellular ferrous iron and ROS, disrupted MMP and increased MDA levels. Taken together, the results of the present study suggest miR-15a induces ferroptosis by regulating GPX4 in prostate cancer cells, which provides evidence for investigating the therapeutic strategies of prostate cancer.

Potential Binding Sites for Taurine on the Insulin Receptor: A Molecular Docking Study.

Taurine has been reported to improve the action of insulin in normal, pre-, and diabetic conditions. However, the mechanism by which this amino acid ameliorates insulin sensitivity is not yet completely understood. Insulin acts on target tissues by interacting with the extracellular portion of a tyrosine kinase receptor, whose structure is known as the ectodomain (ECD) of the insulin receptor (IR). Some studies indicate that taurine can bind to the IR, which would contribute to its beneficial actions on glucose homeostasis. However, the binding mode of the amino acid on the IR ECD is unknown. Herein, using in silico experiments, we aimed to verify whether taurine may be an agonist of the IR and also to demonstrate the potential binding sites of taurine on the IR ECD. Molecular modeling predicted that taurine might interact with the three largest pocket sites for ligands present in the IR ECD. Taurine demonstrated high-energy interactions with these pockets, showing the highest affinity and highest molecular interaction with pocket 1, followed by decreasing energies and binding to pockets 2 and 3 of the IR ECD. The taurine interaction sites on the IR were not the same as the insulin interaction sites. Thus, these data indicate that taurine may be an agonist of the IR ECD, acting with high affinity at pocket 1 of the ECD. The predicted binding sites observed in this study probably constitute the regions of interaction of taurine on the IR and contribute to the mechanism by which taurine ameliorates insulin signaling pathway activation, thereby improving glucose homeostasis and the other cellular functions that are regulated by this intracellular signaling cascade.

Peripheral Blood Levels of miR-448 and SIRT1 in Patients with Deep Venous Thrombosis and Their Relationship.

The goal of this study was to investigate the changes in peripheral blood levels of miR-448 and silent information regulator 1 (SIRT1) in patients with deep venous thrombosis (DVT) and to analyze their relationship. A total of 112 patients treated from January 2019 to June 2020 were divided into DVT group (n = 40) and non-DVT group (n = 72). Fasting venous blood was extracted to separate serum and peripheral blood mononuclear cells (PBMCs). Enzyme-linked immunosorbent assay (ELISA) was employed to measure serum SIRT1 protein, and qPCR was utilized to detect miR-448 expression in PBMCs. The clinical data, serum indicators, and expressions of SIRT1 and miR-448 were compared, and the correlations of miR-448 and SIRT1 with DVT were analyzed using a multivariate Cox regression model. TargetScan Release 7.1 was used to predict the possibility of binding sites between miR-448 and SIRT1 mRNA 3'-untranslated region (3'-UTR), and dual-luciferase reporter assay was used to determine the targeting of miR-448 and SIRT1. HeLa cells were divided into overexpression, inhibition, and blank control groups. The cells were harvested 24 hours after transfection, followed by detection of SIRT1 mRNA expression by qPCR and measurement of supernatant SIRT1 protein expression by ELISA. Serum SIRT1 protein level was lower and miR-448 expression in PBMCs was higher in DVT group than those in non-DVT group (p < 0.05). DVT group had a larger number of patients with vascular diseases and history of venous thrombosis than that of non-DVT group (p < 0.05). miR-448 was an independent risk factor for postoperative DVT, and SIRT1 was a protective factor (p < 0.01). There were potential complementary base binding sites between miR-448 and SIRT1 mRNA 3'-UTR. Dual-luciferase reporter assay verified the targeted regulation between miR-448 and SIRT1. HeLa cell SIRT1 mRNA expression and supernatant SIRT1 protein expression were lower in overexpression group while higher in inhibition group than those in blank control group (p < 0.05). Serum SIRT1 protein level decreases while miR-448 expression in PBMCs increases in patients with DVT, and miR-448 inhibits SIRT1 expression through binding to SIRT1 mRNA 3'-UTR with complementary bases, thus inducing inflammatory response to participate in the formation of DVT. Targeting miR-448 to regulate cytokine expression may become an effective target and approach for the treatment of DVT. miR-488 combined with SIRT1 has a high predictive value for the occurrence of DVT.

