Negative Regulator Of Apoptosis Research Articles

Overexpression of hsa_circ_0061817 Can Inhibit the Proliferation and Invasion of Lung Cancer Cells Based on Active Compounds.

This study was aimed at investigating the expression level of hsa_circ_0061817 in lung adenocarcinoma cells and its effect on cell proliferation and invasion and the possible mechanism of hsa_circ_0061817 in lung adenocarcinoma. The overexpression plasmids of hsa_circ_0061817 (OE-hsacirc_0061817) were transfected into human lung A549 cells and mouse LLC-LUC cells, respectively. The cell viability was detected by CCK-8, and the cell proliferation was detected by cell clone formation assay and EdU assay. Transwell test was used to detect the ability of cell invasion, and apoptosis was detected by flow cytometry. WB was applied to determine the expression of apoptosis and epithelial mesenchymal transition- (EMT-) related proteins and also target proteins for observation the effect of OE-hsa_circ_0061817 on the growth of A549 cells in nude mice. Bioinformatics method was used to predict the binding microRNA (miRNA) of hsa_circ_0061817 and construct the regulatory network of competitive endogenous RNA (ceRNA) and functional analysis of miRNA target genes. Compared with PLO-ciR group, the cell viability, proliferation, and invasive ability of A549 and LLC-LUC were significantly reduced in OE-hsa_circ_00061817 group, while the apoptosis increased in OE-hsa_circ_00061817 group compared to PLO-ciR group. WB results showed that the expression of caspase 3, caspase 7, caspase 9, and E-cadherin increased significantly, while the expression levels of vimentin and N-cadherin decreased severely. Most importantly, OE-hsa_circ_00061817 inhibited the growth of A549 tumor-bearing nude mice. According to TargetScan and mirBase databases, hsa_circ_0061817 may competitively bind hsa_mir-181b-3p, hsa-mir-337-3p, hsa-mir-421, and hsa-mir-548d-3p. The results of functional enrichment showed that miRNA target genes were involved in many cancer-related biological processes, including negative regulation of apoptosis, gene expression, transcriptional imbalance in cancer, transforming growth factor-β, and P53 signal pathway. Over expression of hsa_circ_0061817 inhibits the proliferation of lung adenocarcinoma A549 and LLC-LUC cells and may reduce the invasive ability of lung adenocarcinoma cells by weakening the process of EMT, which provides a new target for the prevention and treatment of lung adenocarcinoma.

Bioinformatic analysis of differentially expressed proteins in the dorsal raphe nucleus of rats after continuous treatment with olanzapine

To analyze the differentially expressed proteins in the dorsal raphe nucleus of rats treated with olanzapine and explore the possible mechanism of metabolic disorders in the early stage of olanzapine treatment. Twenty male and 20 female SD rats were both randomized equally into olanzapine group and control group for daily treatment with olanzapine and saline for 4 weeks, respectively. One hour after the last treatment, the dorsal raphe nucleus of the rats was dissected for proteomic analysis using iTRAQ combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). GO, KEGG pathway, COG, pathways and protein interaction network analyses of the differentially expressed proteins were performed. Several target genes were selected from the proteomic list, and their expression levels in the dorsal raphe nucleus of another 24 mice with identical grouping and treatment using real time real-time quantitative PCR and Western blotting. A total of 214 differentially expressed proteins were identified in the dorsal raphe nucleus of olanzapine-treated mice, including 72 unregulated and 142 downregulated proteins. GO analyses showed that the differentially expressed proteins were enriched in cellular process, biological regulation, metabolic process, response to stimulus, multicellular organismal process, bindings, catalytic activity, molecular function regulator and transcription regulator activity. KEGG analysis suggested that these proteins were enriched in fluid shear stress and atherosclerosis, serotonergic synapse, butanoate metabolism, thyroid hormone synthesis and IL-17 signaling pathway. The differentially expressed proteins Cav1, Hsp90b1, Canx, Gnai1, MAPK9, and LOC685513 were located at the nodes of the protein-protein interaction network in close relation with metabolic disorders. In olanzapine-treated mice, the expression of Hmgcs2, a negative regulator of apoptosis, was significantly down-regulated in the dorsal raphe nucleus, where the expressions of Pla2g4e, Slc6a4 and Gnai1 involved in serotonergic synapse were significantly upregulated. In the early stage of treatment, olanzapine may contribute to the occurrence of metabolic disorders in rats by regulating the expressions of Cav1, Hsp90b1, Canx, Gnai1, MAPK9, LOC685513 (Gng14) and 5-HTR2 synapse-related proteins in the dorsal raphe nucleus.

