CDK4 Research Articles

KDM4B Histone Demethylase Inhibition Attenuates Tumorigenicity of Malignant Melanoma Cells by Overriding the p53-Mediated Tumor Suppressor Pathway.

Despite significant advances in the treatment of cutaneous melanoma (hereaftermelanoma), the prognosis remains less favorable due to therapeutic resistance, which is presumably linked to epigenetic dysregulation. We hypothesized that the histone lysine demethylase KDM4B could play a pivotal role in controlling therapy-resistant melanoma. To validate our hypothesis, we retrieved RNA sequencing data from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA)program and observed upregulation of KDM4B in both primary and metastatic melanoma, which was associated with poor survival. To explore its role, we used murine B16, human SK-MEL-5, and G-361 melanoma cells as in vitro models of melanoma. We found that KDM4B inhibition using NCGC00244536 increased global levels of H3K9me3 and downregulated the expressions of cell cycle progression-related genes Cdk1, Cdk4, Ccnb1, and Ccnd1. Moreover, genetic ablation of KDM4B or its chemical inhibition using NCGC00244536 reduced p53 production by upregulating MDM2, which enhances the proteolytic degradation of p53. Interestingly, despite the reduction of p53, these interventions augmented apoptosis and senescence-induced cell death by activating pathways downstream of p53, as evidenced by reduced levels of pro-survival Bcl-2 and Bcl-xL proteins and increased production of pro-apoptotic cleaved caspase-3, caspase-7, Bax, and the senescence inducer Cdkn1a. Compared to the FDA-approved anti-melanoma agent dacarbazine, NCGC00244536 exhibited more pronounced cytotoxic and antiproliferative effects in melanoma cells. Importantly, NCGC00244536 demonstrated minimal cytotoxicity to low Kdm4b-expressing mouse embryonic fibroblasts. In conclusion, our findings suggest that KDM4B inhibition can override the antitumor effect of p53, and potentially serve as a therapeutic strategy for melanoma.

CDK4 is co-amplified with either TP53 promoter gene fusions or MDM2 through distinct mechanisms in osteosarcoma

Amplification of the MDM2 and CDK4 genes on chromosome 12 is commonly associated with low-grade osteosarcomas. In this study, we conducted high-resolution genomic and transcriptomic analyses on 33 samples from 25 osteosarcomas, encompassing both high- and low-grade cases with MDM2 and/or CDK4 amplification. We discerned four major subgroups, ranging from nearly intact genomes to heavily rearranged ones, each harbouring CDK4 and MDM2 amplification or CDK4 amplification with TP53 structural alterations. While amplicons involving MDM2 exhibited signs of an initial chromothripsis event, no evidence of chromothripsis was found in TP53-rearranged cases. Instead, the initial disruption of the TP53 locus led to co-amplification of the CDK4 locus. Additionally, we observed recurring promoter swapping events involving the regulatory regions of the FRS2, PLEKHA5, and TP53 genes. These events resulted in ectopic expression of partner genes, with the ELF1 gene being upregulated by the FRS2 and TP53 promoter regions in two distinct cases.

Benchmarking of Approaches for Gene Copy-Number Variation Analysis and Its Utility for Genetic Aberration Detection in High-Grade Serous Ovarian Carcinomas.

Objective: The goal of this study was to compare the results of CNV detection by three different methods using 13 paired carcinoma samples, as well as to perform a statistical analysis of the agreement. Methods: CNV was studied using NanoString nCounter v2 Cancer CN Assay (Nanostring), Illumina Infinium CoreExome microarrays (CoreExome microarrays) and digital droplet PCR (ddPCR). Results: There was a good level of agreement (PABAK score > 0.6) between the CoreExome microarrays and the ddPCR results for finding CNVs. There was a moderate level of agreement (PABAK values ≈ 0.3-0.6) between the NanoString Assay results and microarrays or ddPCR. For 83 out of 87 target genes studied (95%), the agreement between the CoreExome microarrays and NanoString nCounter was characterized by PABAK values < 0.75, except for MAGI3, PDGFRA, NKX2-1 and KDR genes (>0.75). The MET, HMGA2, KDR, C8orf4, PAX9, CDK6, and CCND2 genes had the highest agreement among all three approaches. Conclusions: Therefore, to get a better idea of how to genotype an unknown CNV spectrum in tumor or normal tissue samples that are very different molecularly, it makes sense to use at least two CNV detection methods. One of them, like ddPCR, should be able to quantitatively confirm the results of the other.

