Gain-of-function mutations and overexpression of KIT are characteristic features of gastrointestinal stromal tumor (GIST). Dysregulation in miRNA expression may lead to KIT overexpression and tumorigenesis.miRNA microarray analysis and real-time PCR were used to determine the miRNA expression profiles in a cohort of 69 clinical samples including 50 CD117IHC+/KITmutation GISTs and 19 CD117IHC-/wild-type GISTs. GO enrichment and KEGG pathway analyses were performed to reveal the predicted targets of the dysregulated miRNAs. Of the dysregulated miRNAs whose expression was inversely correlated with that of KIT miRNAs were predicted by bioinformatics analysis and confirmed by luciferase reporter assay. Cell counting kit-8 (CCK-8) and flow cytometry were used to measure the cell proliferation, cycle arrest and apoptosis. Wound healing and transwell assays were used to evaluate migration and invasion. A xenograft BALB/c nude mouse model was applied to investigate the tumorigenesis in vivo. Western blot and qRT-PCR were used to investigate the protein and mRNA levels of KIT and its downstream effectors including ERK, AKT and STAT3.Of the six miRNAs whose expression was inversely correlated with that of KIT, we found that miR-148b-3p was significantly downregulated in the CD117IHC+/KITmutation GIST cohort. This miRNA was subsequently found to inhibit proliferation, migration and invasion of GIST882 cells. Mechanistically, miR-148b-3p was shown to regulate KIT expression through directly binding to the 3'-UTR of the KIT mRNA. Restoration of miR-148b-3p expression in GIST882 cells led to reduced expression of KIT and the downstream effectors proteins ERK, AKT and STAT3. However, overexpression of KIT reversed the inhibitory effect of miR-148b-3p on cell proliferation, migration and invasion. Furthermore, we found that reduced miR-148b-3p expression correlated with poor overall survival (OS) and disease-free survival (DFS) in GIST patients.miR-148b-3p functions as an important regulator of KIT expression and a potential prognostic biomarker for GISTs.
Circular RNAs (circRNAs) is a novel class of non-coding RNAs that regulate gene expression during cancer progression. Circ_0092314 is a newly discovered circRNA that was upregulated in pancreatic cancer (PAAD) tissues. However, the detailed functions and underlying mechanisms of circ_0092314 in PAAD cells remain unclear.We first determined the expression of circ_0092314 in PAAD and normal tissues and further investigated the functional roles of circ_0092314 in regulating epithelial-mesenchymal transition (EMT) of PAAD cells. We also assessed the regulatory action of circ_0092314 on the microRNA-671 (miR-671) and its target S100P.Circ_0092314 was markedly upregulated in PAAD tissues and cells, and its overexpression was closely correlated with worse prognosis of PAAD patients. Functionally, circ_0092314 promotes proliferation, invasion and EMT in vitro and tumor growth in vivo. Mechanistically, we demonstrated that circ_0092314 directly binds to miR-671 and relieve its suppression of the downstream target S100P, which induces EMT and activates the AKT signaling pathway. The tumor-promoting effects caused by overexpression of circ_0092314 could be revered by re-expression of miR-671 in PAAD cells.Overall, our study demonstrates that circ_0092314 exerts critical roles in promoting the EMT features of PAAD cells, and provides insight into how elevated expression of circ_0092314 might influence PAAD progression.
Glioblastoma multiforme (GBM) is a common and fatal disease of the central nervous system. GBM cell lines are fundamental tools used in GBM research. The establishment of novel continuous GBM cell lines with clear genetic backgrounds could facilitate the exploration of molecular mechanisms and the screening and evaluation of antitumor drugs in GBM studies. In the present study, a novel primary glioblastoma cell line was established, named GWH04, from a patient with GBM, and its STR genotype and various tumor parameters were examined. The STR information of GWH04 was identical to that of the original primary tumor tissue. Compared with existing cell lines, GWH04 had a similar in vitro proliferation rate as the U87 cell line, but a faster rate than the GL15 cell line, and substantial soft agar clone‑formation capacity and subcutaneous and intracranial tumorigenic capacity. For drug sensitivity test, half maximal inhibitory concentration assays for multiple drugs were performed in these three cell lines, and GWH04 cells were found to be resistant to temozolomide. Aneuploid karyotype with a median of 84 chromosomes was possessed by GWH04, as well as chromosomal structural abnormalities, such as broken chromosomes, double centromere chromosomes, homogeneous staining regions, and double microbodies. Gene sequencing further revealed the mutational status of genes TP53, PTEN, PDGFRA, ERBB2, BRCA1, NF1, and MLH1 and the promoter region of telomerase reverse transcriptase (C228T) in this cell line. Altogether, these results indicated that GWH04 will be a useful tool for human GBM studies both in vitro and in vivo.
