The inhibitory effect of the AKT inhibitor Uprosertib on the proliferation and migration of CRC cell lines (HT29 and SW480).

This study presents a multifunctional RNA regulation strategy that enables RNA molecules to undergo both bioorthogonal ligation and cleavage reactions within the same system. Using guide RNA (gRNA) as an example, we demonstrate on-demand inactivation via click chemistry and subsequent reactivation by light exposure. Applied to CRISPR-mediated, site-specific RNA methylation, this technology enables continuous, multistep programmable control, overcoming the one-way limitation of traditional methods. Results show that gRNA can stably tolerate both azide and photodegradable groups, facilitating efficient targeting of M3M14-dCas9 and dCas13b-M3M14 methylation systems to introduce m6A at specific RNA sites. Small molecules can turn off methylation through in situ click chemistry, while 365-nm light exposure rapidly restores gRNA function, allowing precise control over RNA methylation. This strategy highlights the efficiency and flexibility of progressive bioorthogonal RNA modulation and paves the way for multisite, dynamic regulation in complex biological systems.

Aspartate-specific cysteine protease (Caspase)-8 plays a critical role in initiating pyroptosis by mediating cleavage and activation of gasdermin-D (Gsdmd) during Yersinia infection. However, the role of Caspase-8 in pyroptosis after myocardial infarction (MI) and its underlying mechanism remains elusive. Here we show that Caspase-8 is essential for cardiomyocyte pyroptosis post MI and Caspase-8/Gsdmd signaling pathway is activated in hearts of male infarcted mice. The inhibition of Caspase-8 in male mice rescues the decline of cardiac function and cardiomyocyte pyroptosis following MI. Dnmt3a is further shown to impact DNA methylation on the CpG island of Caspase-8 promoter and regulate Caspase-8 expression. Dnmt3a overexpression protects myocardium from MI in male mice by inhibiting cardiomyocyte pyroptosis. Moreover, reduction of Dnmt3a in MI is attributed to the increased ubiquitination. Our study identifies a regulatory axis, Dnmt3a/Caspase-8/Gsdmd, which drives ischemic heart injury by promoting cardiomyocyte pyroptosis, and suggests potential therapeutic targets for heart diseases.

Pathway enrichment analysis and validation of AKT3, SNAIL, and CDH2 expression in CROM patient samples and organoids.

Delayed re-epithelialisation is a key feature of chronic and recurrent cutaneous wounds, but the metabolic mechanisms that coordinate keratinocyte migration and epidermal barrier restoration remain incompletely defined. Here, we investigated whether NUCB2/Nesfatin-1 regulates keratinocyte-driven wound repair through cholesterol biosynthesis. Transcriptomic profiling revealed coordinated downregulation of cholesterol biosynthetic genes in NUCB2-deficient keratinocytes. Concurrently, gene signatures associated with cell motility and epithelial plasticity were also suppressed. Functionally, NUCB2 depletion impaired CCK-8-based metabolic/proliferative activity, scratch-wound closure, Transwell migration and epithelial marker expression, whereas exogenous Nesfatin-1 partially restored these defects. Mechanistic analyses showed that NUCB2/Nesfatin-1 enhanced mTORC1 downstream signalling, increased nuclear SREBP2 abundance and HMGCR expression, and promoted intracellular cholesterol accumulation. Cholesterol supplementation or HMGCR overexpression partially restored cholesterol availability and migratory capacity in NUCB2-deficient keratinocytes, whereas rapamycin attenuated Nesfatin-1-associated SREBP2-HMGCR induction, cholesterol accumulation and migration. In a murine full-thickness excisional wound model, local Nesfatin-1 administration accelerated wound closure and enhanced early re-epithelialisation, accompanied by increased SREBP2 and HMGCR expression at the wound-edge epidermis. Complementary local Nucb2 silencing delayed wound closure. These findings define a NUCB2/Nesfatin-1-mTORC1 downstream signalling-SREBP2-HMGCR axis that supports cholesterol-dependent keratinocyte migration and epidermal repair.

