Key Target Genes Research Articles

Biomarker profiling to determine clinical impact of microRNAs in cognitive disorders

Alzheimer’s disease (AD) and post-stroke cognitive impairment (PSCI) are the leading causes of progressive dementia related to neurodegenerative and cerebrovascular injuries in elderly populations. Despite decades of research, patients with these conditions still lack minimally invasive, low-cost, and effective diagnostic and treatment methods. MicroRNAs (miRNAs) play a vital role in AD and PSCI pathology. As they are easily obtained from patients, miRNAs are promising candidates for the diagnosis and treatment of these two disorders. In this study, we performed complete sequencing analysis of miRNAs from 24 participants, split evenly into the PSCI, post-stroke non-cognitive impairment (PSNCI), AD, and normal control (NC) groups. To screen for differentially expressed miRNAs (DE-miRNAs) in patients, we predicted their target genes using bioinformatics analysis. Our analyses identified miRNAs that can distinguish between the investigated disorders; several of them were novel and never previously reported. Their target genes play key roles in multiple signaling pathways that have potential to be modified as a clinical treatment. In conclusion, our study demonstrates the potential of miRNAs and their key target genes in disease management. Further in-depth investigations with larger sample sizes will contribute to the development of precise treatments for AD and PSCI.

Identification of active compounds in Vernonia anthelmintica (L.) willd by targeted metabolome MRM and kaempferol promotes HaCaT cell proliferation and reduces oxidative stress.

Introduction: Vernonia anthelmintica (L.) Willd. is a traditional treatment for vitiligo in Xinjiang. However, its therapeutic mechanism remains unclear owing to its complex composition and limited research on its chemical profile. Methods: We employed a targeted metabolome approach, combining selective reaction monitoring/multiple response monitoring (SRM/MRM) with high-performance liquid chromatography and MRM mass spectrometry to quantitatively analyze the flavonoid constituents of Vernonia anthelmintica. We also used network pharmacology and molecular docking to identify potential vitiligo-linked compounds and targets of V. anthelmintica seeds. Additionally, we assessed HaCaT cell proliferation by AAPH-induced, alongside changes in SOD activity and MDA content, following treatment with V. anthelmintica components. Finally, flow cytometry was used to detect apoptosis and ROS levels. Results and Discussion: We identified 36 flavonoid compounds in V. anthelmintica seeds, with 14 compounds exhibiting druggability. AKT1, VEGFA, ESR1, PTGS2, and IL2 have been identified as key therapeutic target genes, with PI3K/AKT signaling being an important pathway. Notably, kaempferol, one of the identified compounds, exhibited high expression in network pharmacology analysis. Kaempferol exhibited a strong binding affinity to important targets. Further, kaempferol enhanced HaCaT cell viability, inhibited apoptosis, reduced MDA levels, suppressed ROS activity, and upregulated SOD activity, increase the expression of cellular antioxidant genes, including HO-1, GCLC, GCLM, Nrf2, NQO1 and Keap1, providing significant protection against oxidative stress damage in vitro. Here, we present the first comprehensive study integrating SRM/MRM approaches and network analysis to identify active flavonoid compounds within V. anthelmintica (L.) Willd. Moreover, we revealed that its active ingredient, kaempferol, offers protection against AAPH-induced damage in keratinocytes, highlighting its potential as a clinical resource.

The miR159a-DUO1 module regulates pollen development by modulating auxin biosynthesis and starch metabolism in citrus.

Achieving seedlessness in citrus varieties is one of the important objectives of citrus breeding. Male sterility associated with abnormal pollen development is an important factor in seedlessness. However, our understanding of the regulatory mechanism underlying the seedlessness phenotype in citrus is still limited. Here, we determined that the miR159a-DUO1 module played an important role in regulating pollen development in citrus, which further indirectly modulated seed development and fruit size. Both the overexpression of csi-miR159a and the knocking out of DUO1 in Hong Kong kumquat (Fortunella hindsii) resulted in small and seedless fruit phenotypes. Moreover, pollen was severely aborted in both transgenic lines, with arrested pollen mitotic I and abnormal pollen starch metabolism. Through additional cross-pollination experiments, DUO1 was proven to be the key target gene for miR159a to regulate male sterility in citrus. Based on DNA affinity purification sequencing (DAP-seq), RNA-seq, and verified interaction assays, YUC2/YUC6, SS4 and STP8 were identified as downstream target genes of DUO1, those were all positively regulated by DUO1. In transgenic F. hindsii lines, the miR159a-DUO1 module down-regulated the expression of YUC2/YUC6, which decreased indoleacetic acid (IAA) levels and modulated auxin signaling to repress pollen mitotic I. The miR159a-DUO1 module reduced the expression of the starch synthesis gene SS4 and sugar transport gene STP8 to disrupt starch metabolism in pollen. Overall, this work reveals a new mechanism by which the miR159a-DUO1 module regulates pollen development and elucidates the molecular regulatory network underlying male sterility in citrus.

