Experimental cell research最新文献

Ferroptosis is principally initiated by dysregulation of iron metabolism and excessive accumulation of ROS, which exacerbates myocardial injury during acute myocardial infarction (AMI). Previous studies have indeed demonstrated the significant involvement of long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) exerts its pleiotropic effects in the pathophysiology of myocardial infarction, heart failure and atherosclerosis by modulating inflammation, apoptosis, and oxidative stress. However, whether and how NEAT1 mediates myocardial ferroptosis remain unknown. In this study, we found that NEAT1 expression was significantly elevated in hypoxic HL-1 cells and AMI mice, while silencing of NEAT1 alleviated lipid peroxidation and myocardial ferroptosis both in vitro and in vivo. Mechanistically, NEAT1 directly sponged miR-450b-5p and negatively regulated its expression. In addition, miR-450b-5p directly targeted Acyl-CoA synthase long-chain family member 4 (ACSL4). Notably, inhibition of miR-450b-5p reversed the role of NEAT1 in AMI mice. Collectively, these findings newly illustrated that NEAT1 acts as a competitive endogenous RNA (ceRNA) of miR-450–5p in AMI. Especially, silencing of NEAT1 effectively ameliorated myocardium ischemia by suppression of ferroptosis via miR-450–5p/ACSL4 pathway, which providing a brand-new therapeutic strategy for myocardial ischemia injury.

N6-methyladenosine (m⁶A) modification plays an important role in RNA molecular functions, therefore affecting the initiation and development of hepatocellular carcinoma (HCC). Herein, multiple datasets were applied to conduct a comprehensive analysis of DEGs within HCC and the analysis revealed significant dysregulation of numerous genes. Functional and signaling pathway enrichment analyses were performed. Further, TP53RK binding protein (TPRKB) emerged as a significant factor, exhibiting high expression level within HCC tissue samples and cells which could predict HCC patients’ poor OS. Knockdown investigations of TPRKB in vitro demonstrated the effect of TPRKB knockdown on attenuating the aggressiveness of HCC cells by suppressing the viability, colony formation, invasive ability, and migratory ability, inducing cell cycle arrest, and facilitating the apoptosis of HCC cells. Investigations in vivo revealed that TPRKB knockdown significantly suppressed tumor growth in mice model. Additionally, the study identified methyltransferase 5, N6-adenosine (METTL5) as a potential regulator of TPRKB expression via m⁶A modification, positively regulating TPRKB expression by enhancing TPRKB mRNA stability. The dynamic effects of METTL5 and TPRKB upon the phenotypes of HCC cells further confirmed that TPRKB overexpression partially abolished the anti-cancer effects of METTL5 knockdown upon the aggressiveness of HCC cells. Conclusively, our findings uncover that TPRKB, significantly overexpressed in HCC, exerts a critical effect on promoting tumor aggressiveness, and its expression shows to be positively regulated by METTL5 via m⁶A methylation. These insights deepen the understanding of HCC pathogenesis and open new avenues for targeted therapies, highlighting that METTL5-TPRKB axis is an underlying new therapeutic target in HCC management.

