Cytotechnology最新文献

Laryngeal carcinoma is one of the malignancies in the head and neck region with high incidence and mortality. Despite advances in therapeutic modalities, the 5-year survival rate remains low. Wilms tumor 1-associated protein (WTAP) has been reported to regulate cancer progression, however, its role and mechanism in regulating laryngeal carcinoma development remain unclear. In this study, the expressions of WTAP, collagen triple helix repeat containing 1 (CTHRC1), and YTH N6-methyladenosine RNA binding protein F1 (YTHDF1) and other molecules were detected by quantitative real-time polymerase chain reaction or western blotting. Cell viability and colony formation rate were determined by cell counting kit-8 assay and cell colony formation assay. Cell migration and invasion were investigated by transwell assay. The relationship between CTHRC1 and YTHDF1 was identified by RNA immunoprecipitation assay. The results showed that WTAP and CTHRC1 were upregulated in laryngeal carcinoma tissues and cells. WTAP or CTHRC1 silencing inhibited the proliferation, migration and invasion of laryngeal carcinoma cells. WTAP knockdown inhibited CTHRC1 mRNA stability by suppressing CTHRC1 m6A modification and YTHDF1 from recognizing CTHRC1 m6A sites. Moreover, CTHRC1 overexpression attenuated WTAP knockdown-mediated effects on laryngeal carcinoma cell phenotypes and the expression of β-catenin, C-myc and cyclinD1. Thus, WTAP facilitated CTHRC1 mRNA stability in an m6A-dependent manner to activate the Wnt/β-catenin pathway and promote laryngeal carcinoma cell malignant phenotypes.

Sea cucumbers are both versatile marine organisms and an Asian marine food known to have several medicinal effects. We evaluated the anti-allergic potential of some major purified holostane-type saponins from the body wall of the black sea cucumber, Holothuria atra. Six saponin compounds were isolated, holothurin B (1), holothurin A (2), 24-dehydro echinoside A (3), desholothurin A1 (4), desholothurin A (5), and des 24-dehydro echinoside A (6). The structures were identified based on spectroscopic methods and by comparison with the literature. Each compound’s inhibitory activity toward the release of β-hexosaminidase was evaluated. Among the six compounds, holothurin B (1) showed the strongest inhibition of the degranulation at all tested concentrations in a dose-dependent manner, compared to the positive control, quercetin. We also observed that holothurin B (1) was able to alleviate the inflammatory mediators interleukin (IL)-6, IL-13, and tumor necrosis factor-alpha (TNF-α). Holothurin B (1) also inhibited the Ca²⁺ influx stimulated by the calcium ionophore A23187, by suppressing the expression of inositol-1,4,5-triphosphate receptor (IP3R) mRNA. These results suggest that (i) holothurin B (1) has good anti-allergy activity without cytotoxicity at effective concentrations, and (ii) this compound could be a lead compound for the treatment of allergic diseases and associated inflammation. We also performed a molecular docking study for the tested compounds to correlate their binding modes and affinity for the IP3R with the in vitro results. The results concluded that the holostane-type saponins could be used as anti-allergy agents, which may be attributed to their holostane group.

Endothelial dysfunction plays a critical role in the pathogenesis of sepsis. This study aims to explore the effect and mechanism of forkhead box A1 (FOXA1) on vascular endothelial cell injury in sepsis. Human umbilical vein endothelial cells (HUVECs) were stimulated by lipopolysaccharide (LPS). Lactate dehydrogenase (LDH) release, cell viability, apoptosis, and inflammatory factors including IL-1β, TNF-α, and IL-6 were measured using LDH kits, CCK-8 assay, flow cytometry, and ELISA respectively. RT-qPCR or Western blot determined the expression of FOXA1 or neuropilin-2 (NRP2) in cells. The binding between FOXA1 and NRP2 was confirmed using ChIP and dual-luciferase assays. Functional rescue experiments were performed to verify the effect of FOXA1 siRNA or NRP2 siRNA on cell injury. LPS treatment induced endothelial cell injury in a concentration-dependent manner. FOXA1 expression was elevated after LPS treatment. FOXA1 silencing reduced LDH release, enhanced cell viability, suppressed apoptosis, and declined inflammation factors. Mechanistically, FOXA1 bound to the NRP2 promoter to suppress the transcription of NRP2. Functional rescue experiments revealed that knockdown of NRP2 offset the protective effect of knockdown of FOXA1 on cell injury. In conclusion, FOXA1 exacerbates LPS-insulted endothelial cell injury in sepsis by repressing the transcription of NRP2.