Expression of GLOD4 in the Testis of the Qianbei Ma Goat and Its Effect on Leydig Cells.

The expression pattern of GLOD4 in the testis and its regulatory effect on testicular cells was explored in goats to enhance our understanding of spermatogenesis and improve reproduction in breeding rams. In this study, we demonstrated the localization of GLOD4 in testicular cells using immunohistochemistry and subcellular localization analyses. Subsequently, we analyzed the GLOD4 expression pattern in four age-based groups (0, 6, 12, and 18 months old) using real-time quantitative polymerase chain reaction (qRT-PCR) and protein blotting. Finally, we performed GLOD4 silencing and overexpression studies in Leydig cells (LCs) and explored the effects on cell proliferation, the cell cycle, steroid hormone secretion and the expression of candidate testosterone hormone-regulated genes. GLOD4 was mainly expressed in Leydig cells, and the subcellular localization results showed that the GLOD4 protein was mainly localized in the cytoplasm and nucleus. Silencing of GLOD4 significantly suppressed the mRNA expression levels of the testosterone secretion-related genes CYP11A1, 3β-HSD, and CYP17A1 and the mRNA expression levels of cell cycle-related genes CDK6, PCNA, and Cyclin E. Moreover, the cell cycle was blocked at the G2/M phase after GLOD4 silencing, which significantly suppressed testosterone secretion. In contrast, GLOD4 overexpression significantly increased the mRNA expression levels of the testosterone secretion-related genes CYP11A1, 3β-HSD, and CYP17A1 and increased the expression of the cell cycle-related genes CDK6, PCNA, and Cyclin E. Moreover, GLOD4 overexpression promoted the cell cycle from G0/G1 phases to enter the S phase and G2/M phases, promoted the secretion of testosterone. Taken together, our experimental results indicate that GLOD4 may affect the development of cells in Qianbei Ma goats of different ages by influencing the cell cycle, cell proliferation, and testosterone hormone synthesis. These findings enhance our understanding of the functions of GLOD4 in goats.

CDK4 gene copy number increase and concurrent genetic changes in acral melanoma of a Chinese cohort

Acral melanoma (AM) is the most common subtype of melanoma in Asian population. Abnormalities in the p16-cyclin D1-CDK4 signalling pathway play a crucial role in the development and progression of AM. However, the alterations of CDK4 gene copy number in AM are underreported. In this study, we investigated CDK4 gene copy number and concurrent molecular changes in a Chinese cohort with AM, in aim of exploring CDK4 gene alterations and its significance in AM. We examined copy number alterations of CDK4 with fluorescence in situ hybridisation (FISH) in 31 patients with AM. Six patients with CDK4 high-level copy number increase were examined by next-generation sequencing to detect concurrent molecular changes. Using FISH, 12 (12/31, 38.7%) cases showed CDK4 copy number increase, with 6 (6/31, 19.4%) low-level copy number increase and 6 (6/31, 19.4%) high-level copy number increase. Five of six CDK4 low-level copy number increase cases were accompanied by polysomy of chromosome 12, while one case was not. Two of six CDK4 high-level copy number increase cases were accompanied by polysomy of chromosome 12, while four cases were not. CDK4 copy number increase was significantly correlated with younger patient age. In six CDK4 high-level copy number increase cases, one case was found to be accompanied by NRAS mutation, one case was accompanied by HER2 mutation, one case was accompanied by BCL2L11 mutation ​and one case was accompanied by BRAF, HER2 and BCL2L11 mutations. Our study confirmed the presence of CDK4 copy number increase in AM cases. Detecting CDK4 copy number increase by FISH can be reliable in the diagnosis of AM. Some CDK4 copy number increases are the results of polysomy of chromosome 12. CDK4 high-level copy number increase coexists with other pathogenic mutations in AM. CDK4 appears to be a promising target for AM treatment and is expected to be combined with other targeted therapies.

