Analytical Cellular Pathology最新文献

Early reperfusion into the myocardium after ischemia causes myocardial ischemia-reperfusion (I/R) injury and ferroptosis was involved. Ischemia activates the expression of a series of oxidative stress genes and their downstream regulatory genes, including ferroptosis-related genes such as nuclear factor E2-related factor 2 (Nrf2), glutathione peroxidase 4 (GPX4), and SLC7A11. This study adopted primary cardiomyocytes and I/R in rats to evaluate the ferroptosis and changing of Nrf2-SLC7A11/heme oxygenase-1 (HO-1) in vitro and in vivo. Online analysis tools were used to predict the possible target Kelch-like ECH-associated protein 1 (Keap1) of miR-432-5p. The mimic of miR-432-5p plasmid was constructed to verify the effect of miR-432-5p on ferroptosis. We found that hypoxia/reoxygenation (H/R) in cardiomyocytes and I/R in rats induced lipid peroxidation and ferroptosis in cardiomyocytes. The activation of the Nrf2-SLC7A11/HO-1 pathway protects cardiomyocytes from ferroptosis. Downregulation of miR-432-5p has been confirmed in H/R cardiomyocytes (in vitro) and cardiomyocytes in myocardial infarction rats (in vivo). Upregulation of miR-432-5p inhibited ferroptosis of cardiomyocytes induced by RAS-selective lethal 3 (RSL3), an inhibitor of GPX4 and ferroptosis inducer through decreasing the binding protein of Nrf2, Keap1, which was confirmed by bioinformatics and mutation assay. Knockdown Nrf2 attenuates the protection effect of miR-432-5p on H/R cardiomyocytes. Intravenous delivery of liposome carriers of miR-432-5p remarkably ameliorated cardiomyocyte impairment in the I/R animal model. In conclusion, miR-432-5p inhibits the ferroptosis in cardiomyocytes induced by H/R by activating Nrf2/SLC7A11 axis by degrading Keap1 and is a potential drug target for clinical myocardial infarction treatment.

Interferon regulatory factor 2 (IRF2) participates in the differentiation of immune T cells. Bone marrow mesenchymal stem cell (BM-MSC)-derived exosomes can secret mRNA, miRNAs, and proteins to regulate tumor microenvironment. The present study focused on the miRNA/IRF2 axis in regulating Th1/Th2 ratio and cell apoptosis in acute myeloid leukemia (AML). The flow cytometry analysis was performed to examine the Th1/Th2 ratio and AML apoptosis in vivo and in vitro. The contents of Interferon γ (IFN-γ) and Interleukin-4 (IL-4) were measured using enzyme-linked immunosorbent assay. StarBase was used to predict the potential binding site between miR-222-3p and the 3' untranslated region of IRF2. Luciferase reporter assay was applied for validating the combination of miR-222-3p and IRF2. BM-MSC exosomes were successfully isolated. BM-MSC exosomes increased Th1/Th2 ratio and promoted apoptosis of AML cells. Further analysis showed that IRF2 was targeted by miR-222-3p. Overexpression of miR-222-3p promoted Th1/Th2 ratio and AML cell apoptosis. IRF2 partially reversed the effect that is exerted by miR-222-3p on Th1/Th2 ratio and AML cell apoptosis. Overexpression of miR-222-3p promoted Th1/Th2 ratio and caspase 3 expression in vivo. To sum up, miR-222-3p promotes Th1/Th2 ratio and AML cell apoptosis by regulating IRF2 expression, which provided crucial targets for the treatment of AML.

Objective: To study the effect of congenital dyskeratosis 1 (DKC1) on neuroblastoma and its regulation mechanism.

Methods: The expression of DKC1 in neuroblastoma was analyzed by TCGA database and molecular assay. NB cells were transfected with siDKC1 to observe the effects of DKC1 on proliferation, cloning, metastasis, and invasion, and apoptosis and apoptosis-related proteins. The tumor-bearing mouse model was constructed, shDKC1 was transfected to observe the tumor growth and tumor tissue changes, and the expression of DKC1 and Ki-67 was detected. Screening and identification of miRNA326-5p targeting DKC1. NB cells were treated with miRNA326-5p mimic or inhibitors to detect the expression of DKC1. NB cells were transfected with miRNA326-5p and DKC1 mimics to detect cell proliferation, apoptosis, and apoptotic protein expression.

