Histology and histopathology Vol.36, nº4 (2021)
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- PublicationOpen AccessPropofol protects PC12 cells from cobalt chloride-induced injury by mediating miR-134(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2021) Zhou, Hong-Yi; Jiang, Fan; Cao, Zhong; Shen, Qi-Yun; Feng, Yu-Jing; Hou, Zhen-HuanObjective. Propofol (PRO) was reported to exert a neuroprotective effect by decreasing microRNA134 (miR-134), a brain-specific miRNA, thus, the role of PRO against cobalt chloride (CoCl 2)-induced injury in rat pheochromocytoma cells (PC12) via mediating miR134 was explored. Methods. CoCl 2-induced PC12 cells treated with PRO were transfected with or without miR-134 negative control (NC)/ inhibitor/mimic, and the following detections were then performed using cell counting kit-8 (CCK-8), Annexin V-fluorescein isothiocyanate/ propidium iodide (Annexin V-FITC/PI) and Hoechst 33258 staining. Autophagy was observed by transmission electron microscope (TEM). Mitochondrial membrane potential (MMP) was detected by Rhodamine-123 (Rh123) staining, and reactive oxygen species (ROS) by dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining. Protein and gene expressions were measured by Western blotting and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), respectively. Results. PRO reversed the CoCl 2-induced decrease in the PC12 cell viability and increased miR-134 in a dose-dependent manner. CoCl 2 increased LC3II/I ratio and Beclin-1 expression, but decreased p62 expression, which was abolished by PRO. In addition, an increased cell apoptosis rates triggered by CoCl 2 were reduced by PRO with the down-regulations of Bax and Caspase-3 and the up-regulation of Bcl-2. Furthermore, PRO decreased methylenedioxyamphetamine (MDA), nitric oxide (NO) and ROS in CoCl 2-induced PC12 cells accompanying the increase in glutathione peroxidase (GSH-Px) and MMP. The effects of PRO on autophagy, apoptosis and oxidative stress in CoCl 2-induced PC12 cell were reversed by miR-134 mimic. Conclusion. PRO may mitigate CoCl2-induced autophagy in PC12 cells with decreased apoptosis and improved oxidative stress via mediating miR-134.
- PublicationOpen AccessOptimisation and validation of immunohistochemistry protocols for cancer research(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2021) Ella-tongwiis, Peter; Makanga, Alexander; Shergill, Iqbal; Hughes, Stephen FônBackground. Immunohistochemistry (IHC) has become a valuable laboratory technique for diagnosing, evaluating metastasis and informing treatment selection in several cancers. Standardization however remains a limiting factor in IHC. The main aim of this research study was to optimise, validate and standardize antibodies and IHC protocols for cancer research. Methods. Seven monoclonal mouse and rabbit antibodies were optimised using formalin-fixed paraffin embedded (FFPE) human tissue blocks. 4um sections of FFPE block were stained using the Roche Ventana XT or Ventana ULTRA IHC automated analysers. This study modified manufacturer recommended protocols by using a unique antigen retrieval method, adding an amplification step, varying primary antibody incubation times, as well as using the Roche Ventana Ultraview detection system. Results. Optimum antibody localisation was observed in modified IHC protocols in comparison with manufacturer recommended protocols for antiCEACAM-1, anti-CD31, anti-COX-2, anti-HER-2/neu, anti-S100P, anti-thrombomodulin and anti-VEGFR-3. Majority of antibodies required more than one modification of the initial protocol. For anti-VEGFR-3 optimum staining was observed following 4 protocol modifications. Conclusions. This study has optimised and standardized several tissue-based biomarkers that may be, in the future, used to screen, diagnose and monitor patients with certain cancer, such as bladder cancer. Accurate data on optimised protocols reduce time and resources wasted on experimental protocols, and ultimately help identify biomarkers or biomarker panels, which may be used to select treatment regimens for various cancers.
