Cytotechnology最新文献

Obesity is one of the major health issues of recent times. It is known that obesity increases endoplasmic reticulum (ER) stress, contributing to the development of insulin resistance in type-2 diabetes. This study investigates the relationship of glutamine metabolism with insulin resistance and ER stress under obese conditions. Using differentiated 3T3-L1 adipocytes, we demonstrate that increased glutamine supplementation enhances insulin sensitivity and reduces ER stress. Enhanced glutamine treatment also upregulated key amino acid transporters, Alanine-Serine-Cysteine Transporter 2 (ASCT2), L-type Amino Acid Transporter 1 (LAT1), and Cystine/Glutamate Antiporter (xCT), activating the mammalian target of rapamycin (mTOR) pathway. To investigate the specific role of ASCT2, its expression was reduced in 3T3-L1 fibroblasts via sh-ASCT2 transfection prior to adipogenic differentiation. Interestingly, the levels of other selected glutamine-glutamate transporters, LAT1 and xCT, were also decreased. Knockdown of ASCT2 via shRNA significantly impaired adipocyte differentiation. The significant reduction of intracellular lipid accumulation leads to decreased ER stress and insulin resistance in sh-ASCT2 adipocytes. Metabolomic analysis results revealed that intracellular glutamine and malate levels increased during fat cell differentiation. The glutamine levels decreased in sh-ASCT2 adipocytes, while malate levels remained unchanged. In conclusion, glutamine may enhance insulin sensitivity by modulating ER stress and influencing transporter expression. ASCT2 might play a role in adipocyte differentiation, and evidence indicates that ASCT2 inhibition could be associated with reduced adipogenesis and improved insulin signaling, suggesting its potential relevance as a therapeutic target in obesity-related insulin resistance. Further studies are needed to clarify the context-dependent mechanisms by which glutamine and ASCT2 regulate metabolic stress adaptation.

Microglial polarization and the NLRP3 inflammasome play critical roles in the pathology of ischemic stroke (IS). Regulation of microglial polarization towards M2 type has become a potential therapy approach for M2 type has become a potential therapy approach for treating IS. The current study demonstrated IFITM1 was markedly upregulated in microglia in the Distal middle cerebral artery (dMCAO) model. This research sought to assess the protective potential of IFITM1 knockdown in IS and to study its downstream regulatory mechanisms. The dMCAO mice model and oxygen-glucose deprivation/reoxygenation (OGD/R) BV2 cell model were constructed to imitate IS injury. The GSE148350 dataset was utilized to screen for differentially expressed genes in IS. Immunofluorescence, western blotting, and ELISA were utilized to evaluated activation level of NLRP3 inflammasome. RT-qPCR was applied to evaluated gene expression associated with the M1/M2 phenotypes and glycolysis of microglia. After treatment with dMCAO and OGD/R, IFITM1 expression was significantly upregulated. Knockdown of IFITM1 significantly inhibited M1 markers and promoted M2 markers of microglia. Additionally, knockdown of IFITM1 obviously inhibited the activation of the NLRP3 inflammasome and glycolysis. The expression of c-FOS was markedly raised following OGD/R, but was obviously inhibited by knockdown of IFITM1. Rescue experiments showed that knockdown of IFITM1 inhibited the NLRP3 inflammasome activation and M1 polarization of microglia by suppressing c-FOS expression. Knockdown of IFITM1 alleviated brain infarction, brain edema, and neurological damage in dMCAO mice. These results suggest that the absence of IFITM1 alleviates brain injury induced by dMCAO, inhibiting NLRP3 inflammasome activation and glycolysis of microglia through suppressing c-FOS expression.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00832-5.

Polyphenols have physiological effects, such as antioxidant and anti-inflammatory effects, and have been reported to associated with osteoporosis and inflammatory diseases. Rosmarinic acid is a natural polyphenol contained in Lamiaceae herbs, such as perilla, sage, and sweet basil. Salviaflaside is a glycosidized compound of rosmarinic acid. It was one of the major components of the defatted Perilla frutescens Britton var. japonica Hara (egoma) seed residue extract. In this study, we investigated the anti-osteoporotic effects of the water-soluble layer fraction of egoma residue heated water extract (DPH-W) and salviaflaside on bone marrow-derived macrophages (BMMs). DPH-W reduced the number of tartrate-resistant acid phosphatase (TRAP) positive osteoclasts in BMM treated with receptor-activated nuclear factor kappa B ligand (RANKL). The mRNA expression levels of NFATc1 and CTSK, which are responsible for osteoclast differentiation and bone resorption, were suppressed. Salviaflaside decreased TRAP activity and suppressed the expression of osteoclast differentiation-related genes. Our findings indicate that egoma seed residue and salviaflaside may have potential as a useful therapeutic or prophylactic agent for the suppression of bone loss.