Integrative bioinformatics analysis of transcriptomic data from CD8+ T cells in Systemic Lupus Erythematosus

IntroductionSystemic Lupus Erythematosus (SLE) is a complex, multisystem autoimmune disorder characterized by extensive inflammation that affects nearly all organ systems in the body. It is primarily mediated by auto-antibodies and immune complexes, and it predominantly affects women more than men. This study employs an in-silico approach to identify key genes potentially involved in the pathogenesis of SLE. ObjectivesTo identify key genes potentially involved in SLE pathogenesis using in-silico approach. MethodsHigh-throughput sequencing dataset GSE97264, from the Gene Expression Omnibus (GEO) database, which contains RNA transcriptome data from CD8+ T-cells of 18 SLE patients and 14 healthy controls was utilized for the analysis. Differentially expressed genes (DEGs) were identified using the Bioconductor DESeq2 package in R platform. Gene Ontology (GO) and pathway enrichment analyses were performed using the ToppGene suite. Motif analysis of the genes’ promoter regions was conducted using HOMER software. Protein-protein interaction (PPI) and Reactome functional interaction (FI) networks were created using Cytoscape plugins StringApp and ReactomeFIViz, and analysed to identify hub genes. ResultsOur analysis identified 931 DEGs, with 577 upregulated and 354 downregulated. GO and pathway enrichment analyses indicated that upregulated genes were associated with immune responses, including cytokine production and receptor activation. Motif analysis identified key regulatory motifs linked to immune regulation in upregulated genes and T-cell activation in downregulated genes. PPI and FI networks analyses revealed 29 cell cycle-associated hub genes, with 10 genes—CDK1, TPX2, BIRC5, CCNA2, BUB1, BUB1B, AURKA, KIF2C, PLK1, and CDCA8—common to both biological networks, suggesting their crucial role in SLE pathogenesis. ConclusionThis study suggests that dysregulation of the identified 10 genes may impact immune responses and contribute to the autoimmune-like conditions observed in SLE. Several of these genes are also implicated in other autoimmune diseases, highlighting their potential as SLE biomarkers. Despite their known roles in other immune-related diseases involving CD8+ T cells, their direct association with SLE had not been previously established. This novel finding underscores the potential of these genes as therapeutic targets and may contribute to the development of diagnostic tools.

The role of AdipoQ on proliferation, apoptosis, and hormone Secretion in chicken primary adenohypophysis cells

Adiponectin (AdipoQ), an adipokine secreted by adipocytes, has been reported to exist widely in various cell types and tissues, including the adenohypophysis of chickens. However, the molecular mechanism by which AdipoQ regulates the function of chicken adenohypophysis remains elusive. In this study, we investigated the effects of AdipoQ on proliferation, apoptosis, secretion of related hormones (FSH, LH, TSH, GH, PRL and ACTH) and expression of related genes (FSHβ, LHβ, GnRHR, TSHβ, GH, PRL and ACTH) in primary adenohypophysis cells of chickens by using real-time fluorescent quantitative PCR (RT-qPCR), cell counting kit-8 (CCK-8), flow cytometry, enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) assays. Our results showed that AdipoQ promoted the proliferation of chicken primary adenohypophysis cells, up-regulated the mRNA expression of proliferation-related genes CDK1, PCNA, CCND1 and P21 (P < 0.05), as well as the increased protein expression of CDK1 and PCNA (P < 0.05). Furthermore, AdipoQ inhibited apoptosis of chicken primary adenohypophysis cells, resulting in down-regulation of pro-apoptotic genes Caspase3, Fas, and FasL mRNA expression, and decreased Caspase3 protein expression (P < 0.05). Moreover, there was an up-regulation of anti-apoptotic gene Bcl2 mRNA and protein expression (P < 0.05). Additionally, AdipoQ suppressed the secretion of FSH, LH, TSH, GH, PRL, and ACTH (P < 0.05), as well as the mRNA expression levels of related genes (P < 0.05). Treatment with AdipoRon (a synthetic substitute for AdipoQ) and co-treatment with RNA interference targeting AdipoQ receptors 1/2 (AdipoR1/2) had no effect on the secretion of FSH, LH, TSH, GH, PRL, and ACTH, as well as the mRNA expression levels of the related genes. This suggests that AdipoQ's regulation of hormone secretion and related gene expression is mediated by the AdipoR1/2 signaling axis. Importantly, we further demonstrated that the mechanism of AdipoQ on FSH, LH, TSH and GH secretion is realized through AMPK signaling pathway. In conclusion, we have revealed, for the first time the molecular mechanism by which AdipoQ regulates hormone secretion in chicken primary adenohypophysis cells.