Mutations in human SARS-CoV-2 spike proteins, potential drug binding and epitope sites for COVID-19 therapeutics development.

The comparison of 303,250 human SARS-CoV-2 spike protein sequences with the reference protein sequence Wuhan-Hu-1, showed ∼96.5% of the spike protein sequence has undergone the mutations till date, since outbreak of the COVID-19 pandemic disease that was first reported in December 2019. A total of 1,269,629 mutations were detected corresponding to 1,229 distinct mutation sites in the spike proteins comprising 1,273 amino acid residues. Thereby, ∼3.5% of the human SARS-CoV-2 spike protein sequence has remained invariant in the past two years. Considering different mutations occur at the same mutation site, a total of 4,729 distinct mutations were observed and are catalogued in the present work. The WHO/CDC, U.S.A., classification and definitions for the current variants being monitored (VBM) and variant of concern (VOC) are assigned to the SARS-CoV-2 spike protein mutations identified in the present work along with a list of other amino acid substitutions observed for the variants. All 195 amino acid residues in receptor binding domain (Thr333-Pro527) were associated with mutations in SARS-CoV-2 spike protein sequence including Lys417, Tyr449, Tyr453, Ala475, Asn487, Thr500, Asn501 and Gly502 that make interactions with the ACE-2 receptor ≤3.2 ​Å distance as observed in the crystal structure complex available in the Protein Data Bank (PDB code:6LZG). However, not all these residues were mutated in the same spike protein. Especially, Gly502 mutated only in two spike protein sequences and Tyr449 mutated only in seven spike protein sequences among the spike protein sequences analysed constitute potential sites for the design of suitable inhibitors/drugs. Further, forty-four invariant residues were observed that correspond to ten domains/regions in the SARS-CoV-2 spike protein and some of the residues exposed to the protein surface amongst these may serve as epitope targets to develop monoclonal antibodies.

Differential Expression of DDX3 and microRNAs in Response to Hormone and Cisplatin Against Cervical Cancer

Objectives: To discover micro ribonucleic acids (miRNAs) involved in the regulation of DDX3 expression using sexual hormones in combination with the well-known anticancer medication cisplatin. Methods: SiHa cells were treated with estradiol, dihydrotestosterone, and cisplatin and evaluated the expression of ER beta, Ki67, and DDX3 via quantitative reverse transcription–polymerase chain reaction. We generated a chimeric fusion construct five untranslated region (UTR)–hLUC–3UTR in the pEZX-MT06 miRNA vector under the control of the SV40 promoter. Reporter activity is measured with/without hormones, and their activity is compared with 5'- and 3'-UTR respectively. Various reporter deletion constructs were generated to identify the minimal UTR region in regulating the expression of DDX3. We identified the potential miRNA binding sites on the DDX3 UTR region, and their expression is monitored in cancer patients and cisplatin-treated SiHa cells. Results: Hormones increased the proliferation of SiHa cells and expression of DDX3. The 3'-UTR region 2135–4307bp contains miRNA sites that regulate DDX3 expression. miRNAs hsa-miR-671-5p, hsa-miR-361-5p, hsa-miR-140-5p, hsamiR-564, and hsa-miR-769-5p downregulated in patient samples but upregulated in cisplatin-treated cells. miRNA hsamiR-671-5p and hsa-miR-564 were associated with patient data and cisplatin-treated cancer cells. Conclusion: We discovered that sexual hormones enhanced DDX3 expression in SiHa cells. MiR-671-5p and miR-564 are two potential therapeutic miRNAs that can be used to treat DDX3-related malignancies. Keywords: Cancer patients, Cervical cancer, Cisplatin, DDX3, miRNA

MicroRNA-21 enhances estradiol production by inhibiting WT1 expression in granulosa cells.