Anterior gradient-2 (AGR2) is highly expressed in several tumors and plays an important role in tumor development. However, the biological function of AGR2 in teratomas has not yet been thoroughly studied. In this study, AGR2 was found to be upregulated in teratoma tissues and in human testicular teratoma cell lines by Western blotting and qRT-PCR assays. A DNA Methylation-Specific PCR assay demonstrated that AGR2 upregulation resulted from hypomethylated AGR2 in teratoma cells. NCC-IT and NT2-D1 cells were transfected with pcDNA-AGR2 or sh-AGR2 to obtain AGR2-overexpressed or -silenced cells, and cell proliferation, invasion and glycolysis were determined using CCK-8, 5-ethynyl-2′-deoxyuridine (EdU), Transwell assays, and commercial kits. The results revealed that overexpression of AGR2 promoted teratoma cell proliferation and invasion and elevated glycolysis levels evidencing by the increase in lactate secretion, glucose consumption, ATP levels and the expression of glycolysis-related proteins, while knockdown of AGR2 showed the opposite results. The interactions between AGR2 and annexin A2 (AnXA2), as well as between AnXA2 and epidermal growth factor receptor (EGFR) were verified by co-immunoprecipitation assay. Mechanistic studies revealed that AGR2 interacts with AnXA2 and increases the level of AnXA2 to recruit more AnXA2 to EGFR, there by promoting EGFR expression. A series of rescue experiments showed that knockdown of AnXA2 or EGFR weakened the promotional effects of AGR2 overexpression on the proliferation, invasion, and glycolysis of teratoma cells. Finally, tumorigenicity assays were performed using NT2-D1 cells stably transfected with either LV-NC-shRNA or LV-shAGR2. The results showed that AGR2 knockdown significantly inhibited teratoma tumor growth in vivo. In conclusion, our data suggested that AGR2 facilitates glycolysis in teratomas through promoting EGFR expression by interacting with AnXA2, thereby promoting teratoma cells proliferation and invasion.

α-Actinin-4 (ACTN4) expression levels are correlated with the invasive and metastatic potential of cancer cells; however, the underlying mechanism remains unclear. Here, we identified ACTN4-localized ruffle-edge lamellipodia using live-cell imaging and correlative light and electron microscopy (CLEM). BSC-1 cells expressing EGFP-ACTN4 showed that ACTN4 was most abundant in the leading edges of lamellipodia, although it was also present in stress fibers and focal adhesions. ACTN4 localization in lamellipodia was markedly diminished by phosphoinositide 3-kinase inhibition, whereas its localization in stress fibers and focal adhesions remained. Furthermore, overexpression of ACTN4, but not ACTN1, promoted lamellipodial formation. Live-cell analysis demonstrated that ACTN4-enriched lamellipodia are highly dynamic and associated with cell migration. CLEM revealed that ACTN4-enriched lamellipodia exhibit a characteristic morphology of multilayered ruffle-edges that differs from canonical flat lamellipodia. Similar ruffle-edge lamellipodia were observed in A549 and MDA-MB-231 invasive cancer cells. ACTN4 knockdown suppressed the formation of ruffle-edge lamellipodia and cell migration during wound healing in A549 monolayer cultures. Additionally, membrane-type 1 matrix metalloproteinase was observed in the membrane ruffles, suggesting that ruffle-edge lamellipodia have the ability to degrade the extracellular matrix and may contribute to active cell migration/invasion in certain cancer cell types.

Matrix stiffness is a crucial factor in the tumor microenvironment, impacting tumor progression and development. TET2 is vital for epigenetic regulation in melanoma and is significantly reduced in advanced melanomas compared with nevi and thin melanomas. However, it is unclear how TET2 mediates the effect of matrix stiffness on melanoma cells. This study utilized A2058 cell lines and prepared different stiffness collagen hydrogels to evaluate TET2 overexpression (TET2OE) and mutant (TET2M) melanoma cells' activity, proliferation, and invasion. A2058 melanoma cells' viability and invasion decreased with increased matrix stiffness, with TET2OE cells experiencing a more significant impact than TET2M cells. Methylation analysis revealed that TET2 determines gene methylation levels, influencing cell-ECM interactions. Transcriptome analysis confirmed that TET2 promotes matrix stiffness's effect on melanoma cell fate. This research provides promising directions and opportunities for melanoma treatment.