KRAS/PI3K axis driven GTF3C6 expression and promotes LUAD via FAK pathway

IntroductionEffective targeting drugs for KRAS mutation-mediated Lung Adenocarcinoma (LUAD) are currently are limited. ObjectivesInvestigating and intervening in the downstream key target genes of KRAS is crucial for clinically managing KRAS mutant-driven LUAD. GTF3C6, a newly identified member of the general transcription factor III (GTF3) family, plays a role in the transcription of RNA polymerase III (pol III)-dependent genes. However, its involvement in cancer remains unexplored. MethodsThis study examined the expression, roles, and potential molecular mechanisms of GTF3C6 in LUAD tissues, LSL-KrasG12D/+;LSL-p53−/− LUAD mouse models, and LUAD patients-derived organoid using Western blot, qRT-PCR, immunofluorescence, immunohistochemistry, and gene manipulation assays. ResultsWe present the first evidence that GTF3C6 is highly expressed in LUAD tissues, LSL-KrasG12D/+;LSL-p53−/− LUAD mouse models, and LUAD organoids, correlating with poor clinical prognosis. Furthermore, GTF3C6 was found to promote anchorage-independent proliferation, migration, and invasion of LUAD cells. Mechanistically, KRAS mutation drives GTF3C6 expression through the PI3K pathway, and GTF3C6 knockdown reverses the malignant phenotype of KRAS mutation-driven LUAD cells. Additionally, the FAK pathway emerged as a crucial downstream signaling pathway through which GTF3C6 mediates the malignant phenotype of LUAD. Finally, GTF3C6 knockdown suppresses LUAD organoid formation and inhibits tumor growth in vivo. ConclusionOur findings demonstrate that GTF3C6, driven by KRAS mutation, promotes LUAD development by regulating FAK phosphorylation, suggesting its potential as a biomarker and therapeutic target in KRAS mutant-driven LUAD.

Optimized therapeutic potential of Yinchenhao decoction for cholestatic hepatitis by combined network meta-analysis and network pharmacology

BackgroundCholestatic hepatitis is recognized as a significant contributor to the development of liver fibrosis and cirrhosis. As a well-known classic formula for the treatment of cholestatic hepatitis, Yinchenhao decoction (YCHD) is widely used in countries in Asia, including China, Japan, and Korea. However, in recent years, a risk of liver injury has been reported from Rheum palmatum L. and Gardenia jasmonoides J.Ellis which are the main ingredients of YCHD. Therefore, the question arises whether YCHD is still safe enough for the treatment of cholestatic hepatitis or whether an optimized ratio of ingredients should be applied. These is inevitable questions for the clinical application of YCHD. PurposeTo provide a scientific basis for the clinical application of YCHD through a combination of meta-analysis and network pharmacology and to find the best ratio of components to ensure optimal therapeutic efficacy and safety. At the same time, a deeper understanding of the mechanisms of YCHD was explored. MethodsWe retrieved relevant trials from various databases including PubMed, Web of Science, EMBASE, Cochrane Library, China National Knowledge Infrastructure (CNKI), VIP and Wanfang databases up to August 2023. After screening for inclusion and exclusion criteria, we assessed efficiency, ALT, AST, and TBIL as outcome parameters. The relevant data underwent a network meta-analysis using STATA 16.0 software. Based on network pharmacology, we screened the disease targets, active ingredients, and targets related to YCHD. The targets were visualized using Cytoscape 3.9.1. Then, potential mechanisms were explored based on bioinformatic techniques. ResultsTwenty eligible studies were finally screened and a total of 1,591 patients who fulfilled the inclusion criteria were enrolled in the study. The meta-analysis results indicated that TG-c (treatment group c) [(Artemisia capillaris Thunb. : Gardenia jasminoides J.Ellis : Rheum palmatum L. = 10:5:2–10:5:3) + CT] was the most promising therapeutic approach, demonstrating superior efficacy and notable improvements in both AST and TBIL levels. For ALT, TG-d [(Artemisia capillaris : Gardenia jasminoides : Rheum palmatum = 5:1:1–5:2:1) + CT] exhibited the greatest potential as optimal therapy option. Based on the surface under the cumulative ranking curve (SUCRA) values, TG-c was the best therapy in terms of efficiency and improvement in TBIL levels, while TG-d was the most effective in reducing ALT levels. For AST levels, TG-e [(Artemisia capillaris : Gardenia jasminoides : Rheum palmatum = 5:2:2–5:3:3) + CT] was the most effective therapy. The comprehensive analysis revealed that TG-c exhibited the most pronounced efficacy. Combined network pharmacology, GO enrichment analysis and KEGG pathway enrichment analysis displayed that the key target genes of Artemisia capillaris, Rheum palmatum, and Gardenia jasminoides were closely involved in inflammation response, bile transport, apoptosis, oxidative stress, and regulation of leukocyte migration. Notably, bile secretion dominated the common pathway of the three herbs. On the other hand, Artemisia capillaris exhibited a unique mode of action by regulating the IL-17 signaling pathway, which may play a crucial role in its effectiveness. ConclusionBased on our findings, the optimal TG-C demonstrated the most favorable overall therapeutic efficacy by increasing the dosage of Artemisia capillaris while reducing the dosage of Gardenia jasminoides and Rheum palmatum. This is attributed to the potent ability of Artemisia capillaris. to effectively modulate the IL-17 signaling pathway, thereby exerting a beneficial therapeutic effect. Conversely, Gardenia jasminoides and Rheum palmatum may potentially enhance the activation of the NF-кB signaling pathway, thereby elevating the risk of hepatotoxicity.