Food security is a major concern due to the growing population and climate change. A method for increasing food production is the use of modern biotechnology, such as cell culture, marker-assisted selection, and genetic engineering. Cellular agriculture has enabled the production of cell-cultivated meat in bioreactors that mimic the properties of conventional meat. Furthermore, 3D food printing technology has improved food production by adding new nutritional and organoleptic properties. Marker-assisted selection and genetic engineering could play an important role in producing animals and crops with desirable traits. Therefore, integrating cellular agriculture with genetic engineering technology could be a potential strategy for the production of cell-based meat and seafood with high health benefits in the future. This review highlights the production of cell-cultivated meat derived from a variety of species, including livestock, birds, fish, and marine crustaceans. It also investigates the application of genetic engineering methods, such as CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein), in the context of cellular agriculture. Moreover, it examines aspects such as food safety, regulatory considerations, and consumer acceptance of genetically engineered cell-cultivated meat and seafood.

Colorectal cancer (CRC) remains the third leading cause of cancer-related death worldwide. Here, we aimed to uncover the mechanism underlying the transcription factor fifth Ewing variant protein (FEV) in CRC. Transcriptome differential expression in human CRC and adjacent tissues was analyzed using GSE143939, GSE142279, GSE196006, and GSE200427 datasets, and the intersecting genes were screened by comparing them with the list of transcription factors in the Human TFBD database, followed by KEGG enrichment analysis. FEV expression was significantly reduced in CRC, and upregulation of FEV inhibited cell growth and tumor progression in CRC. The highly expressed genes in CRC were mainly enriched to the Wnt signaling pathway, and WNT2 is the core initiator of the Wnt signaling pathway. Two binding sites for FEV are present on the WNT2 promoter. WNT2 promoted the proliferation, migration, and invasion of CRC cells. FEV repressed WNT2 transcription by binding to the WNT2 promoter. Collectively, our data revealed that a novel FEV/WNT2 axis is critical for CRC progression. Strategies targeting this specific signaling axis might be developed to treat patients with CRC.

KIAA1429 has been reported as a cancer regulator, but its role and mechanism in the progression of oral squamous cell carcinoma (OSCC) remain elusive. The objective of the present research was to figure out the effect of KIAA1429 regulated CA9 on the progression of OSCC. Using qRT-PCR and bioinformatics analysis, we studied the expression levels of KIAA1429 and CA9 in OSCC tissue samples. The functional roles of KIAA1429 and CA9 were assessed using transwell and CCK-8 assays. The regulation among KIAA1429 and CA9 was investigated using MeRIP and western blotting assays. In addition, the m6A level in OSCC was measured utilizing RNA m6A quantification. In OSCC, KIAA1429 and m6A levels were upregulated. We observed that KIAA1429 inhibition declined proliferation, migration, and invasion of OSCC cells and decreased cell growth in vivo. Furthermore, KIAA1429 serves as a crucial upstream regulator of CA9 in OSCC and upregulates CA9 expression through an m6A-dependent mechanism. We observed that CA9 was upregulated in OSCC samples and that low expression of KIAA1429 partially restored the enhanced malignant phenotype caused by CA9 overexpression. Overall, our findings suggest that KIAA1429 and CA9 act as pro-oncogenic factors in OSCC, with KIAA1429 promoting OSCC malignancy through m6A modification-dependent stabilization of CA9 transcripts, which represents a novel regulatory mechanism in OSCC.