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

To identify genes that could provide clues leading to the discovery of drugs to treat IgG4-related disease (IgG4-RD). 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. 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. The CDK1 gene may be a regulator of the pathogenesis of IgG4-RD and provide clues for drug discovery.

Novel liquid biopsy CNV biomarkers in malignant melanoma

Malignant melanoma (MM) is known for its abundance of genetic alterations and a tendency for rapid metastasizing. Identification of novel plasma biomarkers may enhance non-invasive diagnostics and disease monitoring. Initially, we examined copy number variations (CNV) in CDK genes (CDKN2A, CDKN2B, CDK4) using MLPA (gDNA) and ddPCR (ctDNA) analysis. Subsequently, low-coverage whole genome sequencing (lcWGS) was used to identify the most common CNV in plasma samples, followed by ddPCR verification of chosen biomarkers. CNV alterations in CDK genes were identified in 33.3% of FFPE samples (Clark IV, V only). Detection of the same genes in MM plasma showed no significance, neither compared to healthy plasmas nor between pre- versus post-surgery plasma. Sequencing data showed the most common CNV occurring in 6q27, 4p16.1, 10p15.3, 10q22.3, 13q34, 18q23, 20q11.21-q13.12 and 22q13.33. CNV in four chosen genes (KIF25, E2F1, DIP2C and TFG) were verified by ddPCR using 2 models of interpretation. Model 1 was concordant with lcWGS results in 54% of samples, for model 2 it was 46%. Although CDK genes have not been proven to be suitable CNV liquid biopsy biomarkers, lcWGS defined the most frequently affected chromosomal regions by CNV. Among chosen genes, DIP2C demonstrated a potential for further analysis.

Effect of amisulpride on the expression of serotonin receptors, neurotrophic factor BDNF and its receptors in mice with overexpression of the aggregation-prone [R406W] mutant tau protein.

Serotonin 5-HT7 receptors (5-HT7R) are attracting increasing attention as important participants in the mechanisms of Alzheimer's disease and as a possible target for the treatment of various tau pathologies. In this study, we investigated the effects of amisulpride (5-HT7R inverse agonist) in C57BL/6J mice with experimentally induced expression of the gene encoding the aggregation-prone human Tau[R406W] protein in the prefrontal cortex. In these animals we examined short-term memory and the expression of genes involved in the development of tauopathy (Htr7 and Cdk5), as well as biomarkers of neurodegenerative processes - the Bdnf gene and its receptors TrkB (the Ntrk2 gene) and p75NTR (the Ngfr gene). In a short-term memory test, there was no difference in the discrimination index between mice treated with AAV-Tau[R406W] and mice treated with AAV-EGFP. Amisulpride did not affect this parameter. Administration of AAV-Tau[R406W] resulted in increased expression of the Htr7, Htr1a, and Cdk5 genes in the prefrontal cortex compared to AAV-EGFP animals. At the same time, amisulpride at the dose of 10 mg/kg in animals from the AAV-Tau[R406W] group caused a decrease in the Htr7, Htr1a genes mRNA levels compared to animals from the AAV-Tau[R406W] group treated with saline. A decrease in the expression of the Bdnf and Ntrk2 genes in the prefrontal cortex was revealed after administration of AAV-Tau[R406W]. Moreover, amisulpride at various doses (3 and 10 mg/kg) caused the same decrease in the transcription of these genes in mice without tauopathy. It is also interesting that in mice of the AAV-EGFP group, administration of amisulpride at the dose of 10 mg/kg increased the Ngfr gene mRNA level. The data obtained allow us to propose the use of amisulpride in restoring normal tau protein function. However, it should be noted that prolonged administration may result in adverse effects such as an increase in Ngfr expression and a decrease in Bdnf and Ntrk2 expression, which is probably indicative of an increase in neurodegenerative processes.