Urine-derived exosomes and their role in modulating uroepithelial cells to prevent hypospadias

PurposeUrethral hypospadias, a common congenital malformation in males, is closely linked with disruptions in uroepithelial cell (UEC) processes. Evidence exists reporting that urine-derived exosomes (Urine-Exos) enhance UEC proliferation and regeneration, suggesting a potential role in preventing hypospadias. However, the specific influence of Urine-Exos on urethral hypospadias and the molecular mechanisms involved are not fully understood. This study focuses on investigating the capability of Urine-Exos to mitigate urethral hypospadias and aims to uncover the underlying molecular mechanisms. MethodsBioinformatics analysis was performed to identify key gene targets in Urine-Exos potentially involved in hypospadias. Subsequent in vitro and in vivo experiments were conducted to validate the regulatory effects of Urine-Exos on hypospadias. ResultsBioinformatics screening revealed syndecan-1 (SDC1) as a potential pivotal gene for the prevention of hypospadias. In vitro experiments demonstrated that Urine-Exos enhanced the proliferation and migration of UECs by transferring SDC1 and inhibiting cell apoptosis. Notably, Urine-Exos upregulated β-catenin expression through SDC1 transfer, further promoting UEC proliferation and migration. These findings were confirmed in a congenital hypospadias rat model induced by di(2-ethylhexyl) phthalate (DEHP). ConclusionThis study reveals the therapeutic potential of Urine-Exos in hypospadias, mediated by the SDC1/β-catenin axis. Urine-Exos promote UEC proliferation and migration, thereby inhibiting the progression of hypospadias. These findings offer new insights and potential therapeutic targets for the management of congenital malformations.

Abstract 467: Genetic ablation of microRNA-10b improves treatment outcomes in mouse models of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a recalcitrant and lethal disease with an overall 5-year survival rate of less than 12%. PDAC is a heterogeneous disease characterized by highly aggressive cancer cells, extensive desmoplastic reaction, and hypovascularity. These unique features endow PDAC tumors with an array of innate resistance mechanisms against standard-of-care (SOC) chemotherapy: gemcitabine/nab-paclitaxel or FOLFIRINOX. Thus, there is clinical need to develop novel targets that enhance treatment efficacy of SOC chemotherapy. microRNA-10b (miR-10b) expression is frequently upregulated in PDAC and correlates with poor outcome. High levels of miR-10b expression within cancer cells correlates with poor treatment response to gemcitabine-containing neoadjuvant chemotherapy and poor overall clinical outcome. We and others reported that miR-10b exerts cancer cell-intrinsic pro-survival, anti-apoptotic, and pro-metastatic functions via downregulation of key target genes (i.e., BIM, PTEN, TIAM1). We hypothesize that upregulation of miR-10b expression is a chemoresistance mechanism to increase cell survival and inhibit apoptosis in PDAC. To test this hypothesis, we generated a KPC model (KrasG12D-driven, p53-deleted by Pdx1-Cre) with global Mir-10b gene knockout (Mir-10bKO). Animals were recruited when their tumor volume reached 200 mm3 by multi-mouse MR imaging and were randomly assigned to control or treatment groups (100 mg/kg of gemcitabine q4d). Tumor growth was monitored by weekly MR imaging sessions throughout the duration of the treatment. Untreated KPC;Mir-10bKO animals survived several weeks longer than untreated KPC animals (21.7 vs. 18 weeks, p = 0.056), suggesting a weak but detectable tumor promoting role of miR-10b. Remarkably, loss of miR-10b activity enhances treatment response to gemcitabine and significantly prolongs survival in this KPC model. Treated KPC animals lived only 5 weeks longer than untreated KPC (23 vs 18 weeks, p = 0.015), whereas KPC;Mir-10bKO lived 9 weeks longer (27 vs 18 weeks, p = 0.002). Notably, we observed a more durable response in treated female KPC;Mir-10bKO animals (n = 4) compared to any other experimental group (n > 5). We are currently performing tissue and molecular analyses to identify direct target genes involved in chemoresistance and sex-specific modulation of treatment response. Citation Format: Katarzyna Kempinska, Sudhakar Samuel, Saiprashanth Rajesh, Christiane Mallett, Lorenzo Sempere. Genetic ablation of microRNA-10b improves treatment outcomes in mouse models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 467.

