Histology and histopathology Vol.37, nº6 (2022)
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- PublicationOpen AccessComparison of manual and automated digital image analysis systems for quantification of cellular protein expression(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2022) Jagomast, T.; Idel, C.; Klapper, L.; Kuppler, P.; Proppe, L.; Beume, S.; Falougy, M.; Steller, D.; Hakim, S.G.; Offermann, A.; Roesch, M.C.; Bruchhage, K.L.; Perner, S.; Ribbat Idel, J.Objective. Quantifying protein expression in immunohistochemically stained histological slides is an important tool for oncologic research. The use of computer-aided evaluation of IHC-stained slides significantly contributes to objectify measurements. Manual digital image analysis (mDIA) requires a userdependent annotation of the region of interest (ROI). Others have built-in machine learning algorithms with automated digital image analysis (aDIA) and can detect the ROIs automatically. We aimed to investigate the agreement between the results obtained by aDIA and those derived from mDIA systems. Methods. We quantified chromogenic intensity (CI) and calculated the positive index (PI) in cohorts of tissue microarrays (TMA) using mDIA and aDIA. To consider the different distributions of staining within cellular subcompartments and different tumor architecture our study encompassed nuclear and cytoplasmatic stainings in adenocarcinomas and squamous cell carcinomas. Results. Within all cohorts, we were able to show a high correlation between mDIA and aDIA for the CI (p<0.001) along with high agreement for the PI. Moreover, we were able to show that the cell detections of the programs were comparable as well and both proved to be reliable when compared to manual counting. Conclusion. mDIA and aDIA show a high correlation in acquired IHC data. Both proved to be suitable to stratify patients for evaluation with clinical data. As both produce the same level of information, aDIA might be preferable as it is time-saving, can easily be reproduced, and enables regular and efficient output in large studies in a reasonable time period.
- PublicationOpen AccessMaternal exposure to the environmental pollutant "BDE-47" impairs the postnatal development of rat cerebellar cortex by modulating neuronal proliferation, synaptogenesis, NGF and BDNF pathways(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2022) Mandour, Dalia A.; Tolba, Asmaa M; El Bestawy, Emtethal M.. 2,2’,4,4’-Tetrabromodiphenyl ether (BDE47) is an environmental contaminant that crosses the blood placental barrier and interferes with the homeostasis of fetal thyroid hormones. Aim of work. This study was designed to investigate the perinatal effect of BDE-47 exposure on the postnatal development of the rat cerebellar cortex. Materials and methods. This study was carried out on 20 pregnant rats and 36 of their offspring. The pregnant rats were divided equally into control and BDE-47 treated mother groups; supplemented orally with BDE-47 (0.2 mg/kg/day from day 8 of gestation until the day of weaning). The offspring of both mother groups were subdivided, according to their developmental age, into three subgroups; PND14, PND21and PND42. SerumT3, T4 and TSH were assessed for dams and their offspring. Testing the motor coordination of the offspring via the rotarod test was conducted. Sections of the cerebellar cortex from offspring subgroups were stained with hematoxylin and eosin alongside immunohistochemical reactions and optical density of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), proliferating cell nuclear antigen (PCNA) and synaptophysin (SYN) were assessed. Also, the thickness of different layers of the cerebellar cortex was histomorphometrically measured. Results. BDE-47 treated mothers and their offspring subgroups showed a significant decrease in the serum free T3, T4 and increased TSH. The BDE-47 offspring displayed incoordination of the motor activity together with disturbed cytoarchitecture of the cerebellar cortical layers, and impaired migration of its germinative neuronal zones, particularly on PND14 and PND21. Moreover, these offspring displayed a decrease of the immune-expression and optical density of NGF, BDNF in the cerebellar cortical layers with impaired proliferation, and synaptogenesis. Conclusion. Maternal exposure to BDE-47 during pregnancy and lactation effectuated a potential deleterious retarding effect on the postnatal development of the rat cerebellar cortex mostly via modulating neuronal proliferation, synaptogenesis, NGF and BDNF pathways secondary to its hypothyroid effect.
