Experimental and molecular pathology最新文献

Introduction

Irisin plays an important role in regulating tissue stress, cardiac function, and inflammation. Integrin αvβ5 was recently identified as a receptor for irisin to elicit its physiologic function. It remains unknown whether integrin αvβ5 is required for irisin's function in modulating the physiologic response to hemorrhage. The objective of this study is to examine if integrin αvβ5 contributes to the effects of irisin during the hemorrhagic response.

Methods

Hemorrhage was induced in mice by achieving a mean arterial blood pressure of 35–45 mmHg for one hour, followed by two hours of resuscitation. Irisin (0.5  μg/kg) was administrated to assess its pharmacologic effects in hemorrhage. Cilengitide, a cyclic Arg-Gly-Asp peptide (cRGDyK) which is an inhibitor of integrin αvβ5, or control RGDS (1 mg/kg) was administered with irisin. In another cohort of mice, the irisin-induced protective effect was examined after knocking down integrin β5 with nanoparticle delivery of integrin β5 sgRNA using CRSIPR/Cas-9 gene editing. Cardiac function and hemodynamics were measured using echocardiography and femoral artery catheterization, respectively. Systemic cytokine releases were measured using Enzyme-linked immunosorbent assay (ELISA). Histological analyses were used to determine tissue damage in myocardium, skeletal muscles, and lung tissues. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was carried out to assess apoptosis in tissues.

Results

Hemorrhage induced reduction of integrin αvβ5 in skeletal muscles and repressed recovery of cardiac performance and hemodynamics. Irisin treatment led to significantly improved cardiac function, which was abrogated by treatment with Cilengitide or knockdown of integrin β5. Furthermore, irisin resulted in a marked suppression of tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1), muscle edema, and inflammatory cells infiltration in myocardium and skeletal muscles, which was attenuated by Cilengitide or knockdown of integrin β5. Irisin-induced reduction of apoptosis in the myocardium, skeletal muscles, and lung, which were attenuated by either the inhibition of integrin αvβ5, or knockdown of integrin β5.

Conclusion

Integrin αvβ5 plays an important role for irisin in modulating the protective effect during hemorrhage.

Bisphenol A (BPA) is an endocrine-disrupting chemical with a potential role in endocrine cancers. However, the effects of BPA on the salivary glands have been barely explored. We investigated the impact of in vivo sub-chronic exposure to BPA and its in vitro effects on human salivary gland mucoepidermoid carcinoma cell lines. Male and female mice were exposed to BPA (30 mg/kg/day). Sublingual and submandibular salivary glands from an estrogen-deficiency model were also analyzed. BPA concentration in salivary glands was evaluated by gas chromatography coupled to ion trap mass spectrometry. Immunohistochemical analysis using anti-p63 and anti-α-SMA antibodies was performed on mouse salivary gland tissues. Gene expression of estrogen receptors alpha and beta, P63 and α-SMA was quantified in mouse salivary gland and/or mucoepidermoid (UM-HMC-1 and UM-HMC-3A) cell lines. Cell viability, p63 and Ki-67 immunostaining were evaluated in vitro. BPA disrupted the tissue architecture of the submandibular and sublingual glands, particularly in female mice, and increased the expression of estrogen receptors and p63, effects that were accompanied by significant BPA accumulation in these tissues. Conversely, ovariectomy slightly impacted BPA-induced morphological changes. In vitro, BPA did not affect the proliferation of neoplastic cells, but augmented the expression of p63 and estrogen receptors. The present data highlight a potential harmful effect of BPA on salivary gland tissues, particularly in female mice, and salivary gland tumor cells. Our findings suggest that estrogen-dependent pathways may orchestrate the effects of BPA in salivary glands.

Homeobox (HOX) genes encode highly conserved transcription factors that play vital roles in embryonic development. DNA methylation is a pivotal regulatory epigenetic signaling mark responsible for regulating gene expression. Abnormal DNA methylation is largely associated with the aberrant expression of HOX genes, which is implicated in a broad range of human diseases, including cancer. Numerous studies have clarified the mechanisms of DNA methylation in both physiological and pathological processes. In this review, we focus on how DNA methylation regulates HOX genes and briefly discuss drug development approaches targeting these mechanisms.