Complement hyperactivation and thrombotic microangiopathy are closely associated with severe COVID-19. Endothelial dysfunction is a key mechanism underlying thrombotic microangiopathy. To address the relationship between endothelial injury, complement activation and thrombotic microangiopathy of severe COVID-19, we wonder whether, and if so, what and how SARS-CoV-2 factors make endothelial cells (ECs) sensitive to complement-mediated cytotoxicity. We revealed that multiple SARS-CoV-2 proteins enhanced complement-mediated cytotoxicity to ECs via inhibiting membrane complement regulatory proteins (CRPs) and enhancing the deposition of complement recognizing component FCN1. By screening with CRISPR/Cas9-gRNA libraries, we identified that ADAMTS9, SYAP1 and HIGD1A as intrinsic regulators of CD59 on ECs that were inhibited by the SARS-CoV-2 M, NSP16 and ORF9b proteins. IFN-γ, GM-CSF and IFN-α upregulated CD55 and CD59, which IFN-γ antagonized the inhibition of CD55 and CD59 by the three SARS-CoV-2 proteins. So, the deficiency of IFN-γ weakened the protection of CRPs against complement-mediated injury which may be enhanced during infection. Our findings illustrated the regulation of protection against complement-mediated attack on self-cells by SARS-CoV-2 infection and immune responses, providing insights for endothelial injury, thrombotic microangiopathy and potential targets for treating severe COVID-19.
Objective To research the correlations between HER2 overexpression and other prognostic factors that are assessed in clinical practice and the influence on prognosis of HER2 overexpression with breast cancer.Methods The authors evaluated HER2 status in 176 surgical specimens of patients with breast cancer in Tongji Hospital from Jan.2002 to Dec.2003,and correlations were evaluated with the other prognostic factors.And the prognosis was compared between the HER2 overexpeession and negative expression.Results HER2 overexpression was negative correlated with ER status(P=0.031) and PR status(P=0.002),while not correlated with the other prognostic factors.Multivariate analysis showed that HER2 is an independent prognostic factor for DFS(P=0.018).Conclusion HER2 overexpression may be considered a independent prognostic marker and directed in the treatment in primary breast cancer.
Natural killer (NK)/cytotoxic T-cell lymphoma, a new type of cutaneous neoplasm, has been described recently in the World Health Organization/European Organization for Research and Treatment of Cancer classification for cutaneous lymphomas. We report an 11-year-old boy who had had erythematous plaques and blisters on his face and hands for 4 years and infiltrating plaques and necrosis on his extremities for 4 months. Routine clinical and laboratory examinations found no primary nasal involvement. Biopsies taken from nasal mucosa and skin showed that the tumour only involved dermis and subcutaneous tissue, and the infiltrated lymphohistiocytic tumour cells were CD56+, TIA+, CD45RO+ and CD30+. In situ hybridization for EBV-encoded nuclear RNA was positive. Clonal T-cell receptor-gamma2 gene rearrangement was positive. A diagnosis of extranodal NK/T-cell lymphoma, nasal type, was made. This is a rare case, with slow course and survival for >51 months with the presentation only occurring in the skin.
Abstract Background More and more studies showed pneumothorax is a complication of the 2019 novel coronavirus disease (COVID-19). But no autopsy findings of pneumothorax in COVID-19 decedent were reported, and direct relations between pneumothorax and lung pathology in these decedents were not discussed so far. Methods A 62-year-old man with COVID-19 presenting with persistent hypoxemia and suddenly dead, who was treated by mechanical ventilation in the intensive care unit (ICU) for 5 days. A systemic autopsy examination of COVID-19 decedent, including histopathology study, was conducted and the medical record, chest computerized tomography (CT) image were reviewed by forensic pathologists and clinicians. Results Severe pneumothorax, diffuse alveolar damage and airway obstruction were observed. Pneumothorax should be one of the causes of death. Conclusion Pneumothorax, due to SARS-CoV-2 infection, is a fatal complication of COVID-19. Regular examination of chest CT or X-ray and airway management are important to clinical treatment.