Revised Diagnosis From Histiocytic Neoplasm to Optic Chiasm Glioblastoma After Genetic Analysis.

A 46-year-old man presented with left eye blurring. Automated visual field testing showed an incongruous right hemianopia, with sparing of the lower temporal quadrant in the right eye. MRI revealed foci of gadolinium enhancement in the optic chiasm and optic tracts. Serologic testing (including myelin oligodendrocyte glycoprotein and neuromyelitis optica antibodies) and cerebrospinal fluid analysis were negative. Whole-body PET/CT scan found no malignancy. Biopsy of the optic chiasm revealed a moderately cellular neoplasm composed of atypical, discohesive cells with enlarged nuclei, prominent eosinophilic nucleoli, and abundant vacuolated cytoplasm. Immunohistochemical stains for CD68 and S100 were positive, whereas those for GFAP, OLIG2, SOX10, and multiple others were negative, supporting a diagnosis of histiocytic neoplasm. Five weeks later, results became available from next-generation sequencing targeting the coding regions of hundreds of malignancy-associated genes and select introns. Alterations associated with histiocytic neoplasms (i.e. BRAF and MAP2K1 mutations) were absent. However, there was a nonsense mutation in the PTEN gene, a hotspot mutation in the TERT gene promotor, and focal amplifications of the CDK4 and MDM2 genes. Additionally, there was chromosome 6q loss, 7 gain, and 10q loss. Based on these findings, the diagnosis was revised to glioblastoma, IDH-wildtype, CNS WHO grade 4. The patient began treatment with temozolomide while continuing radiation therapy. This case illustrates how next-generation sequencing can at times provide more accurate diagnostic information than standard tissue histopathology.

Cannabinol Regulates the Expression of Cell Cycle-Associated Genes in Motor Neuron-like NSC-34: A Transcriptomic Analysis.

Cannabinoids are reported to have neuroprotective properties and play a role in neurogenesis and neuroplasticity in in vitro and in vivo models. Cannabinol (CBN) is a minor cannabinoid produced by the degradation of Δ9-tetrahydrocannabinol in Cannabis sativa L. and exhibits anti-oxidant, analgesic, anti-bacterial, and anti-inflammatory effects. In this study, we explored the biological effects of 20 µM CBN (6.20 µg/mL) on differentiated NSC-34 cells by MTT assay and next-generation sequencing analysis on the transcriptome. KEGG and Gene Ontology enrichment analyses have been performed to evaluate potential CBN-associated processes. Our results highlighted the absence of any cytotoxic effect of CBN. The comparative transcriptomic analysis pointed out the downregulation of Cdkn2a, Cdkn2c and Cdkn2d genes, which are known to suppress the cell cycle. Ccne2, Cdk2, Cdk7, Anapc11, Anapc10, Cdc23, Cdc16, Anapc4, Cdc27, Stag1, Smc3, Smc1a, Nipbl, Pds5a, Pds5b, and Wapl genes, renowned for their role as cell cycle progression activators, were instead upregulated. Our work suggests that CBN regulates the expression of many genes related to the cell cycle, which are required for axonal maturation, migration, and synaptic plasticity, while not affecting the expression of genes involved in cell death or tumorigenesis.