Background: The role of scavenger receptor class B type 1 (SR-B1) in breast cancer remains largely unknown. Objective: This study aimed to investigate the effects of SR-B1 on breast cancer cell proliferation, migration, and invasion and to elucidate the underlying mechanisms. Methods: Two breast cancer cell lines, MDA-MB-231 and MCF-7, were used in this study. Cells were transfected with SR-B1-specific siRNA (si-SR-B1), whereas cells transfected with scrambled sequences served as controls. Cell proliferation was assessed using CCK-8 and colony formation assays. Cell migration and invasion were evaluated using wound healing and Transwell assays, respectively. Apoptosis was analyzed using flow cytometry. Western blot analysis was performed to examine activation of the PI3K/AKT signaling pathway following SR-B1 knockdown. Results: Knockdown of SR-B1 significantly inhibited the proliferation, migration, and invasiveness of MDA-MB-231 and MCF-7 cells (all p<0.05). Moreover, SR-B1 knockdown promoted apoptosis in these cells. Western blot analysis revealed that the phosphorylation levels of AKT and mTOR were markedly decreased in the si-SR-B1 group compared to those in the controls. Additionally, the expression of downstream targets, including cyclin D1 and P70, was downregulated by SR-B1 silencing. Conclusion: SR-B1 contributes to the enhanced proliferation and migration of breast cancer cells, likely through activation of the PI3K/AKT signaling pathway.

The effect of AKT3 knockdown on the proliferation, migration, invasion, and cytoskeletal structure of CRC cell lines.

Validation that AKT3 overexpression enhances the invasion and migration capabilities of colorectal cancer cells.

BACKGROUND:In tissue engineering applications, human dental pulp stem cells (hDPSCs) are an optimal biological resource due to their ability to regenerate the pulp-dentin complex through odontogenic differentiation. Shikonin (SHI), a red-purple naphthoquinone compound extracted from the root of Lithospermum erythrorhizon, has shown promise in this context. However, comprehensive studies on SHI's capacity to promote odontogenic differentiation in hDPSCs are limited. This study aims to investigate the effects of SHI on the odontogenic differentiation of hDPSCs and explore the underlying mechanisms involved. MATERIALS AND METHODS:In vitro experiments were conducted using cell counting kit‑8 assays (CCK-8), Alkaline phosphatase (ALP) / Alizarin red S (ARS) staining, ALP activity measurements, Quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot analysis. In vivo, subcutaneous scaffolds loaded with hDPSCs were implanted into the dorsum of nude mice, followed by histological and immunohistochemical analyses. RESULTS:Results indicated that SHI, at concentrations up to 0.5 µM, did not significantly affect the proliferation of hDPSCs. However, at a concentration of 0.25 µM, SHI promoted the odontogenic differentiation of hDPSCs in vitro. Correspondingly, in vivo findings demonstrated that SHI enhanced the formation of a mineralized matrix by hDPSCs. Furthermore, inhibition of the MAPK pathway was found to block the SHI-induced upregulation of odontogenic differentiation markers in hDPSCs. CONCLUSION:These findings suggest that SHI may promote odontogenic differentiation of hDPSCs by activating MAPK signaling pathways, contributing to the exploration of new strategies for pulp-dentin regeneration.

Characterization of EMT-like epithelial subsets, including CNV analysis and marker expression profiling.

Stress granules (SGs) are transient, membraneless condensates that assemble dynamically within cells in response to diverse stressors. In recent years, SGs have been found to be closely associated with multiple pathological states and have attracted significant attention, particularly concerning their roles in hepatic pathophysiology. Functioning as critical hubs for post-transcriptional regulation, SGs maintain cellular homeostasis through the sequestration, transport, and translational suppression of mRNA, thereby potentially modulating the initiation and progression of various liver diseases. Our review summarizes the assembly mechanisms of SGs and recent research advances concerning their involvement in diseases including hepatocellular carcinoma, viral hepatitis, acute liver injury and fatty liver disease. It particularly focuses on SGs core RNA-binding proteins and associated regulatory networks. Although research into the impact of SGs on liver diseases remains in a nascent phase, with mechanistic details still elusive, SGs emerge as pivotal molecular nexuses connecting cellular stress responses to pathophysiological states, highlighting their therapeutic potential for liver disorders. This review aims to provide a theoretical foundation for a deeper understanding of the roles of SGs in liver pathologies and to promote their further development in both fundamental research and clinical translation.