Results: DKC1 was highly expressed in NB cells and tissues. The activity, proliferation, invasion, and migration of NB cells were significantly decreased by DKC1 gene knockout, while apoptosis was significantly increased. The expression level of B-cell lymphoma-2 in shDKC1 group was significantly lower than that of the control group, while the expression level of BAK, BAX, and caspase-3 was significantly higher than that of the control group. The results of experiments on tumor-bearing mice were consistent with the above results. The results of miRNA assay showed that miRNA326-5p could bind DKC1 mRNA to inhibit the protein expression, thereby inhibiting the proliferation of NB cells, promoting their apoptosis, and regulating the expression of apoptotic proteins.

Conclusion: miRNA326-5p targeting DKC1 mRNA regulates apoptosis-related proteins to inhibit neuroblastoma proliferation and promote the apoptotic process.

Hepatocellular carcinoma (HCC) is a malignant type of liver cancer that poses severe threat to human health worldwide. Aerobic glycolysis is a hallmark of HCC and facilitates its progression. Solute carrier family 10 member 1 (SLC10A1) and long intergenic non-protein coding RNA 659 (LINC00659) were detected to be downregulated in HCC cells, yet their potential functions underlying HCC progression remained unidentified. In the current work, colony formation and transwell assays were used to detect HCC cells (HepG2 and HuH-7) proliferation and migration in vitro study. The quantitative real-time polymerase chain reaction (qRT-PCR) and western blot assays were used for gene/protein expression determination. Seahorse assay was performed for aerobic glycolysis assessment. RNA immunoprecipitation (RIP) and RNA pull-down assays were conducted for detection of the molecular interaction between LINC00659 and SLC10A1. The results showed that overexpressed SLC10A1 significantly suppressed the proliferation, migration, and aerobic glycolysis in HCC cells. Mechanical experiments further demonstrated that LINC00659 positively regulated SLC10A1 expression in HCC cells by recruiting fused protein in sarcoma (FUS). Our work elucidated that LINC00659 inhibited HCC progression and aerobic glycolysis via the FUS/SLC10A1 axis, revealing a novel lncRNA-RNA-binding protein-mRNA network in HCC, which might provide potential therapeutic targets for HCC.

Acetaminophen has always been at the center of attention as a non-steroidal anti-inflammatory drug, which is generally associated with the serious side effects on liver and the hematological parameters. This study aimed to compare the effect of N-acetyl cysteine (NAC) and thyme extract on rat models of acetaminophen-induced toxicity. The present experimental study was conducted on 48 Wistar rats randomized into six groups, including the control group (no treatment); the Ac group (470 mg/kg of acetaminophen); the Ac + 100Ex, Ac + 200Ex, and Ac + 400Ex groups (acetaminophen + thyme extract at doses of 100, 200, 400 mg/kg); and Ac + NA group (acetaminophen + NAC). After weighing, a blood sample was taken from heart at the end of the period. The measured parameters were hematological, liver biochemical, and oxidative stress profiles. A part of the liver tissue was also fixed for the pathological examinations. The bone marrow was aspirated to check for cellular changes as well. The lowest mean of the final weight and liver weight to body weight ratio was observed in the Ac group. Weight loss was compensated in Ac + NA and Ac + 200Ex groups (P = 0.035). White blood cell (WBC), red blood cell (RBC), Hemoglobin (Hgb), and Hematocrit (HCT) in Ac and Ac + 400Ex groups showed significant differences from those of the other test groups (P < 0.001). Aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) enzymes in Ac + 200Ex and Ac + NA groups showed a significant decrease compared to those of the other treatment groups (P = 0.043). Total antioxidant capacity (TAC) and glutathione peroxidase (GPx) had the lowest levels in Ac and Ac + 400Ex groups, while malondialdehyde (MDA) had the highest content. In this regard, the liver histopathological indices (necrosis, hyperemia, and hemorrhage) in the Ac + 200Ex and Ac + NA groups reached their lowest grades in the treatment groups. The mean number of erythroid and myeloid cells in the Ac group reached the lowest (17.40 ± 3.48). The microscopic appearance of the bone marrow cells was different from normocytosis in the control group to hypocytosis in the Ac and Ac + 400Ex groups. Thymol, as an effective ingredient in thyme extract at a dose of 200 mg/kg compared to NAC, had a unique effect on reducing bone marrow and liver cell-tissue changes due to the acetaminophen toxicity.

