Morpho-histological and Transcriptome Analysis Reveal the Unreduced Sperm Formation Mechanism in cdk1-Depletion Zebrafish.

Cyclin-dependent kinases (Cdks) are major molecules related to cell cycle regulation. Polyploidy can be caused by the production of unreduced gametes, which is often related to the abnormal cell cycle of germ cells. Here, we successfully constructed a cdk1 mutation line (cdk1+/-) in zebrafish, a commonly used model organism. It showed that cdk1 depletion resulted in the generation of both polyploid and aneuploid embryos of WT♀ × cdk1+/-♂ zebrafish. In addition to normal sperms (1N), the depletion of cdk1 in zebrafish also led to the production of some large-head 2N sperms and higher ploidy sperms. Results of bivalent analysis of testis and ultrastructure analysis of spermatogonia suggested that the production of these large-head sperms was due to spermatogonia chromosome doubling in cdk1+/- zebrafish. Transcriptome analysis revealed aberrant expressions of some cell cycle and DNA replication-related genes in the early testis of cdk1+/- zebrafish relative to WT zebrafish. Through STRING correlation analysis, we further proved that cdk1 depletion affected the mitosis process and endoduplication initiation of spermatogonia by regulating expressions of some proteins related to cell cycle (i.e., Espl1 and Pp1) and DNA replication (i.e., Orc1 and Rnaseh2b), thereby leading to the formation of unreduced sperms. This study provides important information on revealing the molecular mechanisms of unreduced gamete formation caused by cdk1 mutation. Meanwhile, it also provides an important reference for the creation of fish polyploid germplasm.

Integrative Bioinformatics Analysis: Unraveling Variant Signatures and Single-Nucleotide Polymorphism Markers Associated with 5-FU-Based Chemotherapy Resistance in Colorectal Cancer Patients.

BACKGROUND: Drug resistance in colorectal cancer (CRC) is modulated by multiple molecular factors, which can be ascertained through genetic investigation. Single nucleotide polymorphisms (SNPs) within key genes have the potential to impair the efficacy of chemotherapeutic agents such as 5-fluorouracil (5-FU). Therefore, the identification of SNPs linked to drug resistance can significantly contribute to the advancement of tailored therapeutic approaches and the enhancement of treatment outcomes in patients with CRC. MATERIAL AND METHOD: To identify dysregulated genes in 5-FU-based chemotherapy responder or non-responder CRC patients, a meta-analysis was performed. Next, the protein-protein interaction (PPI) network of the identified genes was analyzed using the STRING database. The most significant module was chosen for further analysis. In addition, a literature review was conducted to identify drug resistance-related genes. Enrichment analysis was conducted to validate the main module genes and the genes identified from the literature review. The associations between SNPs and drug resistance were investigated, and the consequences of missense variants were assessed using in silico tools. RESULT: The meta-analysis identified 796 dysregulated genes. Then, to conduct PPI analysis and enrichment analysis, we were able to discover 23 genes that are intricately involved in the cell cycle pathway. Consequently, these 23 genes were chosen for SNP analysis. By using the dbSNP database and ANNOVAR, we successfully detected and labeled SNPs in these specific genes. Additionally, after careful exclusion of SNPs with allele frequencies below 0.01, we evaluated 6 SNPs from the HDAC1, MCM2, CDK1, BUB1B, CDC14B, and CCNE1 genes using 8 bioinformatics tools. Therefore, these SNPs were identified as potentially harmful by multiple computational tools. Specifically, rs199958833 in CDK1 (Val124Gly) was predicted to be damaging by all tools used. Our analysis strongly indicates that this specific SNP could negatively affect the stability and functionality of the CDK1 protein. CONCLUSION: Based on our current understanding, the evaluation of CDK1 polymorphisms in the context of drug resistance in CRC has yet to be undertaken. In this investigation, we showed that rs199958833 variant in the CDK1 gene may favor resistance to 5-FU-based chemotherapy. However, these findings need validation in an independent cohort of patients.

Investigating the molecular mechanism of vitexin targeting CDK1 to inhibit colon cancer cell proliferation via GEO chip data mining, computer simulation, and biological activity verification.