In recent years, the physiological and molecular functions of vitamin D (Vit-D) have been deeply investigated. At first, Vit-D was considered a regulator of mineral and skeletal homeostasis. However, due to the extensive-expression pattern of Vit-D receptor (VDR) in almost every non-skeletal cell, Vit-D is considered mainly a multifunctional agent with broad effects on various tissues, notably the immune system. The expression of VDR in immune cells such as dendritic cells, monocyte/macrophage, neutrophils, B cells and T cells has been well demonstrated. Besides, such immune cells are capable of metabolizing the active form of Vit-D which means that it can module the immune system in both paracrine and autocrine manners. Vit-D binding protein (DBP), that regulates the levels and homeostasis of Vit-D, is another key molecule capable of modulating the immune system. Recent studies indicate that dysregulation of Vit-D axis, variations in the DBP and VDR genes, and Vit-D levels might be risk factors for the development of autoimmune disease. Here, the current evidence regarding the role of Vit-D axis on the immune system, as well as its role in the development of autoimmune disease will be clarified. Further insight will be given to those studies that investigated the association between single nucleotide polymorphisms of DBP and VDR genes with autoimmune disease susceptibility.

Mast cells (MCs) are tissue-resident innate immune cells that express the high-affinity receptor for immunoglobulin E and are responsible for host defense and an array of diseases related to immune system. We aimed in this study to characterize the pathways and gene signatures of human cord blood-derived MCs (hCBMCs) in comparison to cells originating from CD34⁻ progenitors using next-generation knowledge discovery methods. CD34⁺ cells were isolated from human umbilical cord blood using magnetic activated cell sorting and differentiated into MCs with rhIL-6 and rhSCF supplementation for 6–8 weeks. The purity of hCBMCs was analyzed by flow cytometry exhibiting the surface markers CD117⁺CD34⁻CD45⁻CD23⁻FcεR1α^dim. Total RNA from hCBMCs and CD34⁻ cells were isolated and hybridized using microarray. Differentially expressed genes were analyzed using iPathway Guide and Pre-Ranked Gene Set Enrichment Analysis. Next-generation knowledge discovery platforms revealed MC-specific gene signatures and molecular pathways enriched in hCBMCs and pertain the immunological response repertoire.

Assessing the radiosensitivity of cells before administering radiation therapy (RT) to individuals diagnosed with breast cancer (BC) can facilitate the selection of appropriate treatment regimens and minimize the incidence of adverse side effects in patients undergoing radiation exposure.

In this research, blood samples were obtained from 60 women who had been diagnosed with Invasive Ductal Carcinoma (IDC) Breast Cancer. The average age of the patients was 47 ± 9.93. Additionally, the study incorporated 20 healthy women, with an average age of 44.43 ± 6.7. A standard G2 assay was conducted to predict the cellular response to radiation. Out of the 60 samples, the G2 assay identified 20 patients with breast cancer who exhibited radiosensitivity. Hence, molecular investigations were ultimately conducted on two equivalent cohorts comprising 20 subjects each, one with and the other without cellular radiosensitivity. The expression levels of miR-149, miR-25, circ-PVT1, and circ-HIPK3 in peripheral blood mononuclear cells (PBMCs) were evaluated using quantitative polymerase chain reaction (qPCR). Receiver Operating Characteristic (ROC) curves were used to evaluate the sensitivity and specificity of the RNAs. An analysis using binary logistic regression was performed to investigate the relationship between RNAs and both BC and cellular radiosensitivity (CR) in patients with BC.

The findings revealed a significant upregulation of Circ-HIPK3 and circ-PVT1 in individuals diagnosed with BC. The levels of Circ-HIPK3 and Circ-PVT1 were found to be directly associated with CR in BC patients. The analysis of the ROC curve demonstrated that circ-HIPK3 and circ-PVT1 exhibit favorable specificity and sensitivity in accurately predicting both BC and CR in patients with BC. The findings from the binary logistic regression analysis demonstrated that circ-HIPK3 and circ-PVT1 were effective predictors of both BC and CR. The ROC curve and binary logistic regression analyses provide evidence that miR-25 is a reliable predictor for BC patients exclusively.

Our research has demonstrated that circ-HIPK3, circ-PVT1, and miR-25 may be involved in BC regulatory processes. The circular RNAs Circ-HIPK3 and circ-PVT1, as well as miR-25, among other significant biomarkers, could potentially serve as promising biomarkers for predicting BC. Furthermore, Circ-HIPK3 and circ-PVT1 have the potential to serve as biomarkers for predicting CR in BC patients.

Circular RNAs are covalently closed and non-coding in eukaryotes, which have tissue- specific and temporal-specific expression patterns whose biogenesis is regulated by transcription and splicing. Most circular RNAs are localized in the cytoplasm. The sequences and protein-binding elements of circular RNAs facilitate circular RNAs in exerting biological functions through complementary base pairing, regulating protein function or self-translation. Recent studies have revealed that N6-Methyladenosine (m6A), a prevalent post-transcriptional modification, can affect the translation, localization, and degradation of circular RNAs. Cutting-edge research into circular RNAs have benefitted from the development of high-throughput sequencing technology. Furthermore, the expansion of novel research methods has promoted progress into circular RNA research.