CLEC3B inhibits proliferation and radioresistance and induces apoptosis in BT549 breast cancer cells

Among all breast cancer subtypes, triple-negative breast cancer (TNBC) has the worst prognosis due to its resistance to treatment, including radiotherapy. The purpose of this study was to screen and validate potential targets related to radioresistance in TNBC. First, a radiation-resistant TNBC cell model, was generated using BT549 cells through fractionated radiation (30 Gy in total, 10 fractions). Then, mRNA sequencing was used to screen potential target genes related to radioresistance. Furthermore, bioinformatics, proteomics and genomics methods were used to screen and validate the key target genes. The results showed that BT549 cells that had undergone fractionated radiation, namely, BT549R cells, had greater proliferation and radioresistance and a lower apoptosis rate than parental BT549 cells. C-type lectin domain family 3 member B (CLEC3B) was identified as a potential key target gene related to radioresistance. The expression of CLEC3B was closely related to the radioresistance of breast cancer and the survival of breast cancer patients. Downregulation of CLEC3B increased cell viability and radioresistance and reduced apoptosis in BT549R cells, which could be reversed by dihydroartemisinin (DHA). The overexpression of CLEC3B with or without DHA treatment inhibited the activity of the PI3K/AKT signaling pathway. DHA increased the expression of CLEC3B in a concentration-dependent manner. In summary, our study illustrated that the downregulation of CLEC3B increased radioresistance in BT549 breast cancer cells and that this effect could be reversed by DHA.

Acanthopanax senticosus improves cognitive impairment in Alzheimer's disease by promoting the phosphorylation of the MAPK signaling pathway.

Acanthopanax senticosus (AS) can improve sleep, enhance memory, and reduce fatigue and is considered as an effective drug for Alzheimer's disease (AD). The therapeutic effect and mechanism need to be further investigated. To confirm the AS play efficacy in alleviating memory impairment in mice, 5×FAD transgenic mice were subjected to an open-field experiment and a novelty recognition experiment. Network pharmacology technique was used to analyze the information of key compounds and potential key targets of AS for the treatment of AD, molecular docking technique was applied to predict the binding ability of targets and compounds, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were also performed on the targets to derive the possible metabolic processes and pathway mechanisms of AS in treating AD. Quantitative real-time PCR (qRT-PCR) and western blot technique were carried out to validate the candidate genes and pathways. In the open-field experiment, compared with the wild-type (WT) group, the number of times the mice in the AD group crossed the central zone was significantly reduced (P< 0.01). Compared with the AD group, the number of times the mice in the AS group crossed the central zone was significantly increased (P< 0.001). In the new object recognition experiment, compared with the WT group, the percentage of times the AD group explored new objects was significantly reduced (P< 0.05). Compared with the AD group, the AS group had an increase in the percentage of time spent exploring new things and the number of times it was explored (P< 0.05). At the same time, the donepezil group had a significantly higher percentage of times exploring new things (P< 0.01). By using network pharmacology technology, 395 common targets of AS and AD were retrieved. The Cytoscape software was used to construct the protein-protein interaction (PPI) network of common targets. Using the algorithm, nine key targets were retrieved: APP, NTRK1, ESR1, CFTR, CSNK2A1, EGFR, ESR2, GSK3B, and PAK1. The results of molecular docking indicate that 11 pairs of compounds and their corresponding targets have a significant binding ability, as the molecular binding energies were less than -7.0. In comparison to the AD group, the mRNA expression of the key target genes was significantly decreased in the AS treatment group (P< 0.001). The KEGG analysis showed that the MAPK signaling pathway was significantly enriched, and Western blot confirmed that the TRAF6 protein decreased significantly (P< 0.0001). Meanwhile, the levels of MAP3K7 and P38 phosphorylation increased, and there was also an increase in the expression of HSP27 proteins. Our study indicates that the multi-component and multi-target properties of AS play an important role in the alleviation of anxiety and memory impairment caused by AD, and the mechanism is involved in the phosphorylation and activation of the MAPK signaling pathway. The results of this study could provide a novel perspective for the clinical treatment of AD.

BmNPV Bm60 is a key target gene used by a resistant strain of Bombyx mori to inhibit BmNPV proliferation

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes significant losses to the silkworm industry. Numerous antiviral genes and proteins have been identified by studying silkworm resistance to BmNPV. However, the molecular mechanism of silkworm resistance to BmNPV is unclear. We analyzed the differences between the susceptible strain 871 and a near-isogenic resistant strain 871C. The survival of strain 871C was significantly greater than that of 871 after oral and subcutaneous exposure to BmNPV. Strain 871C exhibited a nearly 10,000-fold higher LD50 for BmNPV compared to 871. BmNPV proliferation was significantly inhibited in all tested tissues of strain 871C using HE strain and fluorescence analysis. Strain 871C exhibited cellular resistance to BmNPV rather than peritrophic membrane or serum resistance. Strain 871C suppressed the expression of the viral early gene Bm60. This led to the inhibition of BmNPV DNA replication and late structural gene transcription based on the cascade regulation of baculovirus gene expression. Bm60 could also interact with the viral DNA binding protein and alkaline nuclease, as well as host proteins Methylcrotonoyl-CoA carboxylase subunit alpha, mucin-2-like protein, and 30 K-8. Overexpression of 30 K-8 significantly inhibited BmNPV proliferation. These results increase understanding of the molecular mechanism behind silkworm resistance to BmNPV and suggest targets for the breeding of resistant silkworm strains and the controlling pest of Lepidoptera.