In antral follicles, the transition of proliferative granulosa cells to estradiol-producing is critical for proper oocyte maturation. MicroRNAs are noncoding RNAs that play important roles in ovarian follicular development; however, this has yet to be fully characterized. MicroRNA-21 is significantly higher in granulosa cells isolated from large antral follicles compared to those from small antral follicles. To investigate the function of miR-21, porcine granulosa cells were transfected with miR-21 mimic or miR-21 targeted siRNA. Cells with the miR-21 mimic had higher aromatase expression and estradiol production but decreased WT1 expression. Conversely, cells with the miR-21 siRNA secreted less estradiol and had higher WT1 expression. We hypothesized that miR-21 promotes estradiol production by inhibiting WT1 protein synthesis. We found a potential miR-21 binding site in the 3'UTR of the WT1 transcript and performed a dual-luciferase reporter assay using the WT and mutated 3'UTR. Compared to the negative control, the miR-21 mimic induced a significant decrease in luciferase activity in the WT 3'UTR. This decrease was reversed when the 3'UTR was mutated, suggesting miR-21 targets this site to inhibit WT1 expression. We next transfected porcine granulosa cells with WT1 targeted siRNA and observed a significant increase in aromatase expression and estradiol secretion. We propose that miR-21 represses WT1 expression in granulosa cells to potentially promote aromatase expression and estradiol production. This study offers the first report of a microRNA regulating WT1 expression in granulosa cells and reveals the role of miR-21 in WT1's regulation of estradiol production.

MiR-486 alleviates hypoxia/reoxygenation-induced H9c2 cell injury by regulating forkhead box D3.

Myocardial ischemia-reperfusion injury (IRI) is common in myocardial infarction and is the leading cause of death. Therefore, we investigated the effect of miR-486 on myocardial IRI to explore new targets for clinical treatment of IRI. We made a rat myocardial IRI model by obstructing the coronary arteries and detected the change of miR-486 expression in rat myocardial tissue. In addition, we induced injury of rat cardiomyocytes (H9c2 cells) by hypoxia/reoxygenation and transfected H9c2 cells with agomir-miR-486 and antagomir-miR-486 to detect the effects of miR-486 on the viability, inflammation and apoptosis of cardiomyocytes. We also used the Targetscan system to predict the direct target of miR-486 and verified the effect of miR-486 on downstream targets through the Dual-Luciferase reporter assay. HE staining and the detection of myocardial injury markers and inflammatory factors verified the effectiveness of IRI rat model. The expression of miR-486 in myocardium of IRI rats was significantly lower than that of the control group. The overexpression of miR-486 in H9c2 cells increased the viability of H9c2 cells and reduced the levels of inflammation and apoptosis. MiR-486 is predicted to have a potential binding site to forkhead box D3 (FOXD3). The Dual-Luciferase reporter assay proved that miR-486 can bind and degrade FOXD3 mRNA. In addition, the overexpression of FOXD3 was found to attenuate the protective effect of miR-486 on H9c2 cells. MiR-486 protects cardiomyocytes and reduces the levels of inflammation and apoptosis by binding and inhibiting FOXD3 activity. Therefore, miR-486 may become a new target for myocardial IRI therapy.

Genetic variability in 5' untranslated region of BMPR1B gene from prolific Avishaan strain of sheep