Delivery of letrozole-encapsulated niosomes via a 3D bioprinting gelatin–alginate scaffold for potential breast cancer treatment

3D printing technology is a powerful tool in scaffold engineering for biomedical applications, especially in anticancer activities and drug delivery. The present study developed a 3D-printed gelatin–alginate scaffold incorporating letrozole-loaded niosomes (Let/Nio@Gel-AL-SC) as a more effective drug delivery system. The findings showed that the fabricated niosomes appeared spherical. 3D-printed scaffolds exhibited biodegradability and sustained drug-release properties. The drug release from the scaffold was less prominent under acidic conditions than physiological ones. Cytotoxicity analysis showed that the engineered Let/Nio@Gel-AL-SC scaffold exhibited significant cytotoxicity against MCF-7 cancer cells. Gene expression analysis demonstrated a significant decrease in the expression of BCL2, CCND1, MMP2, and CDK4 genes and a notable increase in the expression of BAX and P53 genes, as well as the activity of Caspase 3/7 enzyme following treatment with Let/Nio@Gel-AL-SC. In addition, flow cytometry analysis revealed that Let/Nio@Gel-AL-SC significantly reduced necrosis and dramatically increased apoptosis. Also, the Let/Nio@Gel-AL-SC formulation exhibited a significantly greater increase in ROS values. The incorporation of letrozole-loaded niosomes into 3D printing gelatin/alginate scaffold has enhanced the efficacy of anticancer therapy. This is demonstrated by the sustained release of drugs, which indicates a promising potential for effective anticancer activity. Consequently, this combination holds promise as a potential future cancer therapy strategy.Graphical abstract

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

BackgroundGlioblastoma (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.MethodsBioinformatics 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.ResultsThe 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.ConclusionsThese findings uncover the cellular function of SRSF9 in GBM and highlight its therapeutic potential for GBM.

DYNLL1 accelerates cell cycle via ILF2/CDK4 axis to promote hepatocellular carcinoma development and palbociclib sensitivity.

Disorder of cell cycle represents as a major driver of hepatocarcinogenesis and constitutes an attractive therapeutic target. However, identifying key genes that respond to cell cycle-dependent treatments still facing critical challenges in hepatocellular carcinoma (HCC). Increasing evidence indicates that dynein light chain 1 (DYNLL1) is closely related to cell cycle progression and plays a critical role in tumorigenesis. In this study, we explored the role of DYNLL1 in the regulation of cell cycle progression in HCC. We analysed clinical specimens to assess the expression and predictive value of DYNLL1 in HCC. The oncogenic role of DYNLL1 was determined by gain or loss-of-function experiments in vitro, and xenograft tumour, liver orthotopic, and DEN/CCl4-induced mouse models in vivo. Mass spectrometry analysis, RNA sequencing, co-immunoprecipitation assays, and forward and reverse experiments were performed to clarify the mechanism by which DYNLL1 activates the interleukin-2 enhancer-binding factor 2 (ILF2)/CDK4 signalling axis. Finally, the sensitivity of HCC cells to palbociclib and sorafenib was assessed by apoptosis, cell counting kit-8, and colony formation assays in vitro, and xenograft tumour models and liver orthotopic models in vivo. DYNLL1 was significantly higher in HCC tissues than that in normal liver tissues and closely related to the clinicopathological features and prognosis of patients with HCC. Importantly, DYNLL1 was identified as a novel hepatocarcinogenesis gene from both in vitro and in vivo evidence. Mechanistically, DYNLL1 could interact with ILF2 and facilitate the expression of ILF2, then ILF2 could interact with CDK4 mRNA and delay its degradation, which in turn activates downstream G1/S cell cycle target genes CDK4. Furthermore, palbociclib, a selective CDK4/6 inhibitor, represents as a promising therapeutic strategy for DYNLL1-overexpressed HCC, alone or particularly in combination with sorafenib. Our work uncovers a novel function of DYNLL1 in orchestrating cell cycle to promote HCC development and suggests a potential synergy of CDK4/6 inhibitor and sorafenib for the treatment of HCC patients, especially those with increased DYNLL1.