Pharmacologic Targeting of TFIIH Suppresses KRAS-Mutant Pancreatic Ductal Adenocarcinoma and Synergizes with TRAIL.

Pancreatic ductal adenocarcinoma (PDAC) typically presents as metastatic disease at diagnosis and remains refractory to treatment. Next-generation sequencing efforts have described the genomic landscape, classified molecular subtypes, and confirmed frequent alterations in major driver genes, with coexistent alterations in KRAS and TP53 correlating with the highest metastatic burden and poorest outcomes. However, translating this information to guide therapy remains a challenge. By integrating genomic analysis with an arrayed RNAi druggable genome screen and drug profiling of a KRAS/TP53 mutant PDAC cell line derived from a patient-derived xenograft (PDCL), we identified numerous targetable vulnerabilities that reveal both known and novel functional aspects of pancreatic cancer biology. A dependence on the general transcription and DNA repair factor TFIIH complex, particularly the XPB subunit and the CAK complex (CDK7/CyclinH/MAT1), was identified and further validated utilizing a panel of genomically subtyped KRAS mutant PDCLs. TFIIH function was inhibited with a covalent inhibitor of CDK7/12/13 (THZ1), a CDK7/CDK9 kinase inhibitor (SNS-032), and a covalent inhibitor of XPB (triptolide), which led to disruption of the protein stability of the RNA polymerase II subunit RPB1. Loss of RPB1 following TFIIH inhibition led to downregulation of key transcriptional effectors of KRAS-mutant signaling and negative regulators of apoptosis, including MCL1, XIAP, and CFLAR, initiating caspase-8 dependent apoptosis. All three drugs exhibited synergy in combination with a multivalent TRAIL, effectively reinforcing mitochondrial-mediated apoptosis. These findings present a novel combination therapy, with direct translational implications for current clinical trials on metastatic pancreatic cancer patients. Significance: This study utilizes functional genetic and pharmacological profiling of KRAS-mutant pancreatic adenocarcinoma to identify therapeutic strategies and finds that TFIIH inhibition synergizes with TRAIL to induce apoptosis in KRAS-driven pancreatic cancer.

Transcriptome Analysis Reveals Key Gene Expression Changes in Blue Catfish Sperm in Response to Cryopreservation.

The hybrids of female channel catfish (Ictalurus punctatus) and male blue catfish (I. furcatus) account for >50% of US catfish production due to superior growth, feed conversion, and disease resistance compared to both parental species. However, these hybrids can rarely be naturally spawned. Sperm collection is a lethal procedure, and sperm samples are now cryopreserved for fertilization needs. Previous studies showed that variation in sperm quality causes variable embryo hatch rates, which is the limiting factor in hybrid catfish breeding. Biomarkers as indicators for sperm quality and reproductive success are currently lacking. To address this, we investigated expression changes caused by cryopreservation using transcriptome profiles of fresh and cryopreserved sperm. Sperm quality measurements revealed that cryopreservation significantly increased oxidative stress levels and DNA fragmentation, and reduced sperm kinematic parameters. The present RNA-seq study identified 849 upregulated genes after cryopreservation, including members of all five complexes in the mitochondrial electron transport chain, suggesting a boost in oxidative phosphorylation activities, which often lead to excessive production of reactive oxygen species (ROS) associated with cell death. Interestingly, functional enrichment analyses revealed compensatory changes in gene expression after cryopreservation to offset detrimental effects of ultra-cold storage: MnSOD was induced to control ROS production; chaperones and ubiquitin ligases were upregulated to correct misfolded proteins or direct them to degradation; negative regulators of apoptosis, amide biosynthesis, and cilium-related functions were also enriched. Our study provides insight into underlying molecular mechanisms of sperm cryoinjury and lays a foundation to further explore molecular biomarkers on cryo-survival and gamete quality.

Osteomodulin protects dental pulp stem cells from cisplatin-induced apoptosis in vitro.