Untranslated regions (UTRs) of the mRNAs have significant contribution towards gene regulation and expression. FecB gene is a member of transforming growth factor-p superfamily. A mutation in exon 8 of gene gives the FecB phenotype in sheep. In the present study 5′UTR region (992 bp encompassing 5′UTR and upstream sequences) and exon 8 region (712 bp encompassing exon 8 and partial sequences of upstream and downstream introns) of FecB gene were sequenced and characterized in prolific Avishaan sheep and its parental stock i.e. Garole, Malpura and Patanwadi. Sequence analysis of 992 bp fragment revealed 14 SNPs and one non-synonymous mutation (rs402422707) was observed in exon 1. Promoter was located in nucleotide 40 to 90 in amplified 5′ UTR sequence. Transcription factor binding site prediction identified 65 segments in the amplified 5′UTR and 5′ upstream sequence as potential binding sites for different transcription factors (TFs). In all 37 TFs binding sites were located on these sequences and majority of these sites were identified for SP-1 (12 segments), c/EBPalp (9 segments), NF-1 (4 segments), 2 segments each of RAP-1, HNF-1, c-Ets-1, TEC1, and Pit1a. These potential binding sites for TFs would be helpful in identifying site of interaction to control gene expression in Avishaan sheep.

Synthesis, characterisation and coordination properties of 5-methyl-3-formylpyrazole 4 N-benzylthiosemicarbazone (HMPzNB) Cobalt(III), nickel(II) and copper(II) complexes with a NNS donor system

The title ligand, 5-methyl-3-formylpyrazole 4 N-benzylthiosemicarbazone (abbreviated as HMPzNB) has been synthesised and physicochemically characterised by elemental analysis, IR, PMR and mass spectrum. The ligational behaviour of the pyrazole-derived thiosemicarbazone, HMPzNB, has been examined by isolation and characterisation of its complexes with Co III , Ni II and Cu II with different counterions. Elemental analyses, magnetic and spectral features indicate [Co(MPzNB) 2 ]X (X = Cl - , Br - , NO - 3 , ClO - 4 or BF - 4 ) as the diamagnetic octahedral species with the ligand in the deprotonated thiol form, while [Ni(HMPzNB) 2 ]X 2 (X = Cl - , Br-, NO - 3 or ClO - 4 ) is proposed as octahedral complex with the ligand in the neutral thione form. However, in the neutral condition (pH ∼6) nickel(II) salt affords octahedral bis-nickel(II) complex, Ni(MPzNB) 2 , while copper(II) salt gives [Cu(MPzNB)]X (X = Cl - , Br - , NO - 3 or ClO - 4 ), with the deprotonated ligand. 5-Methyl-3-formylpyrazole 4N-benzylthiosemicarbazone possesses in its structure, the tertiary nitrogen atom ( 2 N) of the pyrazole ring, the azomethine nitrogen and the sulphur atom as potential binding sites; thus the title ligand is expected to behave as a potential NNS tridentate during complex formation with a metal ion.

ODORANT1 targets multiple metabolic networks in petunia flowers.

SUMMARYScent bouquets produced by the flowers of Petunia spp. (petunia) are composed of a complex mixture of floral volatile benzenoid and phenylpropanoid compounds (FVBPs), which are specialized metabolites derived from phenylalanine (Phe) through an interconnected network of enzymes. The biosynthesis and emission of high levels of these volatiles requires coordinated transcriptional activation of both primary and specialized metabolic networks. The petunia R2R3‐MYB transcription factor ODORANT 1 (ODO1) was identified as a master regulator of FVBP production and emission; however, our knowledge of the direct regulatory targets of ODO1 has remained limited. Using chromatin immunoprecipitation followed by sequencing (ChIP‐seq) in petunia flowers, we identify genome‐wide ODO1‐bound genes that are enriched not only in genes involved in the biosynthesis of the Phe precursor, as previously reported, but also genes associated with the specialized metabolic pathways involved in generating phenylpropanoid intermediates for FVBPs. ODO1‐bound genes are also involved in methionine and S‐adenosylmethionine metabolism, which could modulate methyl group supplies for certain FVBPs. Quantitative reverse transcription polymerase chain reaction (qRT‐PCR) and RNA‐seq analysis in an ODO1 RNAi knockdown line revealed that ODO1‐bound targets are expressed at lower levels when ODO1 is suppressed. A cis‐regulatory motif, CACCAACCCC, was identified as a potential binding site for ODO1 in the promoters of genes that are both bound and activated by ODO1, which was validated by in planta promoter reporter assays with wild‐type and mutated promoters. Overall, our work presents a mechanistic model for ODO1 controlling an extensive gene regulatory network that contributes to FVBP production to give rise to floral scent.