Exploration of hypoglycemic peptides from porcine collagen based on network pharmacology and molecular docking.

In recent years, the extraction of hypoglycemic peptides from food proteins has gained increasing attention. Neuropeptides, hormone peptides, antimicrobial peptides, immune peptides, antioxidant peptides, hypoglycemic peptides and antihypertensive peptides have become research hotspots. In this study, bioinformatic methods were used to screen and predict the properties of pig collagen-derived hypoglycemic peptides, and their inhibitory effects on α-glucosidase were determined in vitro. Two peptides (RL and NWYR) were found to exhibit good water solubility, adequate ADMET (absorption, distribution, metabolism, elimination, and toxicity) properties, potentially high biological activity, and non-toxic. After synthesizing these peptides, NWYR showed the best inhibitory effect on α-glucosidase with IC50 = 0.200±0.040 mg/mL, and it can regulate a variety of biological processes, play a variety of molecular functions in different cellular components, and play a hypoglycemic role by participating in diabetic cardiomyopathy and IL-17 signaling pathway. Molecular docking results showed that NWYR had the best binding effect with the core target DPP4 (4n8d), with binding energy of -8.8 kcal/mol. NWYR mainly bonded with the target protein through hydrogen bonding, and bound with various amino acid residues such as Asp-729, Gln-731, Leu-765, etc., thus affecting the role of the target in each pathway. It is the best core target for adjuvant treatment of T2DM. In short, NWYR has the potential to reduce type 2 diabetes, providing a basis for further research or food applications as well as improved utilization of pig by-products. However, in subsequent studies, it is necessary to further verify the hypoglycemic ability of porcine collagen active peptide (NWYR), and explore the hypoglycemic mechanism of NWYR from multiple perspectives such as key target genes, protein expression levels and differences in metabolites in animal models of hyperglycemia, which will provide further theoretical support for its improvement in the treatment of T2DM.

Qi-dan-dihuang decoction ameliorates renal fibrosis in diabetic rats via p38MAPK/AKT/mTOR signaling pathway.

Qi-dan-dihuang decoction (QDD) has been used to treat diabetic kidney disease (DKD), but the underlying mechanisms are poorly understood. This study reveals the mechanism by which QDD ameliorates DKD. The compounds in QDD were identified by high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry (HPLC-Q-TOF-MS). Key targets and signaling pathways were screened through bioinformatics. Nondiabetic Lepr db/m mice were used as control group, while Lepr db/db mice were divided into model group, dapagliflozin group, 1% QDD-low (QDD-L), and 2% QDD-high (QDD-H) group. After 12 weeks of administration, 24 h urinary protein, serum creatinine, and blood urea nitrogen levels were detected. Kidney tissues damage and fibrosis were evaluated by pathological staining. In addition, 30 mmol/L glucose-treated HK-2 and NRK-52E cells to induce DKD model. Cell activity and migration capacity as well as protein expression levels were evaluated. A total of 46 key target genes were identified. Functional enrichment analyses showed that key target genes were significantly enriched in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, in vivo and in vitro experiments confirmed that QDD ameliorated renal fibrosis in diabetic mice by resolving inflammation and inhibiting the epithelial-mesenchymal transition (EMT) via the p38MAPK and AKT-mammalian target of rapamycin (mTOR) pathways. QDD inhibits EMT and the inflammatory response through the p38MAPK and AKT/mTOR signaling pathways, thereby playing a protective role in renal fibrosis in DKD.

Network pharmacology analysis and experimental verification of the antitumor effect and molecular mechanism of isocryptomerin on HepG2 cells.

Isocryptomerin (ISO) is a flavonoid isolated from the natural medicine Selaginellae Herba, which has various pharmacological activities. This study investigated the antitumor effect and underlying molecular mechanism of ISO on hepatocellular carcinoma (HCC) HepG2 cells. The cell viability assay revealed that ISO has a considerable killing effect on HCC cell lines. The apoptosis assay showed that ISO induced mitochondria-dependent apoptosis through the Bad/cyto-c/cleaved (cle)-caspase-3/cleaved (cle)-PARP pathway. The network pharmacological analysis found 13 key target genes, and epidermal growth factor receptor (EGFR), AKT, mitogen-activated protein kinase(MAPK), and reactive oxygen species (ROS) signaling pathways were strongly associated with ISO against HCC. Further verification of the results showed that ISO induced apoptosis by increasing p-p38 and p-JNK expression and decreasing p-EGFR, p-SRC, p-ERK, and p-STAT3 expression. Furthermore, ISO induced G0/G1 phase arrest by downregulating p-AKT, Cyclin D, and CDK 4 expression and upregulating p21 and p27 expression in HepG2 cells. Moreover, ISO inhibited HepG2 cell migration by decreasing p-GSK-3β, β-catenin, and N-cadherin expression and increasing E-cadherin expression. Additionally, ISO promoted ROS accumulation in HepG2 cells, and ISO-induced apoptosis, arrest cell cycle, and inhibition of migration were reversed by an ROS scavenger, N-acetyl- l-cysteine. Overall, ISO induced cell apoptosis and cell cycle arrest and inhibited cell migration by ROS-mediated EGFR, AKT, and MAPK signaling pathways in HepG2 cells.