- PublicationOpen AccessHistory and development of staining methods for skeletal muscle fiber types(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2022) Sawano, Shoko; Mizunoya, WataruThe contractile and metabolic properties of skeletal muscles depend on the composition of muscle fibers. There are two major fiber types: type 1 and type 2. Type 2 fibers are further subdivided into type 2A, 2X, and 2B fibers. Muscle fiber type composition is an important property that affects sports performance and metabolic ability in humans, and meat quality in domestic animals. In this review, we summarize the history of muscle fiber type classification based on various staining methods for skeletal muscle sections. The history illustrates the development of an experimental method to detect myosin heavy chain (MyHC) proteins, which are the most common marker molecules for muscle fiber type. Metabolic enzymes, such as nicotinamide adenine dinucleotide-tetrazolium reductase and succinate dehydrogenase are also described for histochemical staining combined with myosin ATPase staining. We found an improvement in the quality of antibodies used for immunostaining of MyHC, from polyclonal antibodies to monoclonal antibodies (mAbs) and then to mAbs produced by synthetic peptides as antigens. We believe that the information presented herein will assist researchers in selecting optimal staining methods, dependent on the experimental conditions and purposes
- PublicationOpen AccessLncRNA NR2F2-AS1 functions as a tumor suppressor in gastric cancer through targeting miR-320b/PDCD4 pathway(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2022) Luo, Ming; Deng, Shuangya; Han, Tong; Ou, Yanglu; Hu, YongjunGastric cancer is among the most frequently occurring gastrointestinal malignancies with a high mortality rate worldwide. Long non-coding RNAs (lncRNAs) are defined as core regulators in the occurrence and progression of multiple cancers, including gastric carcinoma. Mounting evidence has indicated that NR2F2-AS1 can inhibit several malignant tumors. However, the function and potential mechanism of NR2F2-AS1 remain unclear. In the current study, we found that NR2F2-AS1 was weakly expressed in gastric cancer cells in comparison with normal cells. The study has further disclosed that ectopic of NR2F2-AS1 repressed cell proliferation, migration, invasion and EMT whereas it promoted cell apoptosis in gastric carcinoma. Subsequently, our results confirmed that miR-320b was negatively regulated and that suppression of miR-320b alleviated the malignant behaviors of GC cells. More importantly, PDCD4 was a target of miR320b. Mechanistically, NR2F2-AS1 modulated the expression level of PDCD4 by sponging miR-320b. Finally, rescue assays demonstrated that NR2F2-AS1 down-regulated PDCD4 expression to restrain the development of gastric cancer by competitively binding to miR-320b. On the whole, our study revealed the role of NR2F2-AS1/miR-320b/PDCD4 regulatory network in gastric cancer, suggesting NR2F2-AS1 may represent a novel therapeutic target for patients with gastric carcinoma.
- PublicationOpen AccessCancer stem cell markers CD44v9+/CD133- are associated with low apoptosis in both sporadic and ulcerative colitis-associated colorectal cancers(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2022) Nakagomi, Eriko; Mikami, Tetuo; Funahashi, Kimihiko; Okazumi, Shinichi; Shibuya, Kazutoshi; Hiruta, Nobuyuki; Igarashi, YoshinoriObjective. To elucidate tumor cell behavior associated with cancer stem cell (CSC) marker expression, the expression of CD133, CD44v9, and ALDH1A1, which are considered markers of CSCs, was examined in sporadic and ulcerative colitis (UC)- associated colorectal tumors. Methods. A total of 23 cases of sporadic colorectal cancer and 44 cases of adenoma were collected. Additionally, 22 cancer lesions and 38 dysplasia lesions were selected from 28 colectomy cases of UC with neoplastic lesions. Lesions were examined by immunohistochemistry using primary antibodies against CD133, CD44v9, ALDH1A1, Ki-67, cleaved-Caspase 3, and p53. Results. CD133, CD44v9, and ALDH1A1 showed higher expression in both sporadic and UC-associated tumors than in the normal mucosa. ALDH1A1 expression in sporadic cancer was higher in the right colon than in the left colon (p=0.0089). ALDH1A1 expression in UC-associated cancer was higher in those with longer disease duration than in those with shorter disease duration (p=0.019). The CD44v9+/CD133- region had fewer cleaved-Caspase 3 positive cells in both sporadic and UC-associated cancers. In sporadic cancer, CD133+/ALDH1A1+ regions had fewer apoptotic cells than CD133+/ALDH1A1- regions, while CD133+/ALDH1A1- regions were less proliferative than CD133+/ALDH1A1+ regions in UC-associated cancer. Conclusion. CD44+/CD133- regions were commonly associated with low apoptosis in sporadic and UC-associated cancers; thus, these were considered target areas for CSCs. Additionally, the combination of markers comprising CSCs may differ between sporadic and UC-associated cancers.