LncRNA LIMT (LINC01089) contributes to sorafenib chemoresistance via regulation of miR-665 and epithelial to mesenchymal transition in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is one of the most malignant tumors worldwide and HCC patients often develop drug resisitene. Long non-coding RNAs (LncRNAs) are closely related to cell cycle, growth, development, differentiation, and apoptosis. Abnormally expressed lncRNAs have been proved to mediate drug resistance in tumor cells. However, the effect of LIMT on drug resistance has not been explored in HCC. In this study, we explored the effect of long non-coding RNA LIMT on drug resistance and its underlying mechanism in hepatocellular carcinoma (HCC). Our results showed that LncRNA LINC01089 (LIMT) expression is downregulated in 78.57% (44/56) of 56 HCC tumor tissue samples. LIMT expression is also downregulated in HCC cells compared with that in normal liver LO2 cells. Inhibition of LIMT increases the resistance to sorafenib and promotes cell invasion via regulation of epithelial to mesenchymal transition (EMT) in HCC. StarBase V3.0 was used to predict the potential binding site of miR-665 in . Furthermore, miR-665 participates in sorafenib resistance and also regulates the level of EMT-related proteins in HCC cells. A rescue experiment demonstrated that silencing of eliminats the inhibitory effect of the miR-665 inhibitor on sorafenib resistance in HCC cells. Taken together, our findings revealed that downregulation of LIMT increases the resistance of HCC to sorafenib via miR-665 and EMT. Therefore, LIMT, which serves as a therapeutically effective target, will provide new hope for the treatment of HCC.

Identification of African Swine Fever Virus Inhibitors through High Performance Virtual Screening Using Machine Learning

African swine fever virus (ASFV) is a highly contagious virus that causes severe hemorrhagic viral disease resulting in high mortality in domestic and wild pigs, until few antiviral agents can inhibit ASFV infections. Thus, new anti-ASFV drugs need to be urgently identified. Recently, we identified pentagastrin as a potential antiviral drug against ASFVs using molecular docking and machine learning models. However, the scoring functions are easily influenced by properties of protein pockets, resulting in a scoring bias. Here, we employed the 5′-P binding pocket of AsfvPolX as a potential binding site to identify antiviral drugs and classified 13 AsfvPolX structures into three classes based on pocket parameters calculated by the SiteMap module. We then applied principal component analysis to eliminate this scoring bias, which was effective in making the SP Glide score more balanced between 13 AsfvPolX structures in the dataset. As a result, we identified cangrelor and fostamatinib as potential antiviral drugs against ASFVs. Furthermore, the classification of the pocket properties of AsfvPolX protein can provide an alternative approach to identify novel antiviral drugs by optimizing the scoring function of the docking programs. Here, we report a machine learning-based novel approach to generate high binding affinity compounds that are individually matched to the available classification of the pocket properties of AsfvPolX protein.

Knockdown of ETV4 promotes autophagy-dependent apoptosis in GBM cells by reducing the transcriptional activation of EMP1.