The objective of this study is to explore the antiproliferative activity of the traditional Chinese medicine monomer vitexin on colon cancer HCT-116 cells and its underlying mechanism. The in vitro antiproliferative activity of vitexin on colon cancer HCT-116 cells was evaluated using the CCK-8 assay. Potential drug targets for colon cancer were identified through GEO chip data mining, and molecular docking using Schrödinger software was conducted. Molecular dynamics simulations were employed to deeply analyze the interaction between candidate compounds and target proteins. Flow cytometry was employed to examine the cell cycle. The impact of vitexin on the expression of CDK1/cyclinB proteins in HCT-116 cells was assessed through Western blot analysis, immunofluorescence, and CDK inhibition assay. Vitexin exhibited inhibitory effects on colon cancer HCT-116 cells, with a half inhibitory concentration (IC50) value of 203.27 ± 9.85 µmol/L. The analysis of differential gene expression in GEO and TCGA datasets, along with the GENECARD dataset of related disease genes, identified 91 disease targets, including "CDK1." Vitexin induced cell cycle arrest in the G2/M phase of HCT-116 cells. Molecular docking revealed a strong interaction between Vitexin and CDK1 (Docking score - 9.497), with molecular dynamics simulations confirming the stability of the Vitexin-CDK1 complex and comparable inhibitory effects to Flavopiridol. Vitexin can inhibit the expression of CDK1/cyclin B proteins in HCT-116 cells, with an IC50 of 58.06 ± 3.07 µmol/L. Vitexin may inhibit colon cancer HCT-116 cell proliferation by suppressing CDK1/cyclin B expression, leading to cell cycle arrest in the G2/M phase.

Reynoutria multiflora (Thunb.) Moldenke and its ingredient suppress lethal prostate cancer growth by inducing CDC25B-CDK1 mediated cell cycle arrest.

BACKGROUND: Reynoutria multiflora (Thunb.) Moldenke (Polygonum multiflorum Thunb, PM) is a medicinal plant that was an element of traditional Chinese medicine (TCM) for centuries as a treatment for a wide range of conditions. Recent studies reported that PM suppressed prostate cancer growth in an AR-dependent manner. However, its role and mechanism in the treatment of advanced prostate cancer remain to be explored. This study aims to explore the anti-tumor role and potential mechanism of PM on prostate cancer. METHODS: Cell viability, colony formation, fluorescence-activated cell sorting (FACS), and wound-healing assays were conducted to evaluate the tumor suppression effect of PM on lethal prostate cancer models in vitro. A xenograft mice model was established to detect the impact of PM on tumor growth and evaluate its biosafety in vivo. Integrative network pharmacology, RNA-seq, and bioinformatics were applied to determine the mechanisms of PM in prostate cancer. Molecular docking, cellular thermal shift assay (CETSA), CRISPR-Cas13, RT-qPCR, and WB were collaboratively employed to identify the potential anti-tumor ingredient derived from PM and its corresponding targets. RESULTS: PM significantly suppressed the growth of prostate cancer and sensitized prostate cancer to AR antagonists. Mechanistically, PM induced G2/M-phase cell-cycle arrest by modulating the phosphorylation of CDK1. Additionally, polygalacic acid derived from PM and its structural analog suppress prostate cancer growth by targeting CDC25B, a master regulator of the cell cycle that governs CDK1 phosphorylation. CONCLUSION: PM and its ingredient polygalacic acid suppress lethal prostate cancer growth by regulating the CDC25B-CDK1 axis to induce cell cycle arrest.

Canine oocyte nuclear maturation with Nano-ozone (NZS) supplementation: The alterations of antioxidant, and oxidant status and CDK1, cyclin B1 expressions.

This study aims to evaluate the effects of nano-ozone solution (NZS) on canine oocyte nuclear maturation, associated with the alterations of antioxidant and oxidant status and cyclin-dependent kinase 1 (CDK1), cyclin B1 gene expressions. Oocytes were cultured in four distinct concentrations of NZS (0.5, 1, 2, and 5 µg/mL) and parthenogenetically activated. The rates of oocytes arrested at the Germinal Vesicle (GV), Germinal Vesicle Breakdown (GVBD), Metaphase I (MI), and Metaphase II (MII) stages were statistically different among groups (P < 0.05). The oocytes cultured in 1 µg/mL NZS yielded the best oocyte maturation rate at the MI and MII stages; however, the lowest maturation and high degeneration rates were observed in Group E. The measurements of Malondialdehyde (MDA), reduced Glutathione (GSH), Superoxide Dismutase (SOD), and Ferric Reducing/Antioxidant Power assay (FRAP) were performed from IVM culture media. No statistical difference was observed in SOD and MDA results (P > 0.05). GSH levels were statistically significant between Group A-Group E (p = 0.003), Group B-Group E (p = 0.045), and Group E-Group D (p = 0.021). The culture media in Group D and Group E had high FRAP concentrations and significantly differed between groups (P < 0.05). CDK1, and cyclin B1 genes, which are subunits of maturation-promoting factor (MPF), are upregulated in Group B and Group C, while are downregulated in oocytes of Group E. This study showed that low, controlled doses of NZS (1 µg/mL) supplementation could improve the meiotic competence of canine oocytes and lead to positive response in expressions of CDK1 and cyclin B1 on the gene level.

