Folia histochemica et cytobiologica最新文献

Introduction: Abnormal proliferation of vascular smooth muscle cells (VSMCs) can cause various vascular diseases, such as atherosclerosis, restenosis, and pulmonary hypertension. However, the effect and underlying mechanism of Wnt5a on the proliferation of VSMCs remain unclear. Our study aimed to investigate whether Wnt5a/Ror2 promotes vascular smooth muscle cell proliferation via activating protein kinase C (PKC), thereby effectively alleviating vascular proliferative diseases.

Material and methods: The proliferation of HA-VSMC cell line was evaluated by CCK-8, EdU, and Plate clone formation assays. The Wnt5a gene knockdown and overexpression were carried out by standard methods. The interaction between Wnt5a and Ror2 was explored by co-immunoprecipitation. Western blotting and immunofluorescence were used to determine the expression levels of key proteins in VSMCs.

Results: The present study found that the expression of Wnt5a protein increased significantly in the proliferation of VSMCs stimulated by 10% serum in a time-dependent manner. Furthermore, the proliferative rate of VSMCs overexpressing Wnt5a was dramatically accelerated, whereas Wnt5a knockdown using siWnt5a reversed thisproliferative effect. Wnt5a up-regulated the expression of receptor tyrosine kinase-like orphan receptor 2 (Ror2) by binding to it. Further studies indicated that Wnt5a induces the PKC expression in VSMCs and knockdown of Wnt5a or Ror2 could inhibit PKC phosphorylation.

Conclusions: Wnt5a could effectively promote the proliferation of VSMCs, which might be related to the binding of Wnt5a and Ror2 to activate PKC.

Introduction: Apoptosis is a key process during normal trophoblastic development and, consequently, the whole gestation. However, in trophoblastic differentiation in spontaneous abortions apoptosis has been hardly investigated. Therefore, the aim of the study was to investigate the correlation between apoptotic frequency in trophoblast and spontaneous abortion incidences.

Material and methods: A total of 72 trophoblastic tissue samples were immunohistochemically examined. 42 of 72 derived from first-trimester spontaneous abortions and the remaining 30 from elective terminations during the same trimester of pregnancy. TUNEL assay and M30 marker were used for apoptosis evaluation by immunohistochemistry.

Results: Comparative study of tissues from spontaneous abortions and elective pregnancy terminations demonstrated increased expression of both apoptotic markers in tissues derived from spontaneous abortions compared to normal pregnancies. In addition, statistical analysis correlated maternal age and gravidity with increased spontaneous abortion incidences. Moreover, both M30 and TUNEL staining were significantly correlated with maternal age and primigravidity in spontaneous abortion cases.

Conclusions: Our data proved that elevated apoptotic activity during the first pregnancy trimester is clearly involved in spontaneous abortions. Moreover, two well-established apoptotic markers revealed high statistical significance in the evaluation of post-abortive tissues.

Introduction: Suppressing the phenotype of cancer stem cells (CSCs) is a promising treatment strategy for cancer. P38 mitogen-activated protein kinases (MAPK, p38) play an important role in the occurrence, development, and stemness maintenance of tumors. The aim of the current study was to investigate the effect of p38 on the stemness maintenance of CSCs in pancreatic cancer cell line PANC-1.

Material and methods: PANC-1 human pancreatic cancer cells were treated with 5-fluorouracil (5-FU) at 0.5 IC50, IC50, and 2 IC50 for 24 h. PANC-1 cells were treated for 24 h with 5-FU at 0.5IC50, IC50, and 2IC50 with or without VX-702, p38 phosphorylation inhibitor. Cells were resuspended in DMEM supplemented with 20 ng/ml epidermal growth factor, 2% B27, 5 mg/ml insulin, 20 g/ml basic fibroblast growth factor, and 10 μg/ml transferrin. Cells were seeded in ultra-low adhesion 6-well dishes to observe tumor spheroidization. The expression of CDK2, cyclin B1, cyclin D1, OCT4, SOX2, Nanog, and p38 was measured by Western blot. The mRNA expression of p38, OCT4, Nanog, and SOX2 was measured by RT-PCR. Flow cytometry was performed to evaluate the cell cycle, apoptosis, and proportion of CD44+CD133+ PANC-1 cells.

Results: 5-FU decreased cell viability and increased apoptosis. 5-FU suppressed the stemness maintenance of CSCs in PANC-1 cells, as demonstrated by the inhibition of tumorsphere formation, the decrease in CD44+CD133+ cells' fraction, and downregulation of OCT4, Nanog, and SOX2 expression. In addition, 5-FU inhibited the phosphorylation of p38 in PANC-1 cells. The phosphorylation of p38 was subsequently suppressed by VX-702, p38 mitogen-activated protein kinase inhibitor, which exhibited similar effects as those of 5-FU treatment. The effect of VX-702 on PANC-1 cells was further enhanced by 5-FU treatment. Thus, p38 inhibitor decreased the viability and increased the apoptosis of PANC-1 cells. P38 inhibitor suppressed the stemness maintenance of CSCs in PANC-1 cells, as demonstrated by the inhibition of tumorsphere formation, the decrease in CD44+CD133+ cells, and the downregulation of OCT4, Nanog, and SOX2 expression.