- PublicationOpen AccessOverexpression of lncRNA IRAIN restrains the progression and Temozolomide resistance of glioma via repressing IGF-1R-PI3K-NF-κB signaling pathway(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2022) Guo, Aishun; Fang, Guixia; Lin, Zhenrong; Zheng, Shuishun; Zhuang, Zhijun; Lin, Ruisheng; Lin, YanlingBackground. Increasing studies have found that long noncoding RNAs (lncRNAs) contribute to regulating tumor progression. This study explores the expression characteristics, effects, and related mechanisms of lncRNA IGF1R antisense imprinted nonprotein coding RNA (IRAIN) in glioma. Methods. Quantitative real-time PCR (qRT-PCR) was implemented to testify the IRAIN profile in glioma tissues and paracancerous tissues, and the link between the IRAIN level and the clinicopathological indicators of glioma was analyzed. IRAIN overexpression and knockdown cell models were constructed in glioma cells. Cell proliferation was verified by the colony formation experiment, while flow cytometry was implemented to monitor apoptosis. Transwell assay was performed to examine cell invasion and migration. Western blot (WB) was adopted to compare the profiles of the apoptosis-related proteins (Bax, Bcl2, and Caspase3) and IGF-1R-PI3K-NF-κB pathway. Results. IRAIN was down-regulated in glioma tissues (compared with adjacent normal tissues), and the low IRAIN expression was significantly linked with the larger tumor volume and higher pathological stages. Functionally, overexpressing IRAIN abated glioma cell proliferation, invasion, and migration, promoted apoptosis, and attenuated IGF-1R-PI3K-NF-κB expression and temozolomide (TMZ) resistance, which was also confirmed in the xenograft tumor experiment. The WB result showed that overexpressing IRAIN inactivated the IGF-1R-PI3K-NF-κB pathway. Additionally, the IGF-1R knockdown model was established in U251 cells. Si-IGF-1R induced cell proliferation inhibition, promoted cell death, and reduced cell migration and TMZ resistance, whereas SiIGF-1R+IRAIN group showed no additional effects on glioma cells compared with the Si-IGF-1R group. Conclusion. IRAIN repressed glioma development and TMZ resistance by inactivating the IGF-1R-PI3KNF-κB axis.
- PublicationOpen AccessCirc_0044520 regulates the progression of laryngeal squamous cell carcinoma via the miR-338-3p/ROR2 axis(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2022) Yang, Huijun; Yu, Gang; Wang, Yan; Guo, XingBackground. Laryngeal squamous cell carcinoma (LSCC) is the second most common malignant tumor in head and neck. Circular RNA_0044520 (circ_0044520) expression is increased in LSCC. However, the molecular mechanism of circ_0044520 remains unknown. Methods. The expression of circ_0044520, microRNA-338-3p (miR-338-3p) and receptor tyrosine kinase-like orphan receptor 2 (ROR2) were detected by quantitative real-time fluorescence polymerase chain reaction (qRT-PCR). Cell viability, cell proliferation and apoptosis were detected by Cell Counting Kit-8 (CCK-8) assay, colony formation assay and flow cytometry assay, respectively. Western blot examined the protein levels of PCNA, Cyclin D1, Bax, Bcl-2 and ROR2 cells. The relationship between miR-338-3p and circ_0044520 or ROR2 was verified by Dual-luciferase reporter assays. The xenotransplantation model was established to study the role of circ_0044520 in vivo. Results. The expression of circ_0044520 and ROR2 was increased in LSCC tissues and cells, while the expression of miR-338-3p was decreased. Circ_0044520 can sponge miR-338-3p, and ROR2 is the target of miR338-3p. In vitro complement experiments showed that knockdown of circ_0044520 significantly inhibited malignant behavior of LSCC, while co-transfection of miR-338-3p inhibitor partially up-regulated this change. In addition, miR-338-3p can inhibit the proliferation of LSCC cells, while co-transfection of overexpression of ROR2 can partially reverse this change. Mechanically, circ_0044520 regulates ROR2 expression in LSCC cells by sponging miR-338-3p. In addition, in vivo studies have shown that down-regulation of circ_0044520 inhibits tumor growth. Conclusion. Circ_0044520 silencing plays antiproliferation and pro-apoptosis roles in LSCC cells by regulating the miR-338-3p/ROR2 axis, suggesting that the circ_0044520/miR-338-3p/ROR2 axis may be a potential regulatory mechanism for the treatment of LSCC