HNRNPD regulates the biogenesis of circRNAs and the ratio of mRNAs to circRNAs for a set of genes.

Exonic circular RNAs (ecircRNAs) in animal cells are generated by backsplicing, and the biogenesis of ecircRNAs is regulated by an array of RNA binding proteins (RBPs). HNRNPD is a heterogeneous nuclear ribonucleoprotein family member with both cytoplasmic and nuclear roles, and whether HNRNPD regulates the biogenesis of circRNAs remains unknown. In this study, we examine the role of HNRNPD in the biogenesis of ecircRNAs. The levels of ecircRNAs are primarily increased upon depletion of HNRNPD. HNRNPD preferentially binds to motifs enriched with A and U nucleotides, and the flanking introns of ecircRNAs tend to have more numbers and higher intensity of HNRNPD binding sites. The levels of mRNAs are generally not significantly altered in HNRNPD knockout cells. For a small set of genes, the circRNA:mRNA ratio is substantially affected, and the mRNA levels of some of these genes demonstrate a significant decrease in HNRNPD knockout cells. CDK1 is identified as a key gene modulated by HNRNPD in the context of circRNA biogenesis. HNRNPD suppresses the biogenesis of circCDK1 and favours the generation of CDK1 mRNA, and the CDK1 protein is a critical regulator of the cell cycle and apoptosis. HNRNPD can participate in cellular physiology, including the cell cycle and apoptosis, and plays roles in clear cell renal cell carcinoma (ccRCC) by modulating circRNA biogenesis and the mRNA levels of key genes, such as CDK1.

ERK synchronizes embryonic cleavages in Drosophila.

Extracellular-signal-regulated kinase (ERK) signaling controls development and homeostasis and is genetically deregulated in human diseases, including neurocognitive disorders and cancers. Although the list of ERK functions is vast and steadily growing, the full spectrum of processes controlled by any specific ERK activation event remains unknown. Here, we show how ERK functions can be systematically identified using targeted perturbations and global readouts of ERK activation. Our experimental model is the Drosophila embryo, where ERK signaling at the embryonic poles has thus far only been associated with the transcriptional patterning of the future larva. Through a combination of live imaging and phosphoproteomics, we demonstrated that ERK activation at the poles is also critical for maintaining the speed and synchrony of embryonic cleavages. The presented approach to interrogating phosphorylation networks identifies a hidden function of a well-studied signaling event and sets the stage for similar studies in other organisms.

Submandibular gland tissue RNAseq and spatial transcriptome analyses in IgG4-related disease.

OBJECTIVE: To identify genes that could provide clues leading to the discovery of drugs to treat IgG4-related disease (IgG4-RD). METHODS: Submandibular gland tissue bulk RNAseq analysis of 45 cases with a definite diagnosis of IgG4-RD was integrated with Visium spatial transcriptome analysis of 2 cases to identify pathogenic genes expressed in tertiary lymphoid tissues. RESULTS: Bulk RNAseq and pathway analyses showed upregulation of cell cycle and T cell-related signals in IgG4-RD. Spatial transcriptome analysis identified the cluster corresponding to germinal centers and the top 38 common genes that showed significant variations in expression compared with other clusters. The top 20 genes were extracted by comparing the bulk RNAseq data. Network analysis identified CDK1 as the ge most strongly associated of the top 20 genes. CONCLUSION: The CDK1 gene may be a regulator of the pathogenesis of IgG4-RD and provide clues for drug discovery.

Hypoxia drives HIF2-dependent reversible macrophage cell cycle entry.