BACKGROUND:This study aimed to investigate the role of kynurenine in Colorectal Cancer (CRC) and the underlying mechanism. METHODS:Enzyme-linked immunosorbent assay was employed to assess the kynurenine concentration. Flow cytometry was utilized to analyze the percentages of CD3+CD4+ and CD3+CD8+ T-cells. Immunofluorescence was used to measure the expression of Programmed Death-Ligand 1 (PD-L1). RNA modification levels in CRC cells were analyzed using a dot blot assay. The interaction between NAT10 and PD-L1 was assessed via RNA immunoprecipitation, dual-luciferase reporter, and immunofluorescence assays. A xenograft tumor rat model was established. RESULTS:Results indicated that kynurenine suppressed T-cell activation and promoted immune escape. Besides, kynurenine promoted N-Acetyltransferase 10 (NAT10)-mediated N4-acetylcytidine (ac4C) modification. Moreover, NAT10 inhibition improved T-cell activation and suppressed immune escape. Mechanically, NAT10 is bound with the mRNA of PD-L1. Rescue experiments showed that PD-L1 inhibitor treatment reversed the suppressed T-cell activation and the promoted immune escape induced by NAT10 overexpression. In vivo, studies indicated that NAT10 deficiency reversed the promoted tumor growth induced by kynurenine treatment. CONCLUSION:In conclusion, kynurenine promoted the immune escape of CRC cells via NAT10-mediated ac4C acetylation of PD-L1.

BACKGROUND:HER2-positive breast cancer is an aggressive molecular subtype characterized by high recurrence rates and poor prognosis. Identifying robust biomarkers for prognosis and therapeutic response is essential to improve individualized treatment strategies. METHODS:Based on genome-wide association study (GWAS) data, summary data-based Mendelian randomization (SMR) analysis was used to screen disease-related genes. The diagnostic model of HER2-positive breast cancer was constructed by combining LASSO, random forest and SVM-RFE and the characteristic genes were identified. The prognostic correlation was evaluated through Kaplan-Meier analysis. The SNP association, expression level, immune activity and drug sensitivity of ILF2 were analyzed emphatically. H&E staining and immunohistochemistry were used to verify the expression of ILF2 in tumor tissues. RESULTS:A total of 14 genes associated with HER2-positive breast cancer were identified through SMR analysis, with the most significant SNPs enriched on chromosome 1. Integrating three machine learning algorithms (LASSO, Random Forest, and SVM-RFE), four robust diagnostic feature genes were identified: SLC16A3, ILF2, ARRDC3, and SNAPIN. Among them, ILF2 emerged as a key gene of interest. Kaplan-Meier survival analysis revealed that high expression of ILF2, NEK10, LAMTOR5, and APOBEC3A was significantly associated with poorer prognosis. The top ILF2-associated SNP (rs10908848) showed a strong negative regulatory effect on disease risk. Further functional analyses indicated that high ILF2 expression was associated with suppressed antitumor immune activity and reduced sensitivity to multiple chemotherapeutic agents, suggesting its role in immune evasion and therapy resistance. Immunohistochemical staining confirmed significantly upregulated ILF2 protein expression in HER2-positive breast tumor tissues compared with adjacent normal tissues. CONCLUSION:ILF2 is biologically and clinically important in HER2-positive breast cancer, and its high expression is linked with tumor immune escape and increased resistance to chemotherapeutic agents. Future studies should further explore the mechanism and application of ILF2 as a potential therapeutic target.

Aim or purpose: To determine the biological effects of arecoline on oral submucous fbrosis (OSF). Materials and methods: The differential genes between OSF tissue and normal oral tissue were collected form GSE64216 dataset, analyzed by Gene Expression Omnibus database. Real-time PCR and immunohistochemistry were used to analyze the expression of IL-4 in oral tissue. Enzyme-linked immunosorbent assay was used to analyze the expression of exocrine IL-4 protein in human oral fbroblasts (HOF) pre-treated by arecoline. Cell Counting Kit-8 and transwell assays were used to analyze the proliferation and migration of HOF cells, respectively. After IL-4 knocked down, the proliferation and migration of HOF cells were detected. Flow cytometry was used to analyze the proportion of M2-macrophages. Real-time PCR and immunohistochemistry were used to verify the expression of biomarker proteins of macrophages in OSF tissues. Results: The expression of IL-4 was up-regulated in OSF tissue .Arecoline could enhance the expression of IL-4 gene and protein in HOF cells, and promote the proliferation and migration of HOF cells. While knockdown of IL-4 could inhibit arecoline-induced proliferation and migration in HOF cells. The recombinant human IL-4 (rhIL-4) protein could increase the pro-portion of M2-macrophages. Similarly, the results of real-time PCR and immunohistochemistry showed the expression of ARG1 (Biomarker proteins of M2-macrophage) was up-regulated in OSF tissues. Conclusions: Arecoline promotes activation of fbroblasts and polarization of M2-macrophages by up-regulating the expression of IL-4.