Cytokeratin 20 (CK20) expression is limited to umbrella cells in the normal urothelium. Since CK20 is often upregulated in neoplastic urothelial cells including dysplasia and carcinoma in situ, immunohistochemical CK20 analysis is often used for the assessment of bladder biopsies. CK20 expression is a feature of luminal bladder cancer subtype, but its prognostic relevance is disputed. In this study, we investigated CK20 on >2700 urothelial bladder carcinomas in a tissue microarray format by immunohistochemistry. Cytoplasmic and membranous CK20 staining was seen in 1319 (51.8%) cancers. The fraction of CK20 positive and especially strongly positive cases increased from pTaG2 low grade (44.5% strongly positive) and pTaG2 high grade (57.7%) to pTaG3 high grade (62.3%; p = 0.0006) but was lower in muscle-invasive (pT2–4) carcinomas (51.1% in all pTa vs. 29.6% in pT2–4; p < 0.0001). Within pT2–4 carcinomas, CK20 positivity was linked to nodal metastasis and lymphatic vessel invasion (p < 0.0001 each) and to venous invasion (p = 0.0177). CK20 staining was unrelated to overall patient survival if all 605 pT2–4 carcinomas were jointly analyzed but subgroup analyses revealed a significant association of CK20 positivity with favorable prognosis in 129 pT4 carcinomas (p = 0.0005). CK20 positivity was strongly linked to the expression of GATA3 (p < 0.0001), another feature of luminal bladder cancer. The combined analysis of both parameters showed best prognosis for luminal A (CK20+/GATA3+, CK20+/GATA3-) and worst outcome for luminal B (CK20−/GATA3+) and basal/squamous (CK20−/GATA3-) in pT4 urothelial carcinomas (p = 0.0005). In summary, the results of our study demonstrate a complex role of CK20 expression in urothelial neoplasms including neoexpression in pTa tumors, a subsequent loss of CK20 expression in a subset of tumors progressing to muscle-invasion, and a stage dependent prognostic role in muscle-invasive cancers.

Background

The mRNA splicing is regulated on multiple levels, resulting in the proper distribution of genes' transcripts in each cell and maintaining cell homeostasis. At the same time, the expression of alternative transcripts can change in response to underlying genetic variants, often missed during routine diagnostics.

Aim

The main aim of this study was to define the frequency of aberrant splicing in BRCA1 and BRCA2 genes in blood RNA extracted from ovarian cancer patients who were previously found negative for the presence of pathogenic alterations in the 25 most commonly analysed ovarian cancer genes, including BRCA1 and BRCA2.

Material and methods

Frequency and spectrum of splicing alterations in BRCA1 and BRCA2 genes were analysed in blood RNA from 101 ovarian cancer patients and healthy controls (80 healthy women) using PCR followed by gel electrophoresis and Sanger sequencing. The expression of splicing events was examined using RT-qPCR.

Results

We did not identify any novel, potentially pathogenic splicing alterations. Nevertheless, we detected six naturally occurring transcripts, named BRCA1ΔE9-10, BRCA1ΔE11, BRCA1ΔE11q, and BRCA2ΔE3, BRCA2ΔE12 and BRCA2ΔE17-18 of which three (BRCA1ΔE11q, BRCA1ΔE11 and BRCA2ΔE3) were significantly higher expressed in the ovarian cancer cohort than in healthy controls (p ≤ 0.0001).

Conclusions

This observation indicates that the upregulation of selected naturally occurring transcripts can be stimulated by non-genetic mechanisms and be a potential systemic response to disease progression and/or treatment. However, this hypothesis requires further examination.

Detecting MLH1 promoter methylation is highly relevant to differentiate between possible Lynch syndrome patients or patients with sporadic causes of MLH1/PMS2 deficiency in colorectal (CRC) and endometrial cancers. Here, we aimed to develop a test for assessing MLH1 promoter methylation based in next generation sequencing (NGS), and to evaluate the concordance of MLH1 methylation and BRAF-V600 mutation status in CRC. For that, we performed a series of experiments with DNA from tumor, saliva and commercial control samples and our in house developed amplicon-based NGS test. In patients' samples, MLH1 methylation above 10% was only observed in tumors with MLH1/PMS2 loss. We confirmed the reproducibility and accuracy of MLH1 promoter analysis performing a serial dilution experiment with completely methylated and unmethylated control DNAs and a comparison between two NGS platforms (Ion Proton and Illumina). In MLH1/PMS2 deficient tumors, the MLH1 methylation status was concordant with the BRAF mutation status in 90% (18/20) of the cases. Our amplicon-based NGS test showed a great sensitivity and specificity for detecting MLH1 methylation in CRC samples, with a high agreement with the evaluation of BRAF mutation. This simple and affordable test could be used as a reflex test to identify patients with sporadic causes of MLH1/PMS2 deficiency in CRC, aiding to genetic test referral and identification of Lynch syndrome patients.