Low-oxygen conditions (hypoxia) have been associated primarily with cell-cycle arrest in dividing cells. Macrophages are typically quiescent in G0 but can proliferate in response to tissue signals. Here we show that hypoxia (1% oxygen tension) results in reversible entry into the cell cycle in macrophages. Cell cycle progression is largely limited to G0-G1/S phase transition with little progression to G2/M. This cell cycle transitioning is triggered by an HIF2α-directed transcriptional program. The response is accompanied by increased expression of cell-cycle-associated proteins, including CDK1, which is known to phosphorylate SAMHD1 at T592 and thereby regulate antiviral activity. Prolyl hydroxylase (PHD) inhibitors are able to recapitulate HIF2α-dependent cell cycle entry in macrophages. Finally, tumor-associated macrophages (TAMs) in lung cancers exhibit transcriptomic profiles representing responses to low oxygen and cell cycle progression at the single-cell level. These findings have implications for inflammation and tumor progression/metastasis where low-oxygen environments are common.

mTORC1 activity oscillates throughout the cell cycle, promoting mitotic entry and differentially influencing autophagy induction.

Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a master metabolic regulator that is active in nearly all proliferating eukaryotic cells; however, it is unclear whether mTORC1 activity changes throughout the cell cycle. We find that mTORC1 activity oscillates from lowest in mitosis/G1 to highest in S/G2. The interphase oscillation is mediated through the TSC complex but is independent of major known regulatory inputs, including Akt and Mek/Erk signaling. By contrast, suppression of mTORC1 activity in mitosis does not require the TSC complex. mTORC1 has long been known to promote progression through G1. We find that mTORC1 also promotes progression through S and G2 and is important for satisfying the Chk1/Wee1-dependent G2/M checkpoint to allow entry into mitosis. We also find that low mTORC1 activity in G1 sensitizes cells to autophagy induction in response to partial mTORC1 inhibition or reduced nutrient levels. Together, these findings demonstrate that mTORC1 is differentially regulated throughout the cell cycle, with important phase-specific consequences for proliferating cells.

TDP1 phosphorylation by CDK1 in mitosis promotes MUS81-dependent repair of trapped Top1-DNA covalent complexes.

Topoisomerase 1 (Top1) controls DNA topology, relieves DNA supercoiling during replication and transcription, and is critical for mitotic progression to the G1 phase. Tyrosyl-DNA phosphodiesterase 1 (TDP1) mediates the removal of trapped Top1-DNA covalent complexes (Top1cc). Here, we identify CDK1-dependent phosphorylation of TDP1 at residue S61 during mitosis. A TDP1 variant defective for S61 phosphorylation (TDP1-S61A) is trapped on the mitotic chromosomes, triggering DNA damage and mitotic defects. Moreover, we show that Top1cc repair in mitosis occurs via a MUS81-dependent DNA repair mechanism. Replication stress induced by camptothecin or aphidicolin leads to TDP1-S61A enrichment at common fragile sites, which over-stimulates MUS81-dependent chromatid breaks, anaphase bridges, and micronuclei, ultimately culminating in the formation of 53BP1 nuclear bodies during G1 phase. Our findings provide new insights into the cell cycle-dependent regulation of TDP1 dynamics for the repair of trapped Top1-DNA covalent complexes during mitosis that prevents genomic instability following replication stress.

Erratum: Exploring HMMR as a therapeutic frontier in breast cancer treatment, its interaction with various cell cycle genes, and targeting its overexpression through specific inhibitors.

[This corrects the article DOI: 10.3389/fphar.2024.1361424.].

CDK1 Acts as a Prognostic Biomarker Associated with Immune Infiltration in Pan-Cancer, Especially in Gastrointestinal Tumors.