Stress has become a universal biological phenomenon in the body, which leads to pathophysiological changes. However, the molecular network interactions between endoplasmic reticulum (ER) stress and ferroptosis under stressful conditions are not clear. For this purpose, we screened the gene expression profile of GSE173795 for intersection with ferroptosis genes and screened 68 differentially expressed genes (DEGs) (63 up-regulated, 5 down-regulated), mainly related to lipid and atherosclerosis, autophagy-animal, mitophagy-animal, focal adhesion, DNA replication, proteasome, oocyte meiosis, toll-like receptor signaling pathway, cell cycle, etc. Immune infiltration analysis revealed that stress resulted in decreased B cells memory, T cells CD8 and T cells CD4 memory resting, monocytes, macrophages M2, and increased B cells naive, T cells follicular helper, and macrophages M1. 19 core-DEGs (ASNS, TRIB3, ATF4, EIF2S1, CEBPG, RELA, HSPA5, DDIT3, STAT3, MAP3K5, HIF1A, HNF4A, MAPK14, HMOX1, CDKN1A, KRAS, SP1, SIRT1, EGFR) were screened, all of which were up-regulated DEGs. These biological processes and pathways were mainly involved in responding to ER stress, lipid and atherosclerosis, cellular response to stress, cellular response to chemical stress, and regulation of DNA-templated transcription in response to stress, etc. Spearman analysis did not find MAPK14 to be significantly associated with immune cells. Other core-DEGs were associated with immune cells, including B cells naive, T cells follicular helper, and monocytes. Based on core-DEGs, 283 miRNAs were predicted. Among the 22 miRNAs with highly cross-linked DEGs, 11 had upstream lncRNA, mainly targeting STAT3, SP1, CDKN1A, and SIRT1, and a total of 39 lncRNA were obtained. 85 potential drugs targeting 11 core-DEGs were identified and were expected to be potential immunotherapeutic agents for stress injury. Our experiments also confirmed that Liproxstatin-1 alleviates common cross-linked proteins between ER stress and ferroptosis. In conclusion, our study explored the molecular mechanisms and network interactions among stress-ER stress-ferroptosis from a novel perspective, which provides new research ideas for studying stressful injury.

Malignant bone neoplasms can be represented by osteosarcoma (OS), which accounts for 36% of all sarcomas. To reduce tumor malignancy, extensive efforts have been devoted to find an ideal target from numerous candidates, among which RNA-binding proteins (RBPs) have shown their unparalleled competitiveness. With the special structure of RNA-binding domains, RBPs have the potential to establish relationships with RNAs or small molecules and are considered regulators of different sections of RNA processes, including splicing, transport, translation, and degradation of RNAs. RBPs have considerable significant roles in various cancers, and experiments revealed that there was a strong association of RBPs with tumorigenesis and tumor cell progression. Regarding OS, RBPs are a new orientation, but achievements in hand are noteworthy. Higher or lower expression of RBPs was first found in tumor cells compared to normal tissue. By binding to different molecules, RBPs are capable of influencing tumor cell phenotypes through different signaling pathways or other axes, and researches on medical treatment have been largely inspired. Exploring the prognostic and therapeutic values of RBPs in OS is a hotspot where diverse avenues on regulating RBPs have achieved dramatical effects. In this review, we briefly summarize the contribution of RBPs and their binding molecules to OS oncogenicity and generally introduce distinctive RBPs as samples. Moreover, we focus on the attempts to differentiate RBP's opposite functions in predicting prognosis and collect possible strategies for treatment. Our review provides forwards insight into improving the understanding of OS and suggests RBPs as potential biomarkers for therapies.

Purpose: Long non-coding RNAs (LncRNAs) OIP5-AS1 and miR-25-3p play important roles in myocardial injury, whereas their roles in lipopolysaccharide (LPS)-induced myocardial injury remain unknown. The purpose of our study was to investigate the functional mechanisms of OIP5-AS1 and miR-25-3p in LPS-induced myocardial injury.

Methods: Rats and H9C2 cells were treated with LPS to establish the model of myocardial injury in vivo and in vitro, respectively. The expression levels of OIP5-AS1 and miR-25-3p were determined by quantitative reverse transcriptase-polymerase chain reaction. Enzyme-linked immunosorbent assay was performed to measure the serum levels of IL-6 and TNF-α. The relationship between OIP5-AS1 and miR-25-3p/NOX4 was determined by luciferase reporter assay and/or RNA immunoprecipitation assay. The apoptosis rate was detected by flow cytometry, and cell viability was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Western blot was performed to detect the protein levels of Bax, Bcl-2, caspase3, c-caspase3, NOX4, and p-NF-κB p65/NF-κB p65.