- PublicationOpen AccessFibroblast activation protein-alpha knockdown suppresses prostate cancer cell invasion and proliferation(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2022) An, Jiali; Hou, Dingkun; Wang, Lei; Wang, Lili; Yang, Yuanyuan; Wang, HaitaoBackground. Prostate cancer is one of the most common malignant tumors of the male genitourinary system. Fibroblast activation protein alpha (FAP-α) overexpression has been shown to occur in a wide range of tumors. However, the specific mechanism of FAP-α in the development of prostate cancer has not been reported. Methods. In this study, real-time quantitative PCR (qRT-PCR) was used to detect the relative expression of FAP-α mRNA in prostate cancer cell lines (PC-3, LNCaP, and DU145) and human normal prostate epithelial cell line RWPE-1. Small interfering RNA (siRNA) targeting FAP-α and vectors expressing exogenous FAP-α were transfected to prostate cancer cells (LNCaP and DU145) to investigate the function of FAP-α. BALB/c nude mice were injected with DU145 cells which were transfected with NC-siRNA, FAP-αsiRNA-1, or FAP-α-siRNA-2. Results. Compared to adjacent normal tissues, FAPα protein and mRNA levels in prostate cancer tissues increased significantly (P<0.05). Compared to patients with high FAP-α mRNA levels, patients with low FAP-α mRNA levels had a significantly higher survival rate (χ2=5.050, log-rank P=0.025). Overexpression of FAP-α in LNCaP cells markedly inhibited cell apoptosis, and promoted cell invasion and proliferation. In contrast, knockdown of FAP-α expression in DU145 cells can significantly reduce invasion, proliferation, and promote apoptosis in prostate cancer. Immunofluorescence assay further indicated that down-regulation of FAP-α could suppress the nuclear translocation of β-catenin. An in vivo study found that compared with the NC-siRNA group, the tumor weight and tumor volume in the FAPα-siRNA-1 and FAP-α-siRNA-2 groups were significantly decreased. Conclusions. In conclusion, down-regulation of FAP-α can inhibit the invasion and proliferation of prostate cancer. Our study provides a theoretical basis for the targeted treatment of prostate cancer.
- PublicationOpen AccessMdivi-1: a promising drug and its underlying mechanisms in the treatment of neurodegenerative diseases(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2022) Liu, Xiaoqin; Song, Lijuan; Yu, Jiezhong; Huang, Fang; Li, Yanhua; Ma, CungenMitochondria are energy-producing organelles, and neurons are high energy consumption cells. Therefore, mitochondrial dysfunction is a critical factor in neurodegenerative processes. Mitochondrial division inhibitor-1 (Mdivi-1) is a small chemical inhibitor of mitochondrial division dynamin, which plays multiple roles in mitochondrial dynamics, mitochondrial autophagy, ATP production, the immune response, and Ca2+ homeostasis. Mdivi-1 inhibition of excessive mitochondrial fission exerted cytoprotective effects in neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). Mdivi-1 changed the mRNA expression of the electron transport chain (ETC) and reduced Ca2+ overload against neuronal injury. Elucidation of the molecular mechanism of Mdivi-1 in neurodegenerative diseases will help evaluate its therapeutic potential and promote its application in clinical studies. The present article focused on the multiple effects of Mdivi-1 on mitochondrial function and its potential therapeutic effects in neurodegenerative diseases.