OBJECTIVE: Cyclin-dependent kinase 1 (CDK1) regulates the cell cycle and is highly expressed in most tumors. CDK1 expression has been associated with poor disease prognosis. This study aimed to identify the prognostic value of CDK1 in pan-cancer and investigate the association between CDK1 expression and immune cell infiltration. METHODS: CDK1 expression and its correlation with prognosis in pan-cancer were analyzed using online databases. Immune infiltration was assessed by ESTIMATE and CIBERSORT algorithms. We then evaluated the relationship between CDK1 expression and tumor mutational burden (TMB), microsatellite instability (MSI), or tumor-infiltrating immune cells. In addition, we performed the co-expression analysis of immune-related genes and GO analysis with CDK1 expression in pan-cancer. Finally, we compared the CDK1 expression profile with the immune-related genes in 30 pairs of clinical gastrointestinal tumor samples. RESULTS: Our analysis demonstrated overexpression of CDK1 in most tumor tissues, especially in gastrointestinal tumors. The high expression of CDK1 was associated with poor overall survival, disease-specific survival, disease-free interval, and progression-free interval in kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD), and sarcoma (SARC). Besides, CDK1 expression was significantly associated with TMB in 22 cancer types and MSI in 8 cancer types as well as greater frequencies of MSI-high (MSI-H) status and high tumor mutational burden (TMB-H) in uterine corpus endometrial carcinoma (UCEC), stomach adenocarcinoma (STAD), sarcoma (SARC), rectum adenocarcinoma (READ), mesothelioma (MESO), head and neck squamous cell carcinoma (HNSC), and colon adenocarcinoma (COAD). In addition, CDK1 expression correlated with immune cell infiltrating levels, such as M0, M1, or M2 macrophages, memory CD4 T cells, T follicular helper cells, and naive B cells. Our data showed that CDK1 was remarkably correlated with 47 immune-related and immune checkpoint genes in many cancer types. Furthermore, CDK1 was up-regulated in gastrointestinal tumor samples, especially in gastric cancer and intestinal cancer. CDK1 was positively correlated with IDO1 in gastric cancer and PD-1 in intestinal cancer. CONCLUSION: Taken together, our data demonstrated the roles of CDK1 in oncogenesis and metastasis in pan-cancer. Thus, CDK1 is a potential prognostic biomarker and a target for tumor immunotherapy.

lncRNA FLJ20021 regulates CDK1-mediated PANoptosis in a ZBP1-dependent manner to increase the sensitivity of laryngeal cancer-resistant cells to cisplatin.

In this study, we investigated the role of the newly discovered lncRNA FLJ20021 in laryngeal cancer (LC) and its resistance to cisplatin treatment. We initially observed elevated lncRNA FLJ20021 levels in cisplatin-resistant LC cells (Hep-2/R). To explore its function, we transfected lncRNA FLJ20021 and cyclin-dependent kinase 1 (CDK1) into Hep-2/R cells, assessing their impact on cisplatin sensitivity and PANoptosis. Silencing lncRNA FLJ20021 effectively reduced cisplatin resistance and induced PANoptosis in Hep-2/R cells. Mechanistically, lncRNA FLJ20021 primarily localized in the nucleus and interacted with CDK1 mRNA, thereby enhancing its transcriptional stability. CDK1, in turn, promoted panapoptosis in a ZBP1-dependent manner, which helped overcome cisplatin resistance in Hep-2/R cells. This study suggests that targeting lncRNA FLJ20021 can be a promising approach to combat cisplatin resistance in laryngeal cancer by regulating CDK1 and promoting PANoptosis via the ZBP1 pathway. These findings open up possibilities for lncRNA-based therapies in the context of laryngeal cancer.

Cdk1/p53/p21 feedback loop mechanisms in the pathogenesis of interstitial cystitis/bladder pain syndrome.

PURPOSE: This study aimed to elucidate the role of the Cdk1/p53/p21 feedback loop in the pathogenesis of interstitial cystitis (IC)/bladder pain syndrome (BPS). MATERIALS AND METHODS: An IC/BPS cell model was established. Cell viability was determined using the CCK-8 assay. Flow cytometry was adopted to assess cell apoptosis rates. ELISA was employed to measure secretion levels of inflammatory factors (IL-6, IL-8, and TNF-α). Gene expressions were assessed using PCR, while protein expressions were analyzed through Western blotting analysis. Epithelial permeability was demonstrated using the phenol red leakage experiment and FITC-dextran permeability assay. The interaction between proteins was determined using co-immunoprecipitation, and protein localization was investigated using immunofluorescence. RESULTS: The CCK-8 assay revealed a significantly reduced viability of IC/BPS cells compared to normal epithelial cells (p < 0.05). Elevated levels of IL-6, IL-8, and TNF-α were detected in IC/BPS cells. Changes in the expressions of E-cadherin and ZO-1 were evident, leading to increased epithelial permeability in IC/BPS cells. Furthermore, within IC/BPS cells, Cdk1 phosphorylated p53 in the nucleus. The Cdk1/p53/p21 feedback loop was established to influence urothelial permeability. Both p21 and Cdk1 inhibitors notably reduced the epithelial permeability in IC/BPS cells. CONCLUSION: The Cdk1/p53/p21 feedback loop was instrumental in IC/BPS, acting as a regulator of urothelial permeability. This discovery offered a novel therapeutic approach for IC/BPS management.