Indirubin In Vitro Apoptotic Effect on Chronic Lymphocytic Leukemia Cells

Background: Chronic lymphocytic leukemia (CLL) primarily affects the elderly, with its etiology largely unknown. It is hypothesized that hematopoietic stem cells may acquire mutations over time, such as the BCL-2 mutation, leading to disruptions in the apoptotic process. Dangui Luhui Wan, a mixture of 11 herbs used in Chinese Medicine, has shown antitumor activities across various cancer cell types. Indirubin-3'-monoxime (I3M), derived from Dangui Luhui Wan, functions as a selective inhibitor of cyclin-dependent kinases (CDKs) and can induce apoptosis in cells. Objectives: The objective of this study was to evaluate the effectiveness of I3M against CLL cells in vitro. Methods: Peripheral blood mononuclear cells (PBMCs) from 14 patients were treated with I3M at concentrations ranging from 0.1 μM to 80 μM over periods of 24, 48, and 72 hours. The optimal dose was determined using Annexin V and MTT assays. The expression of apoptotic genes Bcl-2/Bax and CDK1/2 was assessed using real-time PCR. Results: The results indicated that a 20 µM concentration of I3M exhibited the highest cytotoxicity after 48 hours compared to controls (P = 0.005). Post-treatment, a decrease in Bcl-2 gene expression was observed, while changes in the Bax gene were not significant. However, an increase in the Bax/Bcl-2 gene ratio was noted, suggesting involvement of the mitochondrial pathway in I3M's apoptotic mechanism. Notably, I3M inhibited the expression of the CDK2 gene but did not affect CDK1 gene expression. Conclusions: I3M appears to exert anti-tumor effects by inducing apoptosis and inhibiting the CDK2 gene. Further research is required to elucidate the precise mechanism of action of I3M in CLL and potentially other tumor cell lines.

Abstract 1157: Inhibition Of P90rsk Ameliorates Pdgf-bb-induced Vascular Smooth Muscle Cell Phenotypic Change And Neointimal Hyperplasia

Platelet-derived growth factor type BB (PDGF-BB) has been reported to induce vascular smooth muscle cell (VSMC) proliferation and migration, which are major events that are highly linked to vascular diseases such as atherosclerosis and restenosis. Although p90RSK has been shown to regulate multiple cellular processes including cell growth, proliferation, survival and motility through various downstream substrates in different cell types, the role of p90RSK on PDGF-BB-induced VSMC proliferation and migration, and vascular neointima formation remains unknown. In this study, we determine whether inhibition of p90RSK protects from PDGF-BB-induced cellular phenotypic change and investigate the molecular mechanisms underlying the effect of p90RSK inhibition. In cultured primary rat VSMCs, we found that pretreatment with FMK or BI-D1870, specific inhibitors of p90RSK, suppressed PDGF-BB-induced VSMC phenotypic alteration, ECM accumulation, proliferation, and migration. In addition, FMK and BI-D1870 repressed cell cycle regulatory genes cyclin D1 and Cdk4 induced by PDGF-BB, whereas inhibition of p90RSK augmented cyclin-dependent kinase inhibitor p27 downregulated by PDGF-BB in VSMCs, suggesting that p90RSK induces VSMC proliferation via cell cycle regulation of G0/G1 phase. Notably, using the mouse model of partial carotid ligation model, FMK was found to attenuate the neointimal hyperplasia of carotid arteries. These results suggest that p90RSK impedes PDGF-BB induced VSMC phenotypic switching, proliferation, migration, and neointima formation.

Evaluating the Anti-proliferative and Apoptotic Role of Atrial Natriuretic Peptide in Colon Cancer Cell Lines

The use of small peptides and conventional anticancer drugs is gaining importance in oncology as small peptides may help increase chemo or radiation sensitivity. The present study aimed to study the impact of atrial natriuretic peptide (ANP) in reducing colon cancer cell proliferation in primary and metastatic colon cancer cell lines. The proliferation of colon cancer cell lines (SW480 and SW620) was analysed by CCK-8 assay and cell damage was analyzed by lactate dehydrogenase activity. Catalase activity assay was performed to measure the oxidative stress and antioxidant defense mechanisms in SW480 and SW620 cell lines. Subsequently, up or downregulation of cancer-specific gene expression such as BAX, Caspase-3, BCL-2, CDK-6, and PCNA genes were assessed after the treatment of small peptide ANP in SW480 and SW620 colon cancer cell lines. The ANP treatment decreased the colon cancer cell proliferation, upregulated the apoptosis-related gene expression (Caspase-3, BAX), downregulated the anti-apoptotic gene (BCL-2) expression, and proliferation-related genes (CDK-6, PCNA) in SW480 and SW620 colon cancer cell lines, and the differences were found to be statistically significantly. Further, increased levels of catalase in the colon cancer cell lines after ANP-treatment suggested the therapeutic role of ANP. Subsequently, the LDH analysis showed the potential of ANP in inducing colon cancer cell damage. Collectively, the current study clearly shows that ANP is a potential molecule in reducing uncontrolled cancer cell growth. However, additional research using animal models and additional colon cancer cell lines is needed to validate its potential usage in clinical studies.