Glioma is a malignant primary tumor of the brain. In recent years, numerous LncRNAs (Long non-coding RNA) have been demonstrated to be potential targets for glioma, and induction of pyroptosis is one of the important directions to inhibit the malignant process of cancer. The role of LncRNA PSMA-AS1, a novel lncRNA, on glioma remain unclear. The aim of this study was to investigate the effects and mechanisms of LncRNA PSMA-AS1 on glioma. PSMA1-AS1 and its potential targets were analyzed using bioinformatics tools and validated using a dual luciferase reporter gene system. Glioma cells were cultured to detect the expression levels of PSMA1-AS1 and its targets. The levels of PSMA1-AS1 and its targets were regulated by transfection and their effects on the viability and metastatic ability of glioma cells as well as on the level of pyroptosis were examined. PSMA1-AS1 expression was elevated in glioma, where miR-140-3p was a downstream target, and was decreased in glioma cells. Inhibition of PSMA1-AS1 promoted pyroptosis and inhibited glioma cell viability and metastatic ability. Overexpression of miR-140-3p had the same effect as inhibition of PSMA1-AS1, whereas inhibition of miR-140-3p reversed the effect of inhibition of PSMA1-AS1 on glioma cells. In addition, SRSF10 is a downstream binding target of miR-140-3p, and inhibition of SRSF10 also promoted pyroptosis and inhibited glioma cells proliferation and metastasis. In conclusion, our results confirmed that PSMA1-AS1 affects glioma cell proliferation and metastasis by regulating pyroptosis through miR-140-3p/SRSF10 axis, suggesting that LncRNA PSMA1-AS1 may be a potential target for glioma.

Resveratrol-rich extracts from Vitis vinifera cane have gained recognition as natural compounds with significant anticancer potential. This study examined the impact of this extract on A549 lung cancer cells using both 2D monolayer culture and a more physiologically relevant 3D co-culture model incorporating peripheral blood mononuclear cells (PBMCs). In monolayer culture, at 40 µg/mL we evaluated the extract's impact on cell viability, colony formation ability, morphological changes, and oxidative stress markers. A 3D co-culture model was employed to examine the effects of the extract on matrix metalloproteinase ( MMP-2 and MMP-9) activity, c-Jun N-terminal kinase (JNK) signaling pathway, and nitric oxide (NO) production. Our results demonstrated that the extract significantly reduced A549 cell viability (IC50 39.11 ± 0.32 µg/mL) and colony formation while inducing morphological changes and cellular redox imbalance markers malondialdehyde (MDA) (p ≤ 0.001), protein carbonyl (p ≤ 0.0001), Advanced Oxidation Protein Products (AOPP) (p ≤ 0.01), nitric oxide (NO) (p ≤ 0.0001), Superoxide Dismutase (SOD) activity (p ≤ 0.01), and glutathione (GSH) level (p ≤ 0.01). and in monolayer cultures. In the 3D co-culture system, treatment with the extract significantly decreased the growth rate of the 3D structures (P ≤ 0.1), reduced MMP-2 and MMP-9 activities (P ≤ 0.1), and JNK phosphorylation, with an overproduction of nitric oxide (P ≤ 0.001). These findings reveal novel mechanisms by which resveratrol-rich extracts from Vitis vinifera cane may exert anticancer effects by modulating the JNK/MMPs/NO axis in lung cancer cells A549. This extract could represent a promising therapeutic alternative for lung cancer treatment.

Endometrial cancer (EC) is one of the most pervasive gynecological malignancies, with its incidence increasing annually. Although progress has been made, the underlying molecular mechanisms driving EC progression remain poorly understood. N⁶-methyladenosine (m⁶A) RNA modification is instrumental in tumor biology, but the function of the m⁶A "writer" RNA-binding motif protein 15 (RBM15) in EC remains unclear. This study investigates the role of RBM15 through m⁶A-dependent regulation of its downstream target. RBM15 expression was analyzed using the TNMplot database and validated by qRT-PCR in EC tissues and cell lines. Cell functional assays were conducted to assess the impact of RBM15 knockdown. Bioinformatics analytics and MeRIP-qPCR were used to identify m⁶A-modified downstream targets of RBM15. Furthermore, RNA stability assay, correlation analysis, and Western blotting were employed to explore the underlying regulatory mechanisms. Rescue experiments were done to confirm functional interactions. RBM15 was significantly overexpressed in EC, and its silencing impeded EC cell proliferation, migration, and invasion. PBK was identified as a direct downstream target, with RBM15-mediated m⁶A enrichment enhancing mRNA stability. PBK expression was positively correlated with RBM15 levels in patient samples. RBM15 silencing partially attenuated the PBK-driven promotive effects in EC cells. Mechanistically, RBM15 suppressed the p53 pathway by stabilizing PBK, resulting in decreased phospho-p53 protein levels. In summary, RBM15 promotes EC progression by enhancing m⁶A-dependent stabilization of PBK mRNA, which in turn suppresses p53 signaling, revealing a novel RBM15-PBK-p53 axis as a potential treatment target.