Conclusions: These findings indicate that the inhibition of p38 phosphorylation suppresses the stemness maintenance and 5-FU resistance of PANC-1 cells, providing a potential therapeutic target for the prevention and treatment of pancreatic cancer.

Introduction: Abnormal ovarian angiogenesis is a common feature of polycystic ovary syndrome (PCOS), a typical endocrine disorder affecting women of reproductive age. Histone deacetylase 5 (HDAC5) has been documented as a suppressor of angiogenesis. The aim of this study was to explore the effect of HDAC5 on ovarian angiogenesis in a PCOS mouse model.

Material and methods: PCOS was induced in female C57BL/6 mice by 20-day administration of dehydroepiandrosterone (DHEA). HDAC5 was over-expressed in PCOS mice by corresponding adenovirus injection. In total, 120 mice were used in this study. Western-blotting, real-time PCR, hematoxylin and eosin staining, enzyme-linked immunosorbent assay (ELISA), immunohistochemical staining, flow cytometry, and co-immunoprecipitation were respectively used to evaluate the effect of HDAC5 on PCOS mice.

Results: PCOS ovaries showed a compensatory increase in HDAC5 expression, while HDAC5 over-expression alleviated abnormalities in ovarian morphology and serum hormone levels after PCOS modeling. HDAC5 inhibited ovarian angiogenesis in PCOS mice by regulating angiogenesis-related factors, such as VEGFA, platelet-derived growth factors B and D (PDGFB/D), and angiopoietins 1 and 2 (ANGPT1/2) and CD31. HDAC5 over-expression decreased levels of reactive oxygen species (ROS) and malondialdehyde, while promoting activities of catalase and superoxide dismutase in ovaries of PCOS mice, suggesting its suppressive effects on oxidative stress, an inducer of uncontrolled angiogenesis. Moreover, HDAC5 suppressed activation of angiogenesis-related HIF-1α/VEGFA/VEGFR2 signaling in PCOS ovaries partly via inhibiting VEGFR2 acetylation.

Conclusions: This study reveals the protective role of HDAC5 in PCOS by inhibiting ovarian angiogenesis and provides a molecular candidate for PCOS therapy in the future.

Introduction: Evidence has shown that some microRNAs (miRNAs) play a role in tumorigenesis of hepatocellular carcinoma (HCC). Herein, we aimed to evaluate the diagnostic and prognostic values of serum exosomal miR-370-3p and miR-196a-5p in patients with HCC.

Material and methods: Serum exosomes in 90 HCC patients were extracted and identified. Serum exosomal miR-370-3p and miR-196a-5p expression in HCC patients were detected. The diagnostic value of miR-370-3p and miR-196a- 5p, relationship between miR-370-3p and miR-196a-5p expression and clinicopathological features and prognosis of patients with HCC were analyzed. Relationship between miR-370-3p and miR-196a-5p expression and liver function indices such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) in HCC patients were analyzed. The effects of miR-370-3p and miR-196a-5p on Huh-7 HCC cells' proliferation, invasion and migration were determined.

Results: Lower expression of miR-370-3p and higher expression of miR-196a-5p were found in serum exosomes of HCC patients. Serum exosomal miR-370-3p and miR-196a-5p were associated with tumor size, tumor grade and TNM stage as well as prognosis and liver function indices of HCC patients. Overexpressed miR-370-3p or silenced miR-196a-5p suppressed proliferation, invasion and migration of Huh-7 HCC cells.

Conclusions: We suggest that miR-370-3p/miR-196a-5p in serum exosomes of HCC patients could be potential biomarkers for the diagnosis and prognosis of HCC.

Introduction: Breast cancer has been represented a challenging issue worldwide as it is one of the major leading causes of death among women. CD81 gene, a member of the tetraspanin protein family, has been associated with the development of human cancers. Genome editing technologies, particularly the CRISPR-Cas9 system, have shown rapid progress in gene function studies. In this study, we aimed to evaluate the ability of the CRISPR-Cas9 plasmid-based system to modify specific regions of the CD81 gene in the MDA-MB-231 breast cancer cell line.