The hyaluronan-mediated motility receptor (HMMR) is widely expressed across various species and plays a crucial role in cancer progression. However, its role in nasopharyngeal carcinoma (NPC) remains unexplored. Here, we identified a novel HMMR-FAM83D-β-catenin axis, which drives NPC progression through β-catenin signaling. Using bulk RNA sequencing, single-cell RNA-sequencing, and immunohistochemistry, we determined that HMMR is highly expressed in NPC tissues, correlating with poor survival in NPC patients. In vitro assays demonstrated that modulating HMMR expression influenced NPC cell proliferation, migration, and invasion. In vivo, HMMR knockdown significantly inhibited tumor growth and metastasis in NPC models. HMMR was significantly upregulated in NPC tissues and correlated with worse patient survival. Mechanistically, HMMR was found to interact with the Wnt/β-catenin signaling pathway, affecting both pathway activation and β-catenin expression, as evidenced by RNA-seq and western blotting. Co-immunoprecipitation, mass spectrometry, and western blot analyses further revealed that HMMR interacts with Family with Sequence Similarity 83 Member D (FAM83D), stabilizing its expression by inhibiting its ubiquitination. This interaction, in turn, modulates β-catenin levels, driving NPC progression. Our findings reveal a novel HMMR-FAM83D-β-catenin axis that promotes NPC cell progression through Wnt/β-catenin signaling by modulating β-catenin signaling. HMMR could serve as a potential prognostic biomarker and therapeutic target for NPC, deepening our understanding of its role in disease progression.

KRAS is the most commonly mutated oncogene, which is the driver in nearly 23% human cancers, and are especially prevalent in colorectal, lung, and pancreatic tumors. The KRAS signaling pathway is often hyperactivated in cancer cells due to KRAS gene mutations. This hyperactivation leads to uncontrolled cell proliferation, ultimately leading to the development and progression of cancer. There has been an urgent and unmet need to target KRAS mutations in KRAS-driven cancer. Biochemical HTRF assay was used to measure the inhibition of HEC211909 to both GDP-bound and GTP-bound state of various KRAS mutant. CellTiter-Glo assay was performed to determine the effects on proliferation of tumor cell lines. Several KRAS dependent tumor models were employed to study the in vivo pharmacodynamic and anti-tumor effects. HEC211909 is a potent and orally bioavailable small molecule inhibitor of the KRAS oncogene, such as KRAS G12D, KRAS G12C, KRAS G12V, KRAS Q61H, KRAS G13D and KRAS wild-type amplifications. HEC211909 inhibits both active (GTP-bound) and inactive (GDP-bound) forms of multiple KRAS mutant protein with single-digit nanomolar IC50 values. HEC211909 has a strong anti-proliferative activity in a panel of KRAS mutant cancer cell lines and KRAS wild-type amplifications cancer cell lines in vitro with sub-nanomolar IC50 values, as well as significant inhibition of ERK1/2 phosphorylation with sub-nanomolar IC50 values. HEC211909 is highly selective for KRAS vs HRAS or NRAS, and is expected to be well-tolerated in normal tissues. In vivo, HEC211909 demonstrated dose-dependent anti-tumor activity with tumor regressions at 30 or 60 mg/kg given orally twice daily in KRAS-mutant xenograft models, such as PK59(KRAS G12D), HPAC(KRAS G12D), H358(KRAS G12C) xenograft models, and shows good tolerability during 21 days of treatment. HEC211909 is a highly potent and selective orally pan-KRAS(ON/OFF) inhibitor against KRAS G12D, KRAS G12C, KRAS G12V, KRAS Q61H, KRAS G13D mutations and also displays high selectivity over WT HRAS and NRAS, thus providing an expanded therapeutic index. Haiwang Liu, Hongming Xie, Fuxing Tan, Ning Kang, Ming Li, Yingjun Zhang. Preclinical studies of HEC211909, an orally bioavailable, high potent pan-KRAS inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4381.

