Analytical Cellular Pathology最新文献

Epithelial ovarian cancer (EOC) ranks third in the incidence of gynecological malignancies. m6A methylation as RNA modification plays a crucial role in the evolution, migration, and invasion of various tumors. However, the role of m6A methylation in ovarian cancer (OC) only recently has begun to be appreciated. Therefore, we used various bioinformatic methods to screen the public GEO datasets of epithelial ovarian cancer (EOC) for m6A methylation-related regulators. We identified methyltransferase 16 (METTL16) that was dramatically downregulated in EOC as such a regulator. We also identified metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a known target lncRNA of METTL16, in these five GEO datasets. RT-qPCR and immunohistochemical staining confirmed that compared with the normal ovarian tissues and cells, METTL16 was significantly downregulated, while lncRNA MALAT1 was significantly upregulated, in 30 EOC tissues of our own validation cohorts and EOC cell lines, revealing a negative correlation between METTL16 and lncRNA MALAT1. Moreover, our analysis unveiled a correlation between downregulated METTL16 and the known adverse prognostic factors of EOC patients in our own cohorts. The CCK-8, EdU, scratch wound healing, and transwell invasion assays revealed that METTL16 significantly suppressed the proliferating, migrating, and invading abilities of OC cells. The inhibitory effects of METTL16 on the in vivo tumor growth of EOC cells were measured by subcutaneous tumor formation assay in mice. Furthermore, the RIP, RNA stability assay, western blotting, and cytoimmunofluorescence staining showed that METTL16 hindered the growth of EOC cells through promoting the degradation of MALAT1 by binding that, in turn, upregulates β-catenin protein and promotes nuclear transport of β-catenin protein in EOC cells. This study suggests that METTL16 acts as a tumor suppressor gene of EOC by achieving its inhibitory function on the malignant progression of EOC through the METTL16/MALAT1/β-catenin axis that are new targets for EOC diagnosis and therapy.

Background: Cholesterol-rich low-density lipoprotein (LDL) particles have been demonstrated to regulate breast cancer cell proliferation and migration, but their biological function and relevant mechanisms in endometrial carcinoma (EC) remain unclear.

Methods: Serum and tissue samples were collected from EC patients (n = 50) and patients with benign endometrial hyperplasia (n = 50). Ishikawa and RL95-2 cells were stimulated with different concentrations of LDL, followed by treatment with a JAK2 inhibitor (SD-1029). LDL concentrations were determined by ELISA. The in vitro biological behavior of cells was examined using the CCK-8 assay, EdU staining, and Transwell assay. The tumorigenicity of LDL in vivo was examined using a xenograft mouse model. western blotting, immunofluorescence, and immunohistochemistry studies were performed to measure related protein expression.

Results: The LDL concentrations and levels of p-JAK2 and p-STAT3 expression were elevated in the clinical samples. Similar trends in expression were detected in EC cells after LDL stimulation. LDL treatment significantly promoted EC cell proliferation, migration, and invasion, and also upregulated p-JAK2 and p-STAT3 expression in a dose-dependent manner. Moreover, SD-1029 dramatically blocked the LDL-mediated effects on EC cells. Intravenous injection of LDLs promoted tumor growth in the xenograft nude mice, and also increased p-JAK2, p-STAT3, and Ki-67 expression, and downregulated caspase-3 expression.

Conclusions: These findings indicate that LDLs exert an oncogenic effect in EC cells by activating the JAK/STAT signaling pathway, and also suggest the JAK/STAT pathway as a possible therapeutic target for EC.

Background: Ovarian cancer (OC) is the leading cause of gynecological cancer death and the fifth most common cause of cancer-related death in women in America. Programmed cell death played a vital role in tumor progression and immunotherapy response in cancer.

Methods: The prognostic cell death signature (CDS) was constructed with an integrative machine learning procedure, including 10 methods, using TCGA, GSE14764, GSE26193, GSE26712, GSE63885, and GSE140082 datasets. Several methods and single-cell analysis were used to explore the correlation between CDS and the ecosystem and therapy response of OC patients.

Results: The prognostic CDS constructed by the combination of StepCox (n = both) + Enet (alpha = 0.2) acted as an independent risk factor for the overall survival (OS) of OC patients and showed stable and powerful performance in predicting the OS rate of OC patients. Compared with tumor grade, clinical stage, and many developed signatures, the CDS had a higher C-index. OC patients with low CDS score had a higher level of CD8+ cytotoxic T, B cell, and M1-like macrophage, representing a related immunoactivated ecosystem. A low CDS score indicated a higher PD1 and CTLA4 immunophenoscore, higher tumor mutation burden score, lower tumor immune dysfunction and exclusion score, and lower tumor escape score in OC, demonstrating a better immunotherapy response. OC patients with high CDS score had a higher gene set score of cancer-related hallmarks, including angiogenesis, epithelial-mesenchymal transition, hypoxia, glycolysis, and notch signaling.

Conclusion: The current study constructed a novel CDS for OC, which could serve as an indicator for predicting the prognosis, ecosystem, and immunotherapy benefits of OC patients.

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.