Human dental pulp stem cells (hDPSCs) are considered promising multipotent cell sources for tissue regeneration. Regulation of apoptosis and maintaining the cell homeostasis is a critical point for the application of hDPSCs. Osteomodulin (OMD), a member of the small leucine-rich proteoglycan family, was proved an important regulatory protein of hDPSCs in our previous research. Thus, the role of OMD in the apoptosis of hDPSCs was explored in this study. The expression of OMD following apoptotic induction was investigated and then the hDPSCs stably overexpressing or knocking down OMD were established by lentiviral transfection. The proportion of apoptotic cells and apoptosis-relative genes and proteins were examined with flow cytometry, Hoechst staining, Caspase 3 activity assay, qRT-PCR and western blotting. RNA-Seq analysis was used to explore possible biological function and mechanism. Results showed that the expression of OMD decreased following the apoptotic induction. Overexpression of OMD enhanced the viability of hDPSCs, decreased the activity of Caspase-3 and protected hDPSCs from apoptosis. Knockdown of OMD showed the opposite results. Mechanistically, OMD may act as a negative modulator of apoptosis via activation of the Akt/Glycogen synthase kinase 3β (GSK-3β)/β-Catenin signaling pathway and more functional and mechanistic possibilities were revealed with RNA-Seq analysis. The present study provided evidence of OMD as a negative regulator of apoptosis in hDPSCs. Akt/GSK-3β/β-Catenin signaling pathway was involved in this process and more possible mechanism detected needed further exploration. This anti-apoptotic function of OMD provided a promising application prospect for hDPSCs in tissue regeneration.

Short Communication: Maternal obesity alters ovine endometrial gene expression during peri-implantation development.

Exposure to maternal obesity in utero is associated with marked developmental effects in offspring that may not be evident until adulthood. Mechanisms regulating the programming effects of maternal obesity on fetal development have been reported, but little is known about how maternal obesity affects the earliest periods of embryonic development. This work explored how obesity influences endometrial gene expression during the peri-implantation period using a sheep model. Ewes were assigned randomly to diets that produced an obese state or maintained a lean state. After 4 mo, obese and lean ewes were bred and then euthanized at day 14 post-breeding. The uterus was excised, conceptuses were flushed, and endometrial tissue was collected. Isolated RNA from endometrial tissues (n = 6 ewes/treatment) were sequenced using an Illumina-based platform. Reads were mapped to the Ovis aries genome (Oar_4.0). Differential gene expression was determined, and results were filtered (false discovery rate ≤ 0.05 and ≥2-fold change, ≥0.2 reads/kilobase/million reads). Differentially expressed genes (DEGs) were identified (n = 699), with 171 downregulated and 498 upregulated in obese vs. lean endometrium, respectively. The most pronounced gene ontology categories identified were cellular process, metabolic process, and biological regulation. Enrichments were detected within the DEGs for genes involved with immune system processes, negative regulation of apoptosis, cell growth, and cell adhesion. A literature search revealed that 125 DEGs were associated with either the trophoblast lineage or the placenta. Genes within this grouping were involved with wingless/integrated signaling, angiogenesis, and integrin signaling. In summary, these data indicate that the peri-implantation endometrium is responsive to maternal obesity. Transcript profile analyses suggest that the endometrial immune response, adhesion, and angiogenesis may be especially susceptible to obesity. Thus, alterations in uterine transcript profiles during early embryogenesis may be a mechanism responsible for developmental programming following maternal obesity exposure in utero.

P332: MIR-625-5P IS NOVEL CANDIDATE ONCOMIR IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA IMPLICATED IN REGULATION OF APOPTOSIS VIA REPRESSION OF HARAKIRI