ETS variant transcription factor 4 (ETV4) is a common cancer-promoting transcription factor and its expression has been found to be significantly upregulated in glioblastoma multiforme (GBM), as determined via analysis of the Gene Expression Profiling Interactive Analysis (GEPIA) database. In addition, our previous study demonstrated that ETV4 expression was highly positively correlated with epithelial membrane protein 1 (EMP1). The present study aimed to determine whether ETV4 could influence the activation of the PI3K/AKT/mTOR signaling pathway to affect the autophagy and apoptosis of GBM cells by regulating the transcriptional activity of EMP1. In addition to the analysis of the GEPIA database, the expression levels of ETV4 were also investigated in several different GBM cell lines. After interfering with the expression of ETV4, western blotting was used to detect the expression levels of autophagy- and apoptosis-related proteins, and a TUNEL assay was used to detect the levels of cell apoptosis. Dual luciferase reporter and chromatin immunoprecipitation assays were used to verify the potential binding site of ETV4 on EMP1. Western blotting was also used to analyze the expression levels of PI3K/AKT/mTOR signaling pathway-related proteins. The results of the current study revealed that the expression levels of ETV4 were significantly upregulated in GBM cell lines compared with those in normal glial cells. In the GBM cell line, LN-229, ETV4 was discovered to bind to the EMP1 promoter and positively regulate the expression of EMP1. The knockdown of ETV4 expression inhibited the PI3K/AKT/mTOR signaling pathway activity to promote autophagy and apoptosis, and this effect could be partially reversed by overexpressing EMP1. In conclusion, these findings indicated that the knockdown of ETV4 in GBM cells may reduce the transcriptional activation of EMP1 and thereby inhibit PI3K/AKT/mTOR signaling pathway activity to promote autophagy and apoptosis. This provides a novel insight into potential strategies for the treatment of GBM via the induction of autophagy-dependent apoptosis.

Trypsin Genes Are Regulated through the miRNA Bantam and Associated with Drug Sensitivity in the Sea Louse Caligus rogercresseyi.

The role of trypsin genes in pharmacological sensitivity has been described in numerous arthropod species, including the sea louse Caligus rogercresseyi. This ectoparasite species is mainly controlled by xenobiotic drugs in Atlantic salmon farming. However, the post-transcriptional regulation of trypsin genes and the molecular components involved in drug response remain unclear. In particular, the miRNA bantam family has previously been associated with drug response in arthropods and is also found in C. rogercresseyi, showing a high diversity of isomiRs. This study aimed to uncover molecular interactions among trypsin genes and bantam miRNAs in the sea louse C. rogercresseyi in response to delousing drugs. Herein, putative mRNA/miRNA sequences were identified and localized in the C. rogercresseyi genome through genome mapping and blast analyses. Expression analyses were obtained from the mRNA transcriptome and small-RNA libraries from groups with differential sensitivity to three drugs used as anti-sea lice agents: azamethiphos, deltamethrin, and cypermethrin. The validation was conducted by qPCR analyses and luciferase assay of selected bantam and trypsin genes identified from in silico transcript prediction. A total of 60 trypsin genes were identified in the C. rogercresseyi genome, and 39 bantam miRNAs were differentially expressed in response to drug exposure. Notably, expression analyses and correlation among values obtained from trypsin and bantam revealed an opposite trend and potential binding sites with significant ΔG values. The luciferase assay showed a reduction of around 50% in the expression levels of the trypsin 2-like gene, which could imply that this gene is a potential target for bantam. The role of trypsin genes and bantam miRNAs in the pharmacological sensitivity of sea lice and the use of miRNAs as potential markers in these parasites are discussed in this study.

The potential role of hsa_circ_0001079 in androgenetic alopecia via sponging hsa-miR-136-5p.

BackgroundAndrogenetic alopecia (AGA) is an androgen‐dependent polygenic hereditary disease.MethodsDiseased hair follicles from 5 AGA patients and normal hair follicles from 5 healthy individuals were selected as specimens to carry out whole transcriptome sequencing. Multiple high‐expression circular RNAs (circRNAs) were screened from the diseased group. We further verified the presence of the circRNAs in the clinical specimens by real‐time fluorescence quantitative PCR (qRT‐PCR).ResultsIn total, 100 circRNAs were significantly upregulated, and 184 circRNAs were significantly downregulated. The top 10 upregulated circRNAs were hsa_circ_0101041, hsa_circ_0001578, hsa_circ_0135062, hsa_circ_0002980, hsa_circ_0005062, hsa_circ_0072688, hsa_circ_0133954, hsa_circ_0001079, hsa_circ_0005974, hsa_circ_0000489. The top 10 downregulated circRNAs were hsa_circ_0001278, hsa_circ_0031482, hsa_circ_0008285, hsa_circ_0003784, hsa_circ_0077096, hsa_circ_0001148, hsa_circ_0006886, hsa_circ_0000638, hsa_circ_0084521, and hsa_circ_0101074. Among top 10 upregulated circRNAs, hsa_circ_0001079 showed significantly high expression via large‐sample verification and clinical application potential. Based on a database comparison and base pairing analysis, we found that has‐miR‐136‐5p bound to hsa_circ_0001079 and that hsa‐miR‐136‐5p had potential binding sites with Wnt5A.ConclusionIn summary, through high‐throughput sequencing and bioinformatic analysis, a potential diagnostic marker for alopecia and a key circRNA that might adsorb microRNA (miRNA) through a sponging mechanism, thus mediating alopecia, were discovered in this study.