LncRNA BlncAD1 Modulates Bovine Adipogenesis by Binding to MYH10, PI3K/Akt Signaling Pathway, and miR-27a-5p/CDK6 Axis.

Research on adipogenesis will help to improve the meat quality of livestock. Long noncoding RNAs (lncRNAs) are involved in mammalian adipogenesis as epigenetic modulators. In this study, we analyzed lncRNA expression during bovine adipogenesis and detected 195 differentially expressed lncRNAs, including lncRNA BlncAD1, which was significantly upregulated in mature bovine adipocytes. Gain- and loss-of-function experiments confirmed that BlncAD1 promoted the proliferation, apoptosis, and differentiation of bovine preadipocytes. RNA pull-down revealed that the nonmuscle myosin 10 (MYH10) is a potential binding protein of BlncAD1. Then, we elucidated that loss of BlncAD1 caused increased ubiquitination of MYH10, which confirmed that BlncAD1 regulates adipogenesis by enhancing the stability of the MYH10 protein. Western blotting was used to demonstrate that BlncAD1 activated the PI3K/Akt signaling pathway. Bioinformatic analysis and dual-luciferase reporter assays indicated that BlncAD1 competitively absorbed miR-27a-5p. The overexpression and interference of miR-27a-5p in bovine preadipocytes displayed that miR-27a-5p inhibited proliferation, apoptosis, and differentiation. Further results suggested that miR-27a-5p targeted the CDK6 gene and that BlncAD1 controlled the proliferation of bovine preadipocytes by modulating the miR-27a-5p/CDK6 axis. This study revealed the complex mechanisms of BlncAD1 underlying bovine adipogenesis for the first time, which would provide useful information for genetics and breeding improvement of Chinese beef cattle.

CDK1 and CCNA2 play important roles in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a malignant tumor that occurs in oral cavity and is dominated by squamous cells. The relationship between CDK1, CCNA2, and OSCC is still unclear. The OSCC datasets GSE74530 and GSE85195 configuration files were downloaded from the Gene Expression Omnibus (GEO) database and were derived from platforms GPL570 and GPL6480. Differentially expressed genes (DEGs) were screened. The weighted gene co-expression network analysis, functional enrichment analysis, gene set enrichment analysis, construction and analysis of protein-protein interaction (PPI) network, Comparative Toxicogenomics Database analysis were performed. Gene expression heatmap was drawn. TargetScan was used to screen miRNAs that regulate central DEGs. A total of 1756 DEGs were identified. According to Gene Ontology (GO) analysis, they were predominantly enriched in processes related to organic acid catabolic metabolism, centromeric, and chromosomal region condensation, and oxidoreductase activity. In Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the DEGs were mainly concentrated in metabolic pathways, P53 signaling pathway, and PPAR signaling pathway. Weighted gene co-expression network analysis was performed with a soft-thresholding power set at 9, leading to the identification of 6 core genes (BUB1B, CCNB1, KIF20A, CCNA2, CDCA8, CDK1). The gene expression heatmap revealed that core genes (CDK1, CCNA2) were highly expressed in OSCC samples. Comparative Toxicogenomics Database analysis demonstrated associations between the 6 genes (BUB1B, CCNB1, KIF20A, CCNA2, CDCA8, CDK1) and oral tumors, precancerous lesions, inflammation, immune system disorders, and tongue tumors. The associated miRNAs for CDK1 gene were hsa-miR-203a-3p.2, while for CCNA2 gene, they were hsa-miR-6766-3p, hsa-miR-4782-3p, and hsa-miR-219a-5p. CDK1 and CCNA2 are highly expressed in OSCC. The higher the expression of CDK1 and CCNA2, the worse the prognosis.