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of ALL, arising from T-cell precursors. miRNAs are non-coding RNAs, contributing to leukemogenesis by involvement in key cellular functions such as proliferation, apoptosis, and signaling. We previously found in miRNA transcriptome analysis that miR-625-5p is overexpressed in T-ALL patients, thus is a potential oncomiR in T-ALL. Here, we confirm the implication of miR-625-5p in the regulation of apoptosis in T-ALL cells in vitro. We show that it acts via repression of HARAKIRI (HRK). HRK was originally identified as a proapoptotic gene induced by reduced levels of cytokine in hematopoietic cells and repressed by the expression of death-repressor proteins. Although its proapoptotic function is well described, little is known about the post-transcriptional mechanisms that may participate in HRK inactivation. Aims: The aim of this study was to investigate the mechanism of miR-625-5p oncogenic action via unraveling its targetome and functional effects in T-ALL in vitro. Methods: T-ALL JURKAT cells (with high endogenous miR-625-5p level) were transduced for stable inhibition of this miRNA. To evaluate the influence of miR inhibition on growth of T-ALL cells, we used flow cytometry GFP competition assay and CCK8 proliferation assay. To identify the genes mediating the effect of miR-625-5p, we subjected JURKAT cells to Ago2-RIP-seq. We used magnetic beads coated with anti-AGO2 antibody to immunoprecipitate RNA bound to RISC complexes. Ago2-IP and total RNA fractions were sequenced (polyA RNA-seq, 150 bp reads, 60M PE reads/sample, Illumina NovaSeq 6000). Transcripts depleted in Ago2-IP fraction upon miR inhibition were screened for the presence of miR-625-5p binding sites. Luciferase assay was performed to confirm direct interaction between hsa-miR-625-5p and 3’UTR of HRK gene. The changes of HRK protein level upon miR-625-5p inhibition were evaluated via Western Blot. To estimate the apoptosis rate in T-ALL cells we used two flow cytometry assays based on Annexin V and Caspase 3/7 staining. Results: Inhibition of miR-625-5p decreased the growth of JURKAT cells. In RIP-seq analysis we identified 384 transcripts depleted in RISC by at least 20% upon miR-625-5p inhibition and having at least one putative 3’UTR binding site for this miRNA. Overrepresentation analysis performed for these genes indicated their involvement in the apoptotic process. Among transcripts depleted in RIP-seq with predicted binding sites for miR-625-5p, we found HRK gene, previously reported to have proapoptotic activity. We postulate that repression of this gene by overexpressed miR-625-5p contributes to decreased apoptosis and to growth advantage of T-ALL cells. To further confirm that miR-625-5p affects growth of T-ALL cells via negative regulation of apoptosis, we performed apoptotic assays in JURKAT cells and showed that inhibition of this miRNA increased apoptosis rate, in line with our hypothesis on the oncogenic potential of miR-625-5p. We confirmed the direct interaction between miR-625-5p and HRK 3’UTR by luciferase assay. Additionally, in Western blot a subtle (approaching statistical significance) increase of HRK protein upon miR-625-5p inhibition was observed, supporting our notion that this miRNA is indeed a negative regulator of HRK. Image:Summary/Conclusion: miR-625-5p is a novel candidate oncogenic miRNA in T-ALL, implicated in the negative regulation of apoptosis. Post-transcriptional repression of the proapoptotic HRK gene is a putative mechanism contributing to oncogenic properties of this miRNA when overexpressed in T-ALL cells.

ILP-2: A New Bane and Therapeutic Target for Human Cancers

Inhibitor of apoptosis protein-related-like protein-2 (ILP-2), also known as BIRC-8, is a member of the inhibitor of apoptosis protein (IAPs) family, which mainly encodes the negative regulator of apoptosis. It is selectively overexpressed in a variety of human tumors and can help tumor cells evade apoptosis, promote tumor cell growth, increase tumor cell aggressiveness, and appears to be involved in tumor cell resistance to chemotherapeutic drugs. Several studies have shown that downregulation of ILP-2 expression increases apoptosis, inhibits metastasis, reduces cell growth potential, and sensitizes tumor cells to chemotherapeutic drugs. In addition, ILP-2 inhibits apoptosis in a unique manner; it does not directly inhibit the activity of caspases but induces apoptosis by cooperating with other apoptosis-related proteins. Here, we review the current understanding of the various roles of ILP-2 in the apoptotic cascade and explore the use of interfering ILP-2, and the combination of related anti-tumor agents, as a novel strategy for cancer therapy.

High CD34 surface expression in BCP-ALL predicts poor induction therapy response and is associated with altered expression of genes related to cell migration and adhesion.

Minimal residual disease (MRD) constitutes the most important prognostic factor in B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL). Flow cytometry is widely used in MRD assessment, yet little is known regarding the effect of different immunophenotypic subsets on outcome. In this study of 200 BCP‐ALL patients, we found that a CD34‐positive, CD38 dim‐positive, nTdT dim‐positive immunophenotype on the leukemic blasts was associated with poor induction therapy response and predicted an MRD level at the end of induction therapy (EOI) of ≥ 0.001. CD34 expression was strongly and positively associated with EOI MRD, whereas CD34‐negative patients had a low relapse risk. Further, CD34 expression increased from diagnosis to relapse. CD34 is a stemness‐associated cell‐surface molecule, possibly involved in cell adhesion/migration or survival. Accordingly, genes associated with stemness were overrepresented among the most upregulated genes in CD34‐positive leukemias, and protein–protein interaction networks showed an overrepresentation of genes associated with cell migration, cell adhesion, and negative regulation of apoptosis. The present work is the first to demonstrate a CD34‐negative immunophenotype as a good prognostic factor in ALL, whereas high CD34 expression is associated with poor therapy response and an altered gene expression profile reminiscent of migrating cancer stem‐like cells.

MiRNA-29 aggravates myocardial infarction via inhibiting the PI3K/mTOR/HIF1α/VEGF pathway.