Results: OIP5-AS1 was up-regulated, and miR-25-3p was down-regulated in myocardial tissues of LPS-induced rats and LPS-treated H9C2 cells. Knockdown of OIP5-AS1 relieved the myocardial injury in LPS-induced rats. Knockdown of OIP5-AS1 also inhibited the inflammation and apoptosis of myocardial cells in vivo, which was subsequently confirmed by in vitro experiments. In addition, OIP5-AS1 targeted miR-25-3p. MiR-25-3p mimics reversed the effects of OIP5-AS1 overexpression on promoting cell apoptosis and inflammation and on inhibiting cell viability. Besides, miR-25-3p mimics blocked the NOX4/NF-κB signalling pathway in LPS-induced H9C2 cells.

Conclusion: Silencing of lncRNA OIP5-AS1 alleviated LPS-induced myocardial injury by regulating miR-25-3p.

Extensive peritoneal spread and capacity for distant metastasis account for the majority of mortality from epithelial ovarian cancer (EOC). Accumulating evidence shows that interleukin-6 (IL-6) promotes tumor invasion and migration in EOC, although the molecular mechanisms remain to be fully elucidated. Meanwhile, the hypoxic microenvironment has been recognized to cause metastasis by triggering epithelial-mesenchymal transition (EMT) in several types of cancers. Here, we studied the synergy between IL-6 and hypoxia in inducing EMT in two EOC cell lines, A2780 cells and SKOV3 cells. Exogenous recombination of IL-6 and autocrine production of IL-6 regulated by plasmids both induced EMT phenotype in EOC cells characterized by downregulated E-cadherin as well as upregulated expression of vimentin and EMT-related transcription factors. The combined effects of IL-6 and hypoxia were more significant than those of either one treatment on EMT. Suppression of hypoxia-inducible factor-1α (HIF-1α) before IL-6 treatment inhibited the EMT phenotype and invasion ability of EOC cells, indicating that HIF-1α occupies a key position in the regulatory pathway of EMT associated with IL-6. EMT score was found positively correlated with mRNA levels of IL-6, signal transducer and activator of transcription 3 (STAT3), and HIF-1α, respectively, in 489 ovarian samples from The Cancer Genome Atlas dataset. Next, blockade of the abovementioned molecules by chemical inhibitors reversed the alteration in the protein levels of EMT markers induced by either exogenous or endogenous IL-6. These findings indicate a positive feedback loop between IL-6 and HIF-1α, and induce and maintain EMT phenotype through STAT3 signaling, which might provide a novel rationale for prognostic prediction and therapeutic targets in EOC.

In recent years, the involvement of E3 ubiquitin ligase constitutive photomorphogenesis 1 (COP1) in the tumorigenesis of gastric cancer (GC) has been elucidated. However, the exact underlying mechanism remains to be clarified. In the present study, the expression profiles of COP1 in GC were derived from the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) databases, followed by verification via immunohistochemical staining (IHC), Western blotting (WB), and quantitative real-time polymerase chain reaction (qRT-PCR) reaction assays on clinical samples. In vitro, the gain- and loss-of-function experiments of COP1 protein were conducted to explore its role in GC cell lines HGC-27 and SGC-7901. Furthermore, we screened the interaction protein of COP1 by yeast two-hybrid experiment and verified their combination by co-immunoprecipitation (co-IP). We preliminary explored the possible underlying mechanisms of COP1 protein in GC cell lines via WB. COP1 was upregulated in GC tissues compared with the corresponding non-carcinoma tissues. In vitro, the upregulation of COP1 protein promoted the proliferation and migration of GC cells. The yeast two-hybrid experiment and co-IP indicated that Cadherin 18 (CDH18) could constitute a complex with COP1. Moreover, cells with COP1 over-expression showed low levels of CDH18 expression, with the intracellular PI3K/AKT pathway activated and the malignancy of GC cell lines enhanced. Our findings demonstrated that COP1 promoted the GC tumorigenesis by downregulated CDH18 with the involvement of PI3K/AKT signaling pathway in cell lines, suggesting the potential of COP1 as a prognostic biomarker and therapeutic target for GC.