CHARMM-GUI Ligand Designer for Template-Based Virtual Ligand Design in a Binding Site.

Rational drug design involves a task of finding ligands that would bind to a specific target protein. This work presents CHARMM-GUI Ligand Designer that is an intuitive and interactive web-based tool to design virtual ligands that match the shape and chemical features of a given protein binding site. Ligand Designer provides ligand modification capabilities with 3D visualization that allow researchers to modify and redesign virtual ligands while viewing how the protein-ligand interactions are affected. Virtual ligands can also be parameterized for further molecular dynamics (MD) simulations and free energy calculations. Using 8 targets from 8 different protein classes in the directory of useful decoys, enhanced (DUD-E) data set, we show that Ligand Designer can produce similar ligands to the known active ligands in the crystal structures. Ligand Designer also produces stable protein-ligand complex structures when tested using short MD simulations. We expect that Ligand Designer can be a useful and user-friendly tool to design small molecules in any given potential ligand binding site on a protein of interest.

Direct Interaction of ATP7B and LC3B Proteins Suggests a Cooperative Role of Copper Transportation and Autophagy.

Macroautophagy/autophagy plays an important role in cellular copper clearance. The means by which the copper metabolism and autophagy pathways interact mechanistically is vastly unexplored. Dysfunctional ATP7B, a copper-transporting ATPase, is involved in the development of monogenic Wilson disease, a disorder characterized by disturbed copper transport. Using in silico prediction, we found that ATP7B contains a number of potential binding sites for LC3, a central protein in the autophagy pathway, the so-called LC3 interaction regions (LIRs). The conserved LIR3, located at the C-terminal end of ATP7B, was found to directly interact with LC3B in vitro. Replacing the two conserved hydrophobic residues W1452 and L1455 of LIR3 significantly reduced interaction. Furthermore, autophagy was induced in normal human hepatocellular carcinoma cells (HepG2) leading to enhanced colocalization of ATP7B and LC3B on the autophagosome membranes. By contrast, HepG2 cells deficient of ATP7B (HepG2 ATP7B−/−) showed autophagy deficiency at elevated copper condition. This phenotype was complemented by heterologous ATP7B expression. These findings suggest a cooperative role of ATP7B and LC3B in autophagy-mediated copper clearance.

Isoflurane alleviates hypoxia/reoxygenation induced myocardial injury by reducing miR-744 mediated SIRT6

Background The objective of this study was to investigate the role of miR-744 and its target genes in ISO protection against hypoxia/reoxygenation (H/R) induced myocardial injury. Methods Rat cardiomyocytes H9c2 was used to establish an H/R model in vitro, and the level of miR-744 mRNA was detected by fluorescence quantitative PCR. CCK-8 and flow cytometry was used to detected cell viability and apoptosis. Myocardial injury markers CK-MB, cTnI, and LDH were detected by enzyme-linked immunosorbent assay (ELISA). Online bioinformatics software miRDB and miRWalk predicts miR-744 target and its potential binding site, and verifies the target by luciferase reporter assay. Results After H/R induction, miR-744 mRNA level was remarkedly increased, cell viability was deceased, and apoptosis was increased (p < 0.05). Myocardial injury markers CK-MB, cTnI, and LDH expressions were also increased (p < 0.05). However, ISO pretreatment can significantly alleviate the decrease in cell viability induced by H/R, the increase of cell apoptosis, and the increase of myocardial injury markers, and it play a cardioprotective effect (p < 0.05). More importantly, elevated miR-744 remarkedly weakened the protective effect of ISO on H/R-induced myocardial injury, resulting in decreased cell viability, increased apoptosis, and elevated concentration of myocardial injury indicators (p < 0.05). Luciferase reporter assay confirmed that Sirtuins6 (SIRT6) is a potential target of miR-744 and decreased in H/R-induced myocardial injury, and ISO exposure can reverse its level (p < 0.05). Conclusion Our findings provide new insights that ISO pretreatment can remarkedly regulate miR-744 and its downstream target SIRT6 to mitigate myocardial injury induced by H/R.