Lnc ATG9B-4 aggravated the progression of liver cancer by up-regulating cyclin-dependent-kinase 5 (CDK5). It could be inferred that ATG9B-4 indirectly regulates the expression of CDK5 via lncRNA-mediated negative regulation of target genes. Therefore, the specific molecular mechanism by which ATG9B-4 regulates the malignant characteristics of liver cancer cells still needs further study. The differentially expressed genes were identified by mRNA sequencing in liver cancer cells transfected with or without ATG9B-4. Liver cancer cells were transfected with ATG9B-4, ARNTL, or si-CDK5. The expression of aryl basic helix-loop-helix ARNT like 1 (BMAL1, also known as ARNTL), CDK5, and ATG9B-4 was analyzed by real-time quantitative PCR and western blotting. The proliferation and invasion of the transfected cells were respectively analyzed by cell counting kit-8 and wound healing assays, respectively. The ARNTL expression was down-regulated in the liver cancer tissues and liver cancer cells transfected with ATG9B-4. Low ARNTL expression indicated poor overall survival in patients with liver cancer. The optical density of cells transfected with ATG9B-4 and ARNTL was significantly lower than that of cells transfected with ATG9B-4. The wound areas of cells transfected with ATG9B-4 and ARNTL were markedly wider than those of cells transfected with ATG9B-4. The expression of CDK5 was down-regulated in cells transfected with ARNTL. CDK5 knockdown partially attenuated the ATG9B-4-induced increase in proliferation and migration in liver cancer cells. ATG9B-4 deteriorated the proliferation and migration of liver cancer cells in an ARNTL-CDK5 pathway-dependent manner.

To analyze the biological effect and mechanism of areca nut extract (ANE) on human oral keratinocyte (HOK) cells. The effect of gradient concentration of ANE on the proliferation activity of HOK cells was analyzed by cell counting kit-8 (CCK-8) assays. The differentially expressed genes between the ANE group and control group HOK cells were analyzed by second-generation transcriptome sequencing. Real-time PCR and western blot were, respectively, used to analyze the expression of AREG gene and protein in HOK cells. After AREG gene overexpression or knockdown, the proliferation, migration, and expression of proteins related to epithelial–mesenchymal transformation (EMT), MAPK signal pathway in HOK cells were, respectively, detected by CCK-8, wound healing, transwell, and western blot assays. ANE (500 μg/mL) promoted the proliferation and migration of HOK cells, ANE (2 mg/mL) promoted the EMT of HOK cells, and ANE (50 mg/mL) inhibited the proliferation of HOK cells. AREG knockdown inhibited ANE-induced proliferation and migration of HOK cells, while AREG overexpression promoted the proliferation and migration of HOK cells. Western blot assay showed that ANE activated MAPK signal pathway by upregulating AREG protein in HOK cells. ANE promoted HOK cell proliferation, migration, and EMT by mediating AREG–MAPK signaling pathway.

Objective:This study aims to develop a prediction model for lymph node metastasis (LNM) in colorectal cancer (CRC) patients using common clinicopathologic data and a nomogram. The model seeks to uncover correlations between LNM and clinical indicators, providing an effective tool to identify high-risk patients, aiding clinical decision-making, and enhancing patient prognosis. Methods:We conducted a retrospective analysis of CRC patients diagnosed between January 2021 and December 2023 at Zhuzhou Hospital Affiliated to Xiangya School of Medicine, Central South University. Risk predictors for LNM were identified through comparative analysis and Least Absolute Shrinkage and Selection Operator (LASSO) logistic regression. Nomograms were then utilized to predict the probability of metastasis, and their performance was assessed using calibration curves, receiver operating characteristic (ROC) curves, and decision curve analysis. Results:The study comprised 869 CRC patients, with 435 cases allocated to the training set and 434 cases to the validation set. First, 12 potential risk factors for LNM in CRC patients were identified through comparative analysis in the training set. Next, nine independent predictors (T stage, vascular tumor thrombus, PMS2, MSH2, KRAS, BRAF, PIK3CA, leukocyte, and neutrophil) of LNM occurrence were refined using LASSO regression and multivariate logistic regression models. Subsequently, a clinical nomogram was developed based on these independent predictors of LNM. The nomogram exhibited a C-index of 0.751 (95% CI [0.728-0.774]), indicating its robust predictive value, which was further validated in the independent validation set. Conclusion:T stage, vascular tumor thrombus, PMS2, MSH2, KRAS, BRAF, and neutrophil emerged as significant risk factors for LNM in CRC, while leukocytes appeared to be protective. These findings emphasize the importance of comprehensive risk assessment and personalized therapeutic strategies in CRC management.