Prostate cancer (PCa) is threatening the health of millions of people, the pathological mechanism of prostate cancer has not been fully elaborated, and needs to be further explored. Here, we found that the expression of DUSP26 is dramatically suppressed, and a positive connection of its expression with PCa prognosis was also observed. In vitro, overexpression of DUSP26 significantly inhibited the proliferative, migrative, and invasive capacities of PC3 cells, DUSP26 silencing presented opposite results. Tumor formation experiments in subcutaneous nude mice demonstrated that DUSP26 overexpression could significantly suppress PC3 growth in vivo. Moreover, the mechanism of DUSP26 gene and PCa was discovered by RNA-Seq analysis. We found that DUSP26 significantly inhibited MAPK signaling pathway activation, and further experiments displayed that DUSP26 could impair TAK1, p38, and JNK phosphorylation. Interestingly, treatment with the TAK1 inhibitor (iTAK1) attenuated the effect of DUSP26 on PC3 cells. Together, these results suggested that DUSP26 may serve as a novel therapeutic target for PC3 cell type PCa, the underlying mechanism may be through TAK1-JNK/p38 signaling.

Human periodontal ligament cells (hPDLCs) contain multipotent postnatal stem cells that can differentiate into PDL fibroblasts, osteoblasts, and cementoblasts. Interaction between the extracellular environment and stem cells is an important factor for differentiation into other progenitor cells. To identify cell surface molecules that induce PDL fibroblastic differentiation, we developed a series of monoclonal antibodies against membrane/ECM molecules. One of these antibodies, an anti-PDL25 antibody, recognizes approximately a 100 kDa protein, and this antigenic molecule accumulates in the periodontal ligament region of tooth roots. By mass spectrometric analysis, we found that the antigenic molecule recognized by the anti-PDL25 antibody is fibroblast activation protein α (FAPα). The expression level of FAPα/PDL25 increased in TGF-β1-induced PDL fibroblasts, and this protein was localized in the cell boundaries and elongated processes of the fibroblastic cells. Ectopic expression of FAPα induced fibroblastic differentiation. In contrast, expression of representative markers for PDL differentiation was decreased by knock down and antibody blocking of FAPα/PDL25. Inhibition of dipeptidyl peptidase activity by a potent FAPα inhibitor dramatically inhibited PDL fibroblastic marker expression but did not affect in cell proliferation and migration.

Severe conjunctival damage can lead to extensive ocular cicatrisation, fornix shortening, and even ocular surface failure, resulting in significant vision impairment. Conjunctival reconstruction is the primary therapeutic strategy for these clinical conjunctival diseases. However, there have been limited studies on induced differentiation of conjunctival epithelial cells derived from stem cells. In this study, we established a chemical defined differentiation protocol from human embryonic stem cells (hESCs) into conjunctival epithelial cells. hES cell line H1 was used for differentiation, and RT-qPCR, immunofluorescence staining, Periodic-acid-Schiff staining (PAS), and transcriptome analysis were employed to identify the differentiated cells. Here, to imitate the development of the vertebrate conjunctiva, hESCs were induced using a three-step process involving first chetomin was used to induce ocular surface ectoderm, then nicotinamide was used to induce ocular surface epithelial progenitor cells, and finally epidermal growth factor, keratinocyte growth factor and other factors were used to differentiate mature conjunctival epithelial cells. hESC-derived conjunctival epithelial cells expressed mature conjunctival epithelial lineage markers (including PAX6, P63, K13). The presence of goblet cells was confirmed by positive PAS. Transcriptome analysis revealed that hESC-derived conjunctival epithelial cells possessed a more naïve phenotype, and exhibited greater proliferation capacity compared to mature human conjunctival epithelial cells, suggesting their potential as alternative seed cells for conjunctival reconstruction.

Gasotransmitters are endogenous gaseous signaling molecules that can freely pass through cell membranes and transmit signals between cells, playing multiple roles in cell signal transduction. Due to extensive and ongoing research in this field, we have successfully identified many gasotransmitters so far, among which nitric oxide, carbon monoxide, and hydrogen sulfide are best studied. Gasotransmitters are implicated in various diseases related to necroptosis, such as cardiovascular diseases, inflammation, ischemia-reperfusion, infectious diseases, and neurological diseases. However, the mechanisms of their effects on necroptosis are not fully understood. This review focuses on endogenous gasotransmitter synthesis and metabolism and discusses their roles in necroptosis, aiming to offer new insights for the therapeutic approaches to necroptosis-associated diseases.