In silico prediction of candidate gene targets for the management of African cassava whitefly (Bemisia tabaci, SSA1-SG1), a key vector of viruses causing cassava brown streak disease.

Whiteflies (Bemisia tabaci sensu lato) have a wide host range and are globally important agricultural pests. In Sub-Saharan Africa, they vector viruses that cause two ongoing disease epidemics: cassava brown streak disease and cassava mosaic virus disease. These two diseases threaten food security for more than 800 million people in Sub-Saharan Africa. Efforts are ongoing to identify target genes for the development of novel management options against the whitefly populations that vector these devastating viral diseases affecting cassava production in Sub-Saharan Africa. This study aimed to identify genes that mediate osmoregulation and symbiosis functions within cassava whitefly gut and bacteriocytes and evaluate their potential as key gene targets for novel whitefly control strategies. The gene expression profiles of dissected guts, bacteriocytes and whole bodies were compared by RNAseq analysis to identify genes with significantly enriched expression in the gut and bacteriocytes. Phylogenetic analyses identified three candidate osmoregulation gene targets: two α-glucosidases, SUC 1 and SUC 2 with predicted function in sugar transformations that reduce osmotic pressure in the gut; and a water-specific aquaporin (AQP1) mediating water cycling from the distal to the proximal end of the gut. Expression of the genes in the gut was enriched 23.67-, 26.54- and 22.30-fold, respectively. Genome-wide metabolic reconstruction coupled with constraint-based modeling revealed four genes (argH, lysA, BCAT & dapB) within the bacteriocytes as potential targets for the management of cassava whiteflies. These genes were selected based on their role and essentiality within the different essential amino acid biosynthesis pathways. A demonstration of candidate osmoregulation and symbiosis gene targets in other species of the Bemisia tabaci species complex that are orthologs of the empirically validated osmoregulation genes highlights the latter as promising gene targets for the control of cassava whitefly pests by in planta RNA interference.

RNA-seq combined network pharmacology reveals that Fu-Gan-Wan (FGW) inhibits liver fibrosis via NF-κB/CCL2/CCR2 and lipid peroxidation via Nrf2/HMOX1 signaling pathway

Ethnopharmacological relevanceLiver fibrosis is a serious complication of liver disease characterized by excessive collagen deposition, without effective therapeutic agents in the clinic. Fu-Gan-Wan (FGW) is an empirical formula used for the clinical treatment of hepatitis and cirrhosis. It has been shown to reverse experimental liver fibrosis. However, its corresponding mechanisms remain unclear. Aim of the reviewThis study aimed to elucidate the key pathways and target genes of FGW in attenuating liver fibrosis. Materials and methodsThe therapeutic effects of different doses of FGW on liver fibrosis were investigated using a 2 mL/kg 15% CCl4-induced mouse model. Then, RNA-seq combined with network pharmacology was used to analyze the key biological processes and signaling pathways underlying the anti-liver fibrosis exertion of FGW. These findings were validated in a TGF-β1-induced model of activation and proliferation of mouse hepatic stellate cell line JS-1. Finally, the key signaling pathways and molecular targets were validated using animal tissues, and the effect of FGW on tissue lipid peroxidation was additionally observed. ResultsWe found that 19.5 g/kg FGW significantly down-regulated CCl4-induced elevation of hepatic ALT and AST, decreased collagen deposition, and inhibited the expression of pro-fibrotic factors α-SMA, COL1α1, CTGF, TIMP-1, as well as pro-inflammatory factor TGF-β1. Additionally, FGW at doses of 62.5, 125, and 250 μg/mL dose-dependently blocked JS-1 proliferation, migration, and activation. Furthermore, RNA-seq identified the NF-κB signaling pathway as a key target molecular pathway for FGW against liver fibrosis, and network pharmacology combined with RNA-seq focused on 11 key genes. Significant changes were identified in CCL2 and HMOX1 by tissue RT-PCR, Western blot, and immunohistochemistry. We further demonstrated that FGW significantly attenuated CCl4-induced increases in p-p65, CCL2, CCR2, and HMOX1, while significantly elevating Nrf2. Finally, FGW significantly suppressed the accumulation of lipid peroxidation products MDA and 4-HNE and reconfigured the oxidation-reduction balance, including promoting the increase of antioxidants GPx, GSH, and SOD, and the decrease of peroxidation products ROS and GSSG. ConclusionsThis study demonstrated that FGW exhibits potential in mitigating CCl4-induced hepatic fibrosis, lipid peroxidation, and iron metabolism disorders in mice. This effect may be mediated through the NF-κB/CCL2/CCR2 and Nrf2/HMOX1 pathways.