Introduction: MI is defined by the presence of myocardial necrosis, which is caused by acute and persistent ischemia and hypoxia of the coronary artery. In recent years, its incidence rate has been on the rise in China.Methods: GSE34198, GSE97320 and GSE141512 datasets were download for DEG analysis. KEGG pathway analysis, GO analysis, GSEA and PPI network construction were performed. Later, target genes of candidate miRNAs were predicted. Next, echocardiography was conducted to detect the effects of miR-29 on left ventricular structure and cardiac function in vivo, and H&E staining was adopted to study the effects of miR-29 on angiogenesis and fibrosis in vivo. Furthermore, Western blotting was employed to investigate the effects of miR-29 inhibition on the expressions of proteins related to the PI3K\\mTOR\\ HIF-1α\\VEGF pathway.Results: There were 162 DEGs involved in MI. GO analysis revealed that inflammatory responses, negative regulation of apoptosis and innate immune response were the main enriched biological processes. KEGG analysis manifested that DEGs were mainly enriched in the PI3K/Akt signaling pathway, and GSEA demonstrated that they were mainly enriched in the PI3K/Akt/mTOR, HIF and VEGF pathways. Moreover, target gene prediction showed that miR-29 was lowly expressed in MI. According to Masson's trichrome staining, miR-29 inhibition promoted angiogenesis, reduced fibrosis, and increased the protein expressions of p-PI3K, p-mTOR, HIF-1α, and VEGF.Conclusions: MiR-29 may play an important role in the growth and development of MI. After inhibition of miR-29, the PI3K/mTOR/HIF-1α/VEGF pathway is activated to alleviate MI.

The Anticancer Mechanisms of Scutellaria barbata against Lung Squamous Cell Carcinoma

Objective. Scutellaria barbata (S. barbata) is a Chinese traditional medicinal crop with anti-inflammatory as well as anticancer properties. To explore the anticancer mechanisms of functional monomers of S. barbata against lung squamous cell carcinoma (LUSC), a network pharmacology approach and molecular docking were utilized. Methods. The expression profile of genes encoding functional monomer components in S. barbata was obtained from the Traditional Chinese Medicine Systems Pharmacology platform (TCMSP) database. Expression data of LUSC-related genes were acquired from DisGeNET, GeneCards, OMIM, DrugBank, and TTD databases. The target genes of S. barbata that confer anticancer effects against LUSC were obtained by considering the intersecting genes between S. barbata target genes and LUSC-related genes. The potential regulatory pathways enriched in these intersected genes were identified using the KOBAS database, and Gene Ontology (GO) function enrichment analysis was performed using the online tool DAVID. The relationship network of S. barbata functional monomer components-action targets-disease-pathways was established using Cytoscape 3.8.2, and the protein-protein interaction network of those intersected genes was established using the STRING database. Finally, the hub genes were screened by using CytoNCA, a plug-in of Cytoscape, and hub gene expressions in LUSC were evaluated via the Gene Expression Profiling Interactive Analysis (GEPIA) database. AutoDockTools and PyMOL software were employed to verify the molecular docking on disease target proteins and drug functional molecules. Results. In S. barbata, 104 target genes and 20 hub genes encoding functional components against LUSC were screened out, six of which were significantly differentially expressed between LUSC samples and normal tissue samples in the GEPIA database. Here, GO analysis illustrated the involvement of these genes in the signal transduction and positive regulation of transcription from RNA polymerase II promoter and negative regulation of apoptosis, while KEGG pathway enrichment analysis demonstrated that these genes were mainly involved in several pathways, for instance, AGE-RAGE, PI3K-Akt, p53, and MAPK signaling pathway. There are four main functional components docking with six key target proteins, all of which have strong binding activity. Conclusions. We predicted the molecular mechanisms and signaling pathways of genes encoding functional components in S. barbata against LUSC. These discoveries offer novel understanding for further study, laying a scientific foundation for the production of synthetic monomer components of S. barbata.

Bushen Huatan recipe for treatment of polycystic ovary syndrome: therapeutic mechanism based on network pharmacology and molecular docking