SRSF9 promotes cell proliferation and migration of glioblastoma through enhancing CDK1 expression.

BACKGROUND: Glioblastoma (GBM) is a highly aggressive and prevalent brain tumor that poses significant challenges in treatment. SRSF9, an RNA-binding protein, is essential for cellular processes and implicated in cancer progression. Yet, its function and mechanism in GBM need clarification. METHODS: Bioinformatics analysis was performed to explore differential expression of SRSF9 in GBM and its prognostic relevance to glioma patients. SRSF9 and CDK1 expression in GBM cell lines and patients' tissues were quantified by RT-qPCR, Western blot or immunofluorescence assay. The role of SRSF9 in GBM cell proliferation and migration was assessed by MTT, Transwell and colony formation assays. Additionally, transcriptional regulation of CDK1 by SRSF9 was investigated using ChIP-PCR and dual-luciferase assays. RESULTS: The elevated SRSF9 expression correlates to GBM stages and poor survival of glioma patients. Through gain-of-function and loss-of-function strategies, SRSF9 was demonstrated to promote proliferation and migration of GBM cells. Bioinformatics analysis showed that SRSF9 has an impact on cell growth pathways including cell cycle checkpoints and E2F targets. Mechanistically, SRSF9 appears to bind to the promoter of CDK1 gene and increase its transcription level, thus promoting GBM cell proliferation. CONCLUSIONS: These findings uncover the cellular function of SRSF9 in GBM and highlight its therapeutic potential for GBM.

Hyperactivation of p53 contributes to mitotic catastrophe in podocytes through regulation of the Wee1/CDK1/cyclin B1 axis.

Podocyte loss in glomeruli is a fundamental event in the pathogenesis of chronic kidney diseases. Currently, mitotic catastrophe (MC) has emerged as the main cause of podocyte loss. However, the regulation of MC in podocytes has yet to be elucidated. The current work aimed to study the role and mechanism of p53 in regulating the MC of podocytes using adriamycin (ADR)-induced nephropathy. In vitro podocyte stimulation with ADR triggered the occurrence of MC, which was accompanied by hyperactivation of p53 and cyclin-dependent kinase (CDK1)/cyclin B1. The inhibition of p53 reversed ADR-evoked MC in podocytes and protected against podocyte injury and loss. Further investigation showed that p53 mediated the activation of CDK1/cyclin B1 by regulating the expression of Wee1. Restraining Wee1 abolished the regulatory effect of p53 inhibition on CDK1/cyclin B1 and rebooted MC in ADR-stimulated podocytes via p53 inhibition. In a mouse model of ADR nephropathy, the inhibition of p53 ameliorated proteinuria and podocyte injury. Moreover, the inhibition of p53 blocked the progression of MC in podocytes in ADR nephropathy mice through the regulation of the Wee1/CDK1/cyclin B1 axis. Our findings confirm that p53 contributes to MC in podocytes through regulation of the Wee1/CDK1/Cyclin B1 axis, which may represent a novel mechanism underlying podocyte injury and loss during the progression of chronic kidney disorder.

Study on the mechanism of quercetin in Sini Decoction Plus Ginseng Soup to inhibit liver cancer and HBV virus replication through CDK1.