Macrophages perform key functions in tissue homeostasis that are influenced by the local tissue environment. Within the tumor microenvironment, tumor-associated macrophages can be altered to acquire properties that enhance tumor growth. Here, we found that lactate, a metabolite found in high concentration within the anaerobic tumor environment, activated mTORC1 that subsequently suppressed TFEB-mediated expression of the macrophage-specific vacuolar ATPase subunit ATP6V0d2. Atp6v0d2-/- mice were more susceptible to tumor growth, with enhanced HIF-2α-mediated VEGF production in macrophages that display a more protumoral phenotype. We found that ATP6V0d2 targeted HIF-2α but not HIF-1α for lysosome-mediated degradation. Blockade of HIF-2α transcriptional activity reversed the susceptibility of Atp6v0d2-/- mice to tumor development. Furthermore, in a cohort of patients with lung adenocarcinoma, expression of ATP6V0d2 and HIF-2α was positively and negatively correlated with survival, respectively, suggesting a critical role of the macrophage lactate/ATP6V0d2/HIF-2α axis in maintaining tumor growth in human patients. Together, our results highlight the ability of tumor cells to modify the function of tumor-infiltrating macrophages to optimize the microenvironment for tumor growth.
Erythropoietin elicits protective effects in lung tissue injury induced by ischaemic reperfusion and hyperoxia. We investigated the protective roles of erythropoietin in pulmonary inflammation and lung injury during acute endotoxaemia.A total of 32 male Sprague-Dawley rats were randomly assigned to four groups: saline group, erythropoietin + saline group, saline + lipopolysaccharide group and erythropoietin + lipopolysaccharide group. Rats were treated with erythropoietin (3000 U/kg, i.p.) or saline, 30 minutes prior to lipopolysaccharide administration (6 mg/kg, i.v.). Four hours after lipopolysaccharide injection, samples of pulmonary tissue were collected. Optical microscopy was performed to examine pathological changes in lungs. Wet/dry (W/D) ratios, myeloperoxidase activity, malondialdehyde concentrations and tumour necrosis factor-alpha (TNF-alpha) as well as interleukin 1 beta (IL-1beta) levels in lungs were measured. The pulmonary expression of nuclear factor kappaB (NF-kappaB) p65 was evaluated by Western blotting. Differences between the different groups were analysed by one-way analysis of variance (ANOVA).The lung tissues from the saline + lipopolysaccharide group were significantly damaged, which were less pronounced in the erythropoietin + lipopolysaccharide group. The W/D ratio increased significantly in the saline + lipopolysaccharide group (5.75 +/- 0.22) as compared with the saline group (3.85 +/- 0.20) (P < 0.01), which was significantly reduced in the erythropoietin + lipopolysaccharide group (4.50 +/- 0.35) (P < 0.01). Myeloperoxidase activity and malondialdehyde levels increased significantly in the saline + lipopolysaccharide group compared with the saline group, which was reduced in the erythropoietin + lipopolysaccharide group. The TNF-alpha level of pulmonary tissue increased significantly in the saline + lipopolysaccharide group ((9.80 +/- 0.82) pg/mg protein) compared with the saline group ((4.20 +/- 0.42) pg/mg protein, P < 0.01). However, the increase of TNF-alpha level of pulmonary tissue was significantly reduced in the erythropoietin + lipopolysaccharide group ((6.50 +/- 0.66) pg/mg protein, P < 0.01). Similarly, pulmonary IL-1beta levels were elevated markedly in the saline + lipopolysaccharide group in contrast to the saline group, whereas the elevation was much less in the erythropoietin + lipopolysaccharide group. The nuclear localization of p65 increased markedly in the saline + lipopolysaccharide group and this enhancement of nuclear p65 expression was much less in the erythropoietin + lipopolysaccharide group.Erythropoietin attenuates pulmonary inflammation and suppresses TNF-alpha and IL-1beta overproduction during acute endotoxaemia, which is partially mediated by inhibition of NF-kappaB.
Abstract Glioblastoma (GBM) is a common and fatal disease of the central nervous system. It has a worldwide occurrence and affects people from diverse backgrounds. GBM cell lines are fundamental tools used in GBM research. The establishment of novel continuous GBM cell lines with clear genetic backgrounds could facilitate the exploration of molecular mechanisms and the screening and evaluation of antitumor drugs in GBM studies. Here, we established a novel primary glioblastoma cell line, named GWH04, from a patient with glioblastoma, and examined its short-tandem repeat (STR) genotype and various tumor parameters. The STR information of GWH04 was identical to that of the original primary tumor tissue. Compared with existing cell lines, GWH04 had a similar in vitro proliferation rate as U87 cell line, but a faster rate than GL15 cell line, and a substantial soft agar clone formation capacity. GWH04 showed subcutaneous and intracranial tumorigenic capacity, with an aneuploid karyotype and median 84 chromosomes. Gene sequencing further revealed the mutational status of genes TP53 , PTEN , P DGFRA , ERBB2 , BRCA1 , NF1 , and MLH1 and the promoter region of TERT (C228T) in this cell line. Altogether, these results indicate that GWH04 can facilitate human GBM studies both in vitro and in vivo.
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