To explore the pharmacological mechanism of Bushen Huatan (BSHT) recipe in the treatment of polycystic ovary syndrome (PCOS). The active ingredients in the component drugs of the recipe were screened through TCMSP, and their potential targets were predicted by PubChem and Swiss target prediction. Genecards and OMIM were used to screen the therapeutic targets in the treatment of PCOS. The drug targets and disease targets were corrected using Uniprot, and the intersection targets were obtained. The protein-protein interaction (PPI) network was constructed using STRING, and the intersection targets were analyzed with CytoNCA to screen the core targets. DAVID was used for GO enrichment analysis and KEGG pathway enrichment analysis, and the core components and core targets were verified using AutoDock. Animal experiment was performed to verify the results using a female C57BL/6J mouse model of PCOS, treated daily with 1 mg/kg BSHT recipe granule for 35 days, and the ovarian expressions of the core targets and pathways were detected using Western blotting. We identified a total of 125 potential active ingredients from the 14 component drugs in the recipe, 990 drug targets, 4759 PCOS targets and 434 intersection targets. The core active ingredients of the recipe included β -Sitosterol, kaempferol, and quercetin, whose core targets included PIK3CA, PIK3R1, APP, AKT1, and MAPK1. GO enrichment analysis highlighted such processes as drug reaction, negative regulation of apoptosis, and positive regulation of transcription from RNA polymerase Ⅱ promoter. The enriched KEGG pathways included primarily the cancer pathway and PI3K-Akt signaling pathway. Molecular docking showed that the core active ingredients had strong binding ability with the core targets. In the animal experiment, BSHT recipe was shown to improve the symptoms, down-regulate the expressions of PI3K and Akt proteins and up-regulate MAPK1 expression in the ovary of mice with PCOS. The therapeutic mechanism of BSHT recipe for PCOS involves multiple active ingredients, multiple therapeutic targets and multiple pathways.

RNA binding protein HuD promotes autophagy and tumor stress survival by suppressing mTORC1 activity and augmenting ARL6IP1 levels

BackgroundNeuronal-origin HuD (ELAVL4) is an RNA binding protein overexpressed in neuroblastoma (NB) and certain other cancers. The RNA targets of this RNA binding protein in neuroblastoma cells and their role in promoting cancer survival have been unexplored. In the study of modulators of mTORC1 activity under the conditions of optimal cell growth and starvation, the role of HuD and its two substrates were studied.MethodsRNA immunoprecipitation/sequencing (RIP-SEQ) coupled with quantitative real-time PCR were used to identify substrates of HuD in NB cells. Validation of the two RNA targets of HuD was via reverse capture of HuD by synthetic RNA oligoes from cell lysates and binding of RNA to recombinant forms of HuD in the cell and outside of the cell. Further analysis was via RNA transcriptome analysis of HuD silencing in the test cells.ResultsIn response to stress, HuD was found to dampen mTORC1 activity and allow the cell to upregulate its autophagy levels by suppressing mTORC1 activity. Among mRNA substrates regulated cell-wide by HuD, GRB-10 and ARL6IP1 were found to carry out critical functions for survival of the cells under stress. GRB-10 was involved in blocking mTORC1 activity by disrupting Raptor-mTOR kinase interaction. Reduced mTORC1 activity allowed lifting of autophagy levels in the cells required for increased survival. In addition, ARL6IP1, an apoptotic regulator in the ER membrane, was found to promote cell survival by negative regulation of apoptosis. As a therapeutic target, knockdown of HuD in two xenograft models of NB led to a block in tumor growth, confirming its importance for viability of the tumor cells. Cell-wide RNA messages of these two HuD substrates and HuD and mTORC1 marker of activity significantly correlated in NB patient populations and in mouse xenografts.ConclusionsHuD is seen as a novel means of promoting stress survival in this cancer type by downregulating mTORC1 activity and negatively regulating apoptosis.

Systematic Understanding of Mechanism of Danggui Shaoyao San against Ischemic Stroke Using a Network Pharmacology Approach.

Purpose Danggui Shaoyao San (DSS) was developed to treat the ischemic stroke (IS) in patients and animal models. The purpose of this study was to explore its active compounds and demonstrate its mechanism against IS through network pharmacology, molecular docking, and animal experiment. Methods All the components of DSS were retrieved from the pharmacology database of TCM system. The genes corresponding to the targets were retrieved using OMIM, CTD database, and TTD database. The herb-compound-target network was constructed by Cytoscape software. The target protein-protein interaction network was built using the STRING database. The core targets of DSS were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Then, we achieved molecular docking between the hub proteins and the key active compounds. Finally, animal experiments were performed to verify the core targets. Triphenyltetrazolium chloride (TTC) staining was used to calculate the infarct size in mice. The protein expression was determined using the Western blot. Results Compound-target network mainly contained 51 compounds and 315 corresponding targets. Key targets contained MAPK1, SRC, PIK3R1, HRAS, AKT1, RHOA, RAC1, HSP90AA1, and RXRA FN1. There were 417 GO items in GO enrichment analysis (p < 0.05) and 119 signaling pathways (p < 0.05) in KEGG, mainly including negative regulation of apoptosis, steroid hormone-mediated signaling pathway, neutrophil activation, cellular response to oxidative stress, and VEGF signaling pathway. MAPK1, SRC, and PIK3R1 docked with small molecule compounds. According to the Western blot, the expression of p-MAPK 1, p-AKT, and p-SRC was regulated by DSS. Conclusions This study showed that DSS can treat IS through multiple targets and routes and provided new insights to explore the mechanisms of DSS against IS.