Ovarian cancer (OC) is a type of gynecological cancer, characterized by a high mortality rate. MicroRNAs (miRNAs) and cell autophagy are associated with the chemoresistance of tumors. The purpose of this study was to investigate the role of miR-675-5p in cisplatin (DDP) resistance in OC cells and its related mechanism. We collected OC tissues and paracancerous tissues. The OC cell line A2780 and the DDP-resistant cell line A2780/DDP were used for the study. RT-qPCR, Western blot and immunofluorescence were used to detect the expression of related genes and proteins. Cell proliferation and apoptosis were evaluated by CCK-8 assay and flow cytometry. Furthermore, the effect of miR-675-5p on the progression of OC was investigated in a xenograft model of nude mice. In this research, miR-675-5p expression was significantly increased in OC tissues and A2780/DDP cells. The suppression of miR-675-5p in A2780/DDP cells resulted in a significant reduction in DDP resistance. Further study revealed that miR-675-5p inhibited TSC2 expression in OC cells by binding to the TSC2 3'UTR, thereby activating the mTOR signaling pathway and promoting autophagy in A2780/DDP cells. Knocking down miR-675-5p suppressed DDP resistance in A2780/DDP cells through TSC2/mTOR axis, while overexpressing miR-675-5p suppressed DDP sensitivity in A2780 cells through TSC2. The tumor volume was decreased after injection of miR-675-5p-knockdown A2780/DDP cells in vivo. Knocking down miR-675-5p can inhibit the mTOR signaling pathway and autophagy through TSC2, thereby reducing the DDP resistance of OC cells. This may provide a potential therapeutic target for OC.

[This retracts the article DOI: 10.1007/s10616-013-9534-x.].

Bladder cancer (BLCA) is a prevalent malignancy of the urinary tract. Long noncoding RNAs (lncRNAs) exert significant effects on various human cancers by targeting microRNAs (miRs). This study, therefore, probed the action of the LINC00152/miR-103a-3p axis in epithelial-mesenchymal transition (EMT) and progression of BLCA. LINC00152, miR-103a-3p, and ADORA3 expression in clinical BLCA tissues were tested. LINC00152 was knocked down in BLCA cells alone or combined with miR-103a-3p inhibition or ADORA3 overexpression. The level of EMT- and PI3K/AKT pathway-related genes was assessed with Western blot. Cell proliferation, invasion and migration, and apoptosis were examined with CCK8 and EdU, Transwell, and flow cytometry assays, respectively. Binding of miR-103a-3p to LINC00152 and ADORA3 was validated through RIP and dual-luciferase reporter gene assays. The action of LINC00152 in BLCA progression was verified in vivo. LINC00152 and ADORA3 expression was abundant and miR-103a-3p expression was poor in BLCA tissues. LINC00152 knockdown or PI3K/AKT pathway inhibition repressed malignant behaviors and EMT of BLCA cells. Mechanistically, LINC00152 bound specifically to miR-103a-3p, which targeted ADORA3 and then activated the PI3K/AKT pathway. miR-103a-3p inhibition or ADORA3 overexpression nullified the suppressive impacts of LINC00152 knockdown on EMT and malignant behaviors of BLCA cells. Furthermore, LINC00152 knockdown decreased ADORA3 expression and suppressed tumor growth in vivo. LINC00152 knockdown upregulates miR-103a-3p to reduce ADORA3 expression, thus protecting against the malignant biological behaviors and EMT of BLCA cells.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00829-0.

Super-resolution fluorescence microscopy (SRM) has enabled visualization of nanoscale cellular structures, but systematic evaluation of resolution assessment methods across diverse biological structures and SRM modalities remains lacking. Here, we comparatively assessed three resolution metrics-Full Width at Half Maximum (FWHM), decorrelation analysis, and Fourier Ring Correlation (FRC)-across two SRM techniques (Super-resolution Radial Fluctuation, SRRF; Stimulated Emission Depletion, STED) using key subcellular structures: microtubules (filaments), mitochondria (membranes), and nuclear pore protein Nup98 (single particles) in HeLa/U2OS cells. Our results showed decorrelation analysis provided robust resolution estimates across all structures and modalities (confocal/SRRF/STED), exhibiting superior performance for dense nuclear pore complexes where FWHM failed due to overlapping point spread functions. FWHM was effective for discrete structures (microtubules/mitochondria) but unsuitable for densely packed features. NanoJ-SQUIRREL's integrated FRC analysis successfully quantified SRRF resolution for all structures, with resolution-scaled Pearson coefficient (RSP > 0.90) confirming minimal reconstruction artifacts. STED achieved significantly higher resolution than SRRF, but severe photobleaching prevented FRC application. Our study demonstrates decorrelation analysis offers universal robustness across structures and modalities, FWHM is suitable for discrete structures, and NanoJ-SQUIRREL provides standardized artifact validation and FRC-based resolution quantification for computational super-resolution. This study establishes a framework for context-appropriate resolution assessment in cell biology, emphasizing integration of structural features and technical principles.