LncRNA NNT-AS1 regulates proliferation, ECM accumulation and inflammation of human mesangial cells induced by high glucose through miR-214-5p/smad4

BackgroundLncRNA NNT-AS1 (NNT-AS1) has been extensively studied as the causative agent in propagation and progression of lung and bladder cancers, and cholangiocarcinoma. However, its significance in proliferation and inflammation of diabetic nephropathy is enigmatic. This study focuses on the molecular mechanisms followed by NNT-AS1 to establish diabetic nephropathy (DN) and its potential miRNA target.MethodsBioinformatics analysis to identify potential miRNA target of NNT-AS1 and smad4 transcription factor was conducted using LncBase and TargetScan, and was subsequently confirmed by luciferase reporter assay. Relative quantitative expression of NNT-AS1 in human glomerular mesangial cells (HGMCs) was detected through quantitative real-time PCR and WB analysis. Cell proliferation was detected through CCK-8 assay, whereas, ELISA was conducted to evaluate the expression of inflammatory cytokines. Following this, relative expression of miR-214-5p and smad4 were confirmed through qRT-PCR and western blot analysis.ResultsResults from the experiments manifested up-regulated levels of NNT-AS1 and smad4 in the blood samples of DN patients as well as in HGMCs, whereas, downregulated levels of miR-214-5p were measured in the HGMCs suggesting the negative correlation between NNT-AS1 and miR-214-5p. Potential binding sites of NNT-AS1 showed miR-214-5p as its direct target and NNT-AS1 as potential absorber for this microRNA, in turn increasing the expression of transcription factor smad4.ConclusionThe data suggests that NNT-AS1 can be positively used as a potential biomarker and indicator of DN and causes extracellular matrix (ECM) accumulation and inflammation of human mesangial cells.

Mbd2 deficiency alleviates retinal cell apoptosis via the miR-345-5p/Atf1 axis in high glucose injury and streptozotocin-induced diabetic mice

DNA methylation is considered to play an important role in the development of diabetic retinopathy. Here, our goal was to investigate the precise role of methyl-CpG binding domain protein 2 (Mbd2) in the apoptosis of retinal ganglion cells (RGCs) in the early diabetic retina. Mbd2 was significantly upregulated after high glucose (HG) treatment and played a proapoptotic role in RGCs during HG-induced apoptosis. Combining ChIP and gene microarray datasets, the results showed that Mbd2 possessed potential binding sites for miR-345-5p, thereby elevating the expression levels of miR-345-5p via the enhancement of promoter demethylation. Activating transcription factor 1 (Atf1) played an anti-apoptotic role during the process of apoptosis in RGCs and acted as the target gene for miR-345-5p. Furthermore, the number of surviving RGCs in the diabetic retina was increased in Mbd2-knockout mice when compared with wild-type mice and the visual function became better accordingly. Collectively, our data demonstrated that the HG-induced overexpression of Mbd2 in the retina was partly responsible for the apoptosis of retinal neuronal cells through the miR-345-5p/Atf1 axis. Therefore, the targeting of Mbd2 might represent a novel therapeutic strategy for the treatment of neurodegeneration in the early diabetic retina.