Exploring the therapeutic mechanisms and prognostic targets of Biochanin A in glioblastoma via integrated computational analysis and in vitro experiments

Glioblastoma (GBM) is the most aggressive brain tumor and is characterized by a poor prognosis and high recurrence and mortality rates. Biochanin A (BCA) exhibits promising clinical anti-tumor effects. In this study, we aimed to explore the pharmacological mechanisms by which BCA acts against GBM. Network pharmacology was employed to identify overlapping target genes between BCA and GBM. Differentially expressed genes from the Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database were visualized using VolcaNose. Interactions among these overlapping genes were analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins database. Protein–protein interaction networks were constructed using Cytoscape 3.8.1. The Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology enrichment analyses were conducted using the Database for Annotation, Visualization, and Integrated Discovery. Survival analyses for these genes were performed using the GEPIA2 database. The Chinese Glioma Genome Atlas database was used to study the correlations between key prognostic genes. Molecular docking was confirmed using the DockThor database and visualized with PyMol software. Cell viability was assessed via the CCK-8 assay, apoptosis and the cell cycle stages were examined using flow cytometry, and protein expression was detected using western blotting. In all, 63 genes were initially identified as potential targets for BCA in treating GBM. Enrichment analysis suggested that the pharmacological mechanisms of BCA primarily involved cell cycle inhibition, induction of cell apoptosis, and immune regulation. Based on these findings, AKT1, EGFR, CASP3, and MMP9 were preliminarily predicted as key prognostic target genes for BCA in GBM treatment. Furthermore, molecular docking analysis suggested stable binding of BCA to the target protein. In vitro experiments revealed the efficacy of BCA in inhibiting GBM, with an IC50 value of 98.37 ± 2.21 μM. BCA inhibited cell proliferation, induced cell apoptosis, and arrested the cell cycle of GBM cells. Furthermore, the anti-tumor effects of BCA on U251 cells were linked to the regulation of the target protein. We utilized integrated bioinformatics analyses to predict targets and confirmed through experiments that BCA possesses remarkable anti-tumor activities. We present a novel approach for multi-target treatment of GBM using BCA.

Revealing the pharmacological effects of Remodelin against osteosarcoma based on network pharmacology, acRIP-seq and experimental validation

Osteosarcoma (OS) is the most common primary malignant tumor of bone. Remodelin, an inhibitor of the N (4)-Acetylcytidine (ac4C) acetylation modifying enzyme N-acetyltransferase 10 (NAT10), has been shown to have therapeutic effects on cancer in several studies, and our previous studies have confirmed the inhibitory effect of Remodelin on OS cells, however, the mechanism of action has not yet been elucidated. We used network pharmacological analysis to quantify the therapeutic targets of Remodelin against OS. acRIP-seq and RNA-seq were performed to investigate the inhibitory activity of Remodelin on acetylation and its effect on the transcriptome after intervening in OS cells U2OS with Remodelin in vitro. Key target genes were deduced based on their pharmacological properties, combined with network pharmacology results and sequencing results. Finally, the deduced target genes were validated with vitro experiments. Network pharmacological analysis showed that 2291 OS-related target genes and 369 Remodelin-related target genes were obtained, and 116 overlapping genes were identified as Remodelin targets for OS treatment. Sequencing results showed that a total of 13,736 statistically significant ac4C modification peaks were detected by acRIP-seq, including 6938 hypoacetylation modifications and 6798 hyperacetylation modifications. A total of 2350 statistically significant mRNAs were detected by RNA-seq, of which 830 were up-regulated and 1520 were down-regulated. Association analyses identified a total of 382 genes that were Hypoacetylated-down, consistent with inhibition of mRNA acetylation and expression by Remodelin. Five genes, CASP3, ESR2, FGFR2, IGF1 and MAPK1, were identified as key therapeutic targets of Remodelin against OS. Finally, in vitro experiments, CCK-8 and qRT-PCR demonstrated that Remodelin indeed inhibited the proliferation of OS cells and reduced the expression of three genes: ESR2, IGF1, and MAPK1. In conclusion, ESR2, IGF1 and MAPK1 were identified as key therapeutic targets of Remodelin against OS. This reveals the target of Remodelin's pharmacological action on OS and provides new ideas for the treatment of OS.

Network pharmacology-based investigation and experimental validation of the therapeutic potential and molecular mechanism of Danshen Chuanxiongqin injection in acute pancreatitis.

Danshen Chuanxiong Injection (DCI) has demonstrated significant clinical efficacy in the treatment of acute pancreatitis (AP); however, the precise molecular mechanisms underlying its therapeutic effects remain incompletely understood. In this study, we employed network pharmacology analysis to comprehensively investigate the active components, potential targets, and signaling pathways involved in DCI-mediated treatment of AP. We utilized the mouse pancreatic acinar cell line 266-6 to establish an cholecystokinin (CCK)-induced AP cell injury model and evaluated cell viability using the Cell counting kit-8 assay. Western blotting and quantitative PCR were employed to determine the expression levels of key target proteins and genes. Network pharmacology analysis identified a total of 144 active components and 430 potential targets within DCI. By integrating data from public databases, we identified 762 AP-related genes. Among these, we identified 93 potential targets that may be involved in the therapeutic effects of DCI for AP. These targets were significantly enriched in biological processes such as oxidative stress, regulation of cytokine production, leukocyte migration, and the TNF signaling pathway. Molecular docking studies revealed a high binding affinity between the active components and the key targets AKT1 and NFKBA, indicative of potential interaction. Additionally, CCK-induced acinar cell injury led to upregulation of AKT1, NFKBA, and P53 proteins, as well as TNF, IL6, and MMP9 genes. Conversely, treatment with DCI dose-dependently attenuated CCK-induced acinar cell injury and restored the expression levels of the aforementioned proteins and genes. Overall, this study provides a comprehensive understanding of the molecular mechanisms underlying the therapeutic effects of DCI in the treatment of AP. Our findings confirm the protective effect of DCI against CCK-induced acinar cell injury and its regulation of key targets.