Molecular mechanism of GANT61 combined with doxorubicin in the treatment of gliomas based on network pharmacology

Gliomas are common malignant intracranial tumors. Efficacious targeted therapy against gliomas is lacking. GANT61 combined with the chemotherapy drug doxorubicin for treatment of glioma (LN-229) cells, and the effect of their combination, was tested. The molecular mechanism was explored by target prediction, along with functional analysis using the Gene Ontology (GO) database, enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, construction of protein–protein interaction (PPI) networks, and protein expression. Combination of GANT61 plus doxorubicin could inhibit the growth of LN-229 cells effectively. Wound-healing data and expression of migration proteins related to epithelial-to-mesenchymal transition showed that this combination could inhibit the migration of LN-229 cells. Sixty-one targets of drug and disease intersected. Functional analysis revealed negative regulation of apoptosis, positive regulation of cell proliferation, and other biological processes related to apoptosis and proliferation. Pathway-enrichment analysis showed drug combination to be related to the cyclic adenosine monophosphate signaling pathway, pathways in cancer, and Hedgehog signaling pathway. Measurement of expression of several proteins related to these pathways revealed expression of BIRC5, GLi1 and GLi2, MMP3 and MMP9 proteins to decrease, and expression of MDM2 and P53 proteins to decrease and increase, respectively. This study provides a: (a) new direction for targeted therapy of gliomas; (b) theoretical basis for drug research and molecular-mechanism research on gliomas. How to cite: Chen J, Zhang Q, Zhang G et al. Molecular mechanism of GANT61 combined with doxorubicin in the treatment of gliomas based on network pharmacology. Electron J Biotechnol 2022;55. https://doi.org/10.1016/j.ejbt.2021.11.001.

Conditioned Medium from Bone Marrow Mesenchymal Stem Cells Restored Oxidative Stress-Related Impaired Osteogenic Differentiation.

Oxidative stress from high levels of intracellular reactive oxygen species (ROS) has been linked to various bone diseases. Previous studies indicate that mesenchymal stem cells (MSC) secrete bioactive factors (conditioned medium (MSC-CM)) that have antioxidant effects. However, the antioxidant role of MSC-CM on osteogenesis has not been fully studied. We aimed to identify antioxidant proteins in MSC-CM using mass spectrometry-based proteomics and to explore their effects on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSC) exposed to oxidative stress induced by hydrogen peroxide (H2O2). Our analysis revealed that MSC-CM is comprised of antioxidant proteins that are involved in several biological processes, including negative regulation of apoptosis and positive regulation of cell proliferation. Then, hBMSC exposed to H2O2 were treated with MSC-CM, and the effects on their osteogenic differentiation were evaluated. MSC-CM restored H2O2-induced damage to hBMSC by increasing the antioxidant enzyme-SOD production and the mRNA expression level of the anti-apoptotic BCL-2. A decrease in ROS production and cellular apoptosis was also shown. MSC-CM also modulated mRNA expression levels of osteogenesis-related genes, runt-related transcription factor 2, collagen type I, bone morphogenic protein 2, and osteopontin. Furthermore, collagen type I protein secretion, alkaline phosphatase activity, and in vitro mineralization were increased. These results indicate that MSC-CM contains several proteins with antioxidant and anti-apoptotic properties that restored the impaired hBMSC osteogenic differentiation associated with oxidative stress.

Elucidating the Mechanism of Action of Salvia miltiorrhiza for the Treatment of Acute Pancreatitis Based on Network Pharmacology and Molecular Docking Technology.

Using network pharmacology and molecular docking, this study investigated the molecular mechanisms by which the active components in Salvia miltiorrhiza can alleviate acute pancreatitis. Initially, the active components of Salvia miltiorrhiza and the targets collected from the GeneCards database were screened based on the platform of systematic pharmacology analysis of traditional Chinese medicine. Subsequently, the active components were intersected with the disease targets. Also, interactions among the targets were computed using the STRING database. Biological function and pathway enrichment were analyzed using the Cluster Profiler package in the R software. Protein-protein interaction and component target pathway network were constructed using the Cytoscape software. Ultimately, the key targets and their corresponding components in the network were verified using the AutoDock Vina software. The results showed Salvia miltiorrhiza had 111 targets for acute pancreatitis. The biological process (BP) analysis showed that the active components of Salvia miltiorrhiza induced a drug response, positive regulation of transcription by RNA polymerase II promoter, signal transduction, positive regulation of cell proliferation, and negative regulation of apoptosis. Furthermore, the KEGG enrichment analysis screened 118 (P < 0.05) signaling pathways, such as the pathways related to cancer, neuroactive ligand-receptor interaction, PI3K-Akt signaling pathway, and cAMP signaling pathway, to name a few. Finally, molecular docking showed that the active components of Salvia miltiorrhiza had a good binding affinity with their corresponding target proteins. Through network pharmacology, this study predicted the potential pharmacodynamic material basis and the mechanisms by which Salvia miltiorrhiza can treat acute pancreatitis. Moreover, this study provided a scientific basis for mining the pharmacodynamic components of Salvia miltiorrhiza and expanding the scope of its clinical use.