Materials and methods: Using bioinformatics database search, four different single guide RNAs (sgRNAs) to target exon 3 and exon 5 of the CD81 gene were designed. The intended sgRNAs sequences were cloned into the expression plasmid pSpCas9(BB)-2A-GFP (PX458) bearing sgRNA scaffold backbone, Cas9, and EGFP coding sequences, which was confirmed by colony PCR and sequencing. Transfection efficiency was determined by fluorescence microscopy and flow cytometry analysis. Gene editing efficiency was measured qualitatively and quantitatively using the T7E1 and TIDE software, respectively.

Results: Our data show that expression constructs were successfully introduced into MDA-MB-231 cells with an acceptable transfection efficiency. Two sgRNAs that were afforded to introduce significant mutations in their target regions were detected by TIDE software (p-value < 0.05). To the best of our knowledge, CD81 gene editing in these cells has been investigated for the first time in this study using the CRISPR/Cas9 technique.

Conclusions: Taken together, our data show that the CRISPR-Cas9 system can change the genomic sequence in the target area of MDA-MB-231 cells. Along with previous studies, we propose forethought when using T7E1-based quantitative indel estimates, as comparing activities of multiple gRNAs with the T7E1 assay may lead to inaccurate conclusions. Instead, estimating non-homologous end-joining events (NHEJ) by Sanger sequencing and subsequent TIDE analysis is recommended.

Introduction: As one of the basic components of Astragalus, Astragaloside IV (AS-IV) has a protective effect on endothelial injury caused by diabetes. AS-IV stimulated endothelial progenitor cells (EPCs) to secrete exosomes loaded with miR-21. This study aimed to investigate the effects of AS-IV-mediated EPCs exosomal miR-21 (EPC-exos-miR-21) on high glucose (HG) damaged endothelial cells.

Materials and methods: After the isolation of EPCs derived from fetal umbilical cord blood, exosomes of EPCs were obtained by differential centrifugation. The morphology of exosomes was observed by electron microscopy. The particle size distribution of exosomes was detected by Nanoparticle Tracking Analysis. Human umbilical vein endothelial cells (HUVECs) were treated with 33 mM glucose to establish an HG injury model. Flow cytometry and TUNEL assay were used to characterize the surface markers of primary EPCs and the apoptosis of HUVECs. The gene and protein expression were detected by qPCR, immunofluorescence, and Western blotting. A dual luciferase assay was used to verify the targeting relationship of miR-21 with PTEN.

Results: HG environment led to time- and dose-dependent inhibition and enhancement of autophagy and apoptosis in HUVECs. AS-IV stimulated EPCs to secrete exosomes loaded with miR-21. Exosomes secreted by EPCs pretreated with AS-IV [EPC-exo(ASIV)] promoted autophagy and inhibited apoptosis in HG-impaired HUVECs. PTEN is a target of miR-21. MiR-21 carried by EPC-exo(ASIV) repressed PTEN expression in HG-impaired HUVECs. In contrast, p-AKT, p-mTOR, p-PI3K, cleaved PARP and PARP levels were upregulated. Compared to the HG group, the expression of autophagy regulatory genes (ATG5, beclin1 and LC3) was enhanced in the EPC-exo(ASIV) group and EPC-exo(ASIV)-miR-21 mimic group. In contrast, apoptosis-positive regulatory genes (Bax, caspase-3 and caspase-9) were attenuated. Further overexpression of PTEN reversed the expression of these genes.

Conclusions: AS-IV-mediated EPC-exos-miR-21 could enhance autophagy and depress apoptosis in HG-damaged endothelial cells via the miR-21/PTEN axis.

Introduction: Aberrant fucosylation is closely related to malignant transformation, cancer detection, and evaluation of treatment efficacy. The fucosylation process requires GDP-L-fucose, fucosyltransferases, and fucosidases. In gastric cancer (GC), fucosylation alterations were associated with tumor formation, metastasis inhibition, and multi-drug resistance. It is not clear whether tissue-specific transplantation antigen P35B (TSTA3) and alpha-L-fucosidase 2 (FUCA2) have any effect on the development of GC.

Materials and methods: We used immunohistochemistry to assess the expression of TSTA3 and FUCA2 in 71 gastric adenocarcinoma samples and their relationship with clinicopathological parameters.

Results: TSTA3 expression was associated with lower histological grade I and II (P = 0.0120) and intestinal type Lauren classification (P = 0.0120). TSTA3 immunopositivity could predict Lauren's classification. Analysis of mRNA expression in GC validation cohorts corroborates the significant TSTA3 association with histological grade observed in our study. However, no associations were found between TSTA3 staining and overall survival. FUCA2 expression was markedly increased in GC tissues compared with non-tumoral tissues (P < 0.0001) and was associated with surgical staging III and IV (P = 0.0417) and advanced histological grade tumor states (P = 0.0125).

Conclusions: Alterations of FUCA2 and TSAT3 immunoexpression could lay the basis for future studies using cell glycosylation as a biomarker for the planning of therapeutic strategy in primary gastric cancer.