Construction of an exosome-associated miRNA-mRNA regulatory network and validation of FYCO1 and miR-17-5p as potential biomarkers associated with ovarian cancer.

The occurrence and development of several human physiological processes are significantly influenced by the competing endogenous RNA (ceRNA) network. The aim of the present study was to construct a microRNA (miRNA)-mRNA network associated with exosomes in ovarian cancer (OV), and experimental validation of key target genes. By exploring the Gene Expression Omnibus (GEO) database, we analyzed the RNAs from 226 samples to identify differentially expressed miRNAs (DEMs) and genes (DEGs) that showed differential expression as OV progressed. Subsequently, we conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on the DEGs. Furthermore, we constructed a miRNA-mRNA network that pertains to exosomes in OV using DEMs and DEGs. Moreover, we validated the expression levels of mRNAs in the miRNA-mRNA network using Gene Expression Profiling Interactive Analysis (GEPIA2). Ultimately, luciferase reporter assay was used to identify the potential target relationship between FYVE and coiled-coil domain containing 1 (FYCO1) and miRNAs. Our analysis screened a total of 14 DEMs and 101 DEGs, and the DEGs were mainly enriched in DNA replication or repair, amino acid biosynthesis and carbon metabolism. Furthermore, a miRNA-mRNA network was constructed including 3 miRNAs (hsa-miR-17-5p, hsa-miR-20b-5p and hsa-miR-20a-5p) and 2 mRNAs, FYCO1 and purine rich element binding protein A (PURA). Finally, the 2 mRNAs in this miRNA-mRNA network were verified by GEPIA2 using The Cancer Genome Atlas (TCGA) database. Among them, only FYCO1 showed significant different expression of mRNA in OV and normal tissue, while the prognosis of FYCO1 in OV remains controversial due to different database. Interestingly, FYCO1 was identified as the target of hsa-miR-17-5p. By constructing a novel network of miRNA-mRNA, we can gain new understanding of the molecular mechanisms that drive exosomes in OV. Targeting FYCO1, which originates from exosomes, may hold promise as a diagnostic marker for OV.

Artesunate improves glucose and lipid metabolism in db/db mice by regulating the metabolic profile and the MAPK/PI3K/Akt signalling pathway

BackgroundDiabetes is a metabolic disorder characterized by chronic hyperglycaemia. Chronic metabolic abnormalities and long-term hyperglycaemia may result in a wide range of acute and chronic consequences. Previous studies have demonstrated that artesunate(ART) has antidiabetic, anti-inflammatory, antiatherosclerotic, and other beneficial effects, but the specific regulatory mechanism is not completely clear. AimThis study investigated the effects of ART on metabolic disorders in type 2 diabetes mellitus (T2DM) model db/db mice and explored the underlying mechanisms involved. MethodsC57BL/KsJ-db/db mice were used to identify the targets and molecular mechanism of ART. Metabolomic methods were used to evaluate the efficacy of ART in improving T2DM-related metabolic disorders. Network pharmacology and transcriptomic sequencing were used to analyse the targets and pathways of ART in T2DM. Finally, molecular biology experiments were performed to verify the key targets and pathways selected by network pharmacology and transcriptomic analyses. ResultsAfter a 7-week ART intervention (160 mg/kg), the glucose and lipid metabolism levels of the db/db mice improved. Additionally, the oxidative stress indices, namely, the MDA and SOD levels, significantly improved (p<0.01). Linoleic acid and glycerophospholipid metabolism, amino acid metabolism, bile acid synthesis, and purine metabolism disorders in db/db mice were partially corrected after ART treatment. Network pharmacology analysis identified important targets of ART for the treatment of metabolic disorders in T2DM . These targets are involved in key signalling pathways, including the highest scores observed for the PI3K/Akt signalling pathway. Transcriptomic analysis revealed that ART could activate the MAPK signalling pathway and two key gene targets, HGK and GADD45. Immunoblotting revealed that ART increases p-PI3K, p-AKT, Glut2, and IRS1 protein expression and suppresses the phosphorylation of p38, ERK1/2, and JNK, returning HGK and GADD45 to their preartesunate levels. ConclusionTreatment of db/db mice with 160 mg/kg ART for 7 weeks significantly reduced fasting blood glucose and lipid levels. It also improved metabolic imbalances in amino acids, lipids, purines, and bile acids, thereby improving metabolic disorders. These effects are achieved by activating the PI3K/AKT pathway and inhibiting the MAPK pathway, thus demonstrating the efficacy of the drug.