BACKGROUND: To explore the anti-tumor and anti-virus key active ingredients of Sini Decoction Plus Ginseng Soup (SNRS) and their mechanisms. METHODS: The main ingredients of SNRS were analyzed by network pharmacology, and quercetin was identified as the key active ingredient. Then, we obtained the targets of quercetin by using Drugbank, PharmMapper, and SwissTargetPrediction databases. Then, the targets of HBV-related hepatocellular carcinoma (HBV-related HCC) were obtained by using Genecards database. In addition, using the gene expression profiles of HBV-related HCC patients in GEO database and the genes with the greatest survival difference in GEPIA 2 database identified the potential targets of quercetin. In addition, the mechanism of potential genes was studied through GO, KEGG analysis, and PPI network. Using AUC and survival analysis to evaluate the diagnostic and prognostic value of cyclin-dependent kinase 1 (CDK1) and CCNB1. Finally, the effects of quercetin on proliferation of Hep3B and HepG2215 cells and the level of CDK1 and CCNB1 were verified in vitro. ELISA was used to measure the expression levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) after the intervention by quercetin for 24 h and 48 h in HepG2215 cell. RESULTS: The first 10 key ingredients of SNRS were identified, and quercetin was the most key ingredient. The 101 potential quercetin targets were identified for the treatment of HBV-related HCC. GO and KEGG showed that 101 potential target enrichment in cancer and cell cycle regulation. By Venn analysis, CDK1 and CCNB1 were intersection targets, which could be used as potential targets for the action of quercetin on HBV-related HCC. Moreover, the expression of CDK1 and CCNB1 was highly expressed in the high-risk group, while the OS rate was low. The 1-year, 3-year and 5-year area under the curve (AUC) curves of CDK1 and CCNB1 were 0.724, 0.676, 0.622 and 0.745, 0.678, 0.634, respectively. Moreover, experimental results also showed that quercetin inhibited cell proliferation and reduced CDK1 expression in Hep3B and HepG2215 cells. The expressions of HBsAg and HBeAg in HepG2215 cell supernatant and cell gradually decreased with the increase of intervention time of quercetin and CDK1 inhibitor. CONCLUSIONS: Quercetin is a key ingredient of anti-HBV-related HCC activity and inhibits HBV replication in SNRS by inhibiting CDK1.

Paclitaxel inhibits proliferation by negatively regulating Cdk1-cell cycle axis in rat airway smooth muscle cells.

OBJECTIVE: Paclitaxel exhibits outstanding biological activities in inhibiting cell proliferation and inducing cell apoptosis. However, the effects of paclitaxel on airway smooth muscle cells (ASMCs) have not been reported yet. The purpose of this study is to determine the effects of paclitaxel on the proliferation and apoptosis of ASMCs. METHODS: Rat primary ASMCs were isolated and used in all the experiments. Cell Counting Kit-8 assay and Edu assay were used to analyze the cell viability and proliferation, respectively. Flow cytometry was used to detect the cell cycle and apoptosis. Quantitative real-time PCR (qRT-PCR), western blotting, and immunostaining were used to detect the expression of cyclin-dependent kinase 1 (Cdk1). RESULTS: Our study showed that paclitaxel inhibits the proliferation of ASMCs in a dose- and time-gradient-dependent manner. Further study displayed that cell cycle is arrested at G2/M phase. And Cdk1 was dramatically down-regulated by paclitaxel treatment. Cell morphological analysis showed that ASMCs are elliptical with a larger surface area after paclitaxel treatment. Nucleus morphological analysis showed that the nuclei are in a diffuse state after paclitaxel treatment. However, paclitaxel did not induce the apoptosis of ASMCs. CONCLUSIONS: Our study demonstrated that paclitaxel inhibits the proliferation of ASMCs at least partly by negatively regulating Cdk1-cell cycle axis.

Potential molecular mechanisms of Huangqin Tang for liver cancer treatment by network pharmacology and molecular dynamics simulations.

OBJECTIVE: This study aims to investigate the mechanism of Huangqin Tang in treating liver cancer. METHODS: Active ingredients and corresponding targets of Huangqin Tang were obtained from the Traditional Chinese Medicine Systems Pharmacology Database. Differentially expressed genes in liver cancer were identified from mRNA expression data. A protein-protein interaction (PPI) network was constructed using differentially expressed genes and Huangqin Tang targets. Random walk with restart (RWR) analysis was performed on the PPI network. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were conducted. A drug-active ingredient-gene interaction network was established, and molecular docking and molecular dynamics simulations were performed. Finally, the stability of binding between CDK1 and oroxylin was tested according to cellular thermal shift assay (CETSA). RESULTS: 160 active ingredients, 239 targets, and 1093 differentially expressed genes were identified. RWR analysis identified 10 potential targets for liver cancer. Enrichment analysis revealed protein kinase regulator activity and Steroid hormone biosynthesis as significant pathways. Molecular docking suggested a stable complex between oroxylin A and CDK1. CETSA demonstrated that the combination of oroxylin A and CDK1 increased the stability of CDK1, and the combination efficiency was high. CONCLUSION: Huangqin Tang may treat liver cancer by targeting CDK1 with oroxylin A. Protein kinase regulator activity and Steroid hormone biosynthesis pathways may play a role in liver cancer treatment with Huangqin Tang. This study provides insight into the mechanistic basis of Huangqin Tang for liver cancer treatment.