- PublicationOpen AccessPancreatic neuroendocrine neoplasms: Clinicopathological features and pathological staging(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2021) Lam, Alfred King-yin; Ishida, Hirotaka. The nomenclature and classification of pancreatic neuroendocrine neoplasms has evolved in the last 15 years based on the advances in knowledge of the genomics, clinical behaviour and response to therapies. The current 2019 World Health Organization classification of pancreatic neuroendocrine neoplasms categorises them into three groups; pancreatic neuroendocrine tumours (PanNETs) (grade 1 grade 2, grade 3), pancreatic neuroendocrine carcinomas and mixed neuroendocrine-non-neuroendocrine neoplasms (MiNENs) based on the mitotic rate, Ki-67 index, morphological differentiation and/or co-existing tissue subtype. PanNETs are also classified into non-functional NET, insulinoma, gastrinoma, VIPoma, glucagonoma, somatostatinoma, ACTH-producing NET and serotonin producing NET based on hormone production and clinical manifestations. A portion of the cases were associated with genetic syndromes such as multiple neuroendocrine neoplasia 1 (MEN 1), neurofibromatosis and Von Hippel-Lindau syndrome. In view of the distinctive pathology and clinical behaviour of PanNENs, the current 8th AJCC/UICC staging system has separated prognostic staging grouping for PanNETs from the pancreatic neuroendocrine carcinomas or MiNENs. Pancreatic neuroendocrine carcinomas and MiNENs are staged according to the prognostic stage grouping for exocrine pancreatic carcinoma. The new stage grouping of PanNETs was validated to have survival curves separated between different prognostic groups. This refined histological and staging would lead to appropriate selections of treatment strategies for the patients with pancreatic neuroendocrine neoplasms
- PublicationOpen AccessExpression of long-chain noncoding RNA GAS5 in osteoarthritis and its effect on apoptosis and autophagy of osteoarthritis chondrocytes(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2021) Ji, Qinghui; Qiao, Xiaofeng; Liu, Yongxiang; Wang, DaweiObjective. To investigate the expression of long-chain noncoding RNA GAS5 in osteoarthritis(OA) and the effect of silencing GAS5 on autophagy of osteoarthritis chondrocytes (OACs). Methods. OA rat models were constructed by cutting the anterior cruciate ligament, and the expressions of GAS5 in rat cartilage tissues at 4 weeks (early OA) and 12 weeks (late OA) after modeling were detected. The rat chondrocytes were isolated, cultured and transfected with si-GAS5 to silencing GAS5. Then, the changes of apoptosis and autophagy levels of OA chondrocytes were detected by transfection of GFP-LC3 and flow cytometry. Bioinformatic tools were used to analyze the miRNA binding to GAS5 and the downstream target genes, then luciferase reporter assay and GDC-0349 (inhibitor of mTOR) were used to verify their relationships. Results. The expression of GAS5 in cartilage tissue of OA rats was higher than control, which was higher in late OA than that in early OA. After silencing the GAS5, the autophagy ability of OACs was increased and the apoptosis rate was decreased. GAS5 was able to bind to miR-144 and regulate the expressin of mTOR. mTOR inhibitor GDC-0349 could reverse the inhibition of GAS5 on autophagy but could not reverse its effect on apoptosis. Conclusion. GAS5 expresses highly in OA cartilage tissues and increases with the progression of OA. GAS5 inhibits autophagy and promotes the apoptosis of OACs, and the inhibition of autophagy may be related to its regulation of mTOR
- PublicationOpen AccessPDCD4 regulates apoptosis in human peritoneal mesothelial cells and promotes gastric cancer peritoneal metastasis(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2021) Wu, Pei; Wang, Jinou; Mao, Xiaoyun; Xu, Huimian; Zhu, ZhiObjective. Programmed cell death 4 (PDCD4) is a tumor suppressor gene, however, the function and regulatory mechanism remain to be discovered. The connection between tumorigenesis and apoptosis is one of the most important foci of cancer research. Our study aimed to explore the connections between PDCD4-mediated apoptosis of human peritoneal mesothelial cells (HPMC) and peritoneal metastasis in gastric cancer. Methods. The PDCD4 expression in 31 pairs of HPMC and tumor tissues was assessed by immunohistochemistry and RT-PCR. In cell experiments, we monitored gastric cancer cell migration with a Transwell chamber assay when PDCD4 was silenced in HPMC. Subsequently, apoptosis of HPMC was detected by a flow cytometric assay and western blotting. After analyzing cytokines in culture supernatants from gastric cancer with enzyme-linked immunosorbent assays (ELISAs), transforming growth factor-beta 1 (TGF-β1) was abundant in the culture supernatants of gastric cancer. Then, PDCD4 expression in HMrSV5 cells was analyzed by western blotting after retreatment with different concentrations of TGF-β1. Moreover, apoptosis of peritoneal mesothelial cells treated with TGF-β1 was detected according to the above methods. Results. In human metastatic peritoneal tissues, the expression of PDCD4 was significantly lower than that in normal tissues. At the same time, decreased expression of PDCD4 in HPMC was associated with increased migration capacity of gastric cancer cells. Moreover, suppressing the expression of PDCD4 promoted apoptosis in mesothelial cells which may be regulated by TGF-β secreted from gastric cancer cells. Conclusions. These data suggested that decreased expression of PDCD4 significantly promoted apoptosis in human peritoneal mesothelial cells, thus inducing peritoneal metastasis, and that TGF-β1 secreted from gastric cancer cells may have played a crucial role.