Synovial mesenchymal stem cells (sMSCs) have great potential for cartilage repair, but their therapeutic design to avoid adverse effects associated with unknown factors remains a challenge. In addition, because long-term preservation is indispensable to maintain high quality levels until implantation, it is necessary to reduce their fluctuations. This study aimed to investigate the properties and feasibility of novel scaffold-free tissue-engineered constructs using serum-free media and to develop long-term preservation methods. sMSCs were cultured in serum-free media, seeded at high density in a monolayer, and finally developed as a sheet-like construct called “gMSC1”. The properties of frozen gMSC1 (Fro-gMSC1) were compared with those of refrigerated gMSC1 (Ref-gMSC1) and then examined by their profile. Chondrogenic differentiation potential was analyzed by quantitative real-time polymerase chain reaction and quantification of glycosaminoglycan content. Xenografts into the cartilage defect model in rats were evaluated by histological staining. gMSC1 showed nearly similar properties independent of the preservation conditions. The animal experiment demonstrated that the defect could be filled with cartilage-like tissue with good integration to the adjacent tissue, suggesting that gMSC1 was formed and replaced the cartilage. Furthermore, several chondrogenesis-related factors were significantly secreted inside and outside gMSC1. Morphological analysis of Fro-gMSC1 revealed comparable quality levels to those of fresh gMSC1. Thus, if cryopreserved, gMSC1, with no complicated materials or processes, could have sustained cartilage repair capacity. gMSC1 is a prominent candidate in novel clinical practice for cartilage repair, allowing for large quantities to be manufactured at one time and preserved for a long term by freezing.

Atherosclerosis is a chronic inflammatory vascular disease. It was confirmed that activation of ferroptosis could induce the development of AS. Meanwhile, Krüppel-like factor 7 was reported to be involved in AS. Nevertheless, the detailed function of KLF7 in ferroptosis during AS has not been not explored. To mimic AS in vitro, human microvascular endothelial cells (HMEC-1) were exposed to 100 μg/mL ox-LDL. Cell viability was tested using MTT assay, and commercial kits were applied to examine the ferroptosis. Flow cytometry was applied for testing lipid ROS level. The relation between KLF7 and AlkB homolog 5 (ALKBH5) was explored using dual luciferase and ChIP assays. Furthermore, MeRIP was used to test the m6A modification level of ACSL4. KLF7 and ALKBH5 overexpression reversed ox-LDL-induced ferroptosis (characterized by up-regulated MDA, iron, Fe²⁺, lipid ROS and ACSL4, and down-regulated GSH and GPX4) in HMEC-1 cells. In addition, KLF7 transcriptionally activated ALKBH5. ALKBH5 decreased the level of ACSL4 by inhibiting the m6A modification of ACSL4. Furthermore, upregulation of KLF7 restored ox-LDL-induced ferroptosis in HMEC-1 cells via upregulating ALKBH5. KLF7 repressed ox-LDL-induced ferroptosis in HMEC-1 cells through promoting ALKBH5 mediated m6A demethylation of ACSL4. Our study might supply a new therapeutic strategy for AS treatment.

Colorectal cancer (CRC) is a common tumor type, and liver metastasis reduces the long-term survival in CRC patients. Natural killer (NK) cells play an important role in anti-tumor immunity. The aim of this study was to investigate the mechanism of miR-214-5p on NK cells in CRC liver metastasis. We collected clinical samples of CRC liver metastasis and nonmetastatic tissues and purchased the human NK cell lines NK92 and liver metastatic CRC cells KM12L4 for research. RT‒qPCR, Western blot, CCK-8, Transwell, and flow cytometry methods were used to evaluate the effect of miR-214-5p/USP27X/Bim pathway regulating NK cell activity on CRC liver metastasis. In addition, we also investigated the potential targets and regulatory mechanisms of the signaling pathway of miR-214-5p. In this study, we found that miR-214-5p was downregulated in CRC liver metastasis tissues. After transfection of miR-214-5p mimic, the activity of NK cells was significantly enhanced, and the proliferation and migration ability of CRC liver metastasis cells were inhibited, while inducing tumor cell apoptosis. Further research proved that USP27X is a potential target for miR-214-5p and upregulates Bim level through deubiquitination. In addition, miR-214-5p mimic reduced the level of USP27X and Bim, thereby enhancing the antitumor effect of NK cells. In conclusion, our research results show that miR-214-5p promotes the antitumor effect of NK cells by regulating the USP27X/Bim pathway, thereby inhibiting CRC liver metastasis. This finding reveals the important role of miR-214-5p in regulating the immune function of NK cells, and provides new ideas for developing new immunotherapy strategies.