Cloning, Expression, and Purification of the Human Synthetic Survivin Protein in Escherichia coli Using Response Surface Methodology (RSM).

Survivin is one of the novel members of the apoptosis inhibitor protein family in humans. The main activity of the Survivin protein is to suppress caspases activity resulting in negative regulation of apoptosis. Survivin protein can be a potential target for the treatment of cancers between cancerous and normal cells. In the present research, the synthetic Survivin gene with PelB secretion signal peptide was cloned into a prokaryotic expression vector pET21a. The recombinant plasmid pET21a-PelB-Surv was expressed in Escherichia coli (E.coli) BL21, and the relative molecular mass of expressed protein was calculated 34,000g/mol, approximately. The recombinant protein was purified through chromatography column and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Response surface methodology (RSM) was used to design 20 experiments for optimization of IPTG concentration, post-induction period, and cell density of induction (OD600). The optimum levels of the selected parameters were successfully determined to be 0.28mM for IPTG concentration, 10h for post-induction period, and 3.40768 for cell density (OD600). These findings resulted in 4.14-fold increases in the Survivin production rate of optimum expression conditions (93.6363mg/ml).

Modeling Notch-Induced Tumor Cell Survival in the Drosophila Ovary Identifies Cellular and Transcriptional Response to Nuclear NICD Accumulation.

Notch is a conserved developmental signaling pathway that is dysregulated in many cancer types, most often through constitutive activation. Tumor cells with nuclear accumulation of the active Notch receptor, NICD, generally exhibit enhanced survival while patients experience poorer outcomes. To understand the impact of NICD accumulation during tumorigenesis, we developed a tumor model using the Drosophila ovarian follicular epithelium. Using this system we demonstrated that NICD accumulation contributed to larger tumor growth, reduced apoptosis, increased nuclear size, and fewer incidents of DNA damage without altering ploidy. Using bulk RNA sequencing we identified key genes involved in both a pre- and post- tumor response to NICD accumulation. Among these are genes involved in regulating double-strand break repair, chromosome organization, metabolism, like raptor, which we experimentally validated contributes to early Notch-induced tumor growth. Finally, using single-cell RNA sequencing we identified follicle cell-specific targets in NICD-overexpressing cells which contribute to DNA repair and negative regulation of apoptosis. This valuable tumor model for nuclear NICD accumulation in adult Drosophila follicle cells has allowed us to better understand the specific contribution of nuclear NICD accumulation to cell survival in tumorigenesis and tumor progression.

Cancer-associated fibroblasts are associated with poor prognosis in solid type of lung adenocarcinoma in a machine learning analysis

Cancer-associated fibroblasts (CAFs) participate in critical processes in the tumor microenvironment, such as extracellular matrix remodeling, reciprocal signaling interactions with cancer cells and crosstalk with infiltrating inflammatory cells. However, the relationships between CAFs and survival are not well known in lung cancer. The aim of this study was to reveal the correlations of CAFs with survival rates, genetic alterations and immune activities. This study reviewed the histological features of 517 patients with lung adenocarcinoma from The Cancer Genome Atlas (TCGA) database. We performed gene set enrichment analysis (GSEA), network-based analysis and survival analysis based on CAFs in four histological types of lung adenocarcinoma: acinar, papillary, micropapillary and solid. We found four hallmark gene sets, the epithelial-mesenchymal transition, angiogenesis, hypoxia, and inflammatory response gene sets, that were associated with the presence of CAFs. CAFs were associated with tumor proliferation, elevated memory CD4+T cells and high CD274 (encoding PD-L1) expression. In the pathway analyses, CAFs were related to blood vessel remodeling, matrix organization, negative regulation of apoptosis and transforming growth factor-β signaling. In the survival analysis of each histological type, CAFs were associated with poor prognosis in the solid type. These results may contribute to the development of therapeutic strategies against lung adenocarcinoma cases in which CAFs are present.