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Objective To investigate the MRI features of solitary fibrous tumors(SFT)in the spinal canal.Methods MRI images of 5 cases with pathologically proved STF in the spinal canal were analyzed retrospectively.Results Of 5 lesions,there were 2 in the cervical spine,3 in the thoracic spine;1 in the epidural space and 4 in the subdural extramedullary space.On MRI plain scan,3 lesions showed homogeneous iso-intense signal on T1WI and hypo-intense signal on T2WI,2 lesions showed heterogeneous signal,1 showed patchy hypo-intense signal on T1WI and T2WI at the upper edge of lesion,which had been confirmed as hemorrhage and the other lesion showed internal cystic variation.All of the 5 lesions enhanced on enhancement scan,with moderate enhancement in 2 lesions and significant enhancement in 3 lesions.Cystic and hemorrhagic area were not enhanced.The"dural tail sign"was showed in 3 cases.Conclusion The diagnosis of SFT should be considered when a lesion shows a localized solitary mass in the spinal canal with hypo-intense on T2WI and moderate to significant enhancement.
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PURPOSE: Long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) has been implicated as an oncogene in the development and progression of osteosarcoma. This study aims to explore the mechanism of NEAT1 in osteosarcoma. MATERIALS AND METHODS: Expressions of NEAT1 and miR-194 in osteosarcoma tissues and cells were detected by quantitative real-time PCR. The effects of NEAT1 knockdown or miR-194 overexpression on cell proliferation, invasion, and apoptosis were determined by 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide (MTT) assay, transwell invasive assay, and flow cytometry analysis, respectively. Luciferase reporter assay was performed to observe the possible interaction between NEAT1 and miR-194. RESULTS: NEAT1 was upregulated and miR-194 was downregulated in osteosarcoma tissues and cells. Knockdown of NEAT1 or overexpression of miR-194 suppressed proliferation and invasion and induced apoptosis of osteosarcoma cells in vitro. Luciferase reporter assay validated that NEAT1 could interact with miR-194 and negatively modulated its expression. Furthermore, inhibition of miR-194 reversed the suppression of proliferation and invasion and the promotion of apoptosis induced by NEAT1 depletion in osteosarcoma cells. CONCLUSION: Knockdown of NEAT1 suppressed proliferation and invasion and induced apoptosis in osteosarcoma cells by inhibiting miR-194 expression.
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Apoptosis , Carcinogenesis , Cell Proliferation , Flow Cytometry , In Vitro Techniques , Luciferases , Oncogenes , Osteosarcoma , Real-Time Polymerase Chain Reaction , RNA, Long NoncodingABSTRACT
Background Studies confirmed that hydroxycamptothecin cause the apoptosis of human Tenon capsule fibroblasts (HTFs) by protein kinase R-like endoplasmic reticulum stress kinase (PERK) single pathway.Autophagy and apoptosis are programmed cell death following stress reaction,so they remain a close association.However,the effect of hydroxycamptothecin on the autophagy of HTFs and its mechanism are still unclear.Objective This study was to explore the promoting effect of PERK signal pathway on hydroxycamptothecin inducing the autophagy of HTFs.Methods This study procedure was approval by Ethic Committee of Nanjing Medical University.Human Tenon capsule tissue was obtained from fresh adult donors.HTFs were cultured and passaged by explant-culture method and identified by immunofluorescence for vimentin and keratin.pLVX-PERK lentiviral packed by 293T cells was transfected into HTFs to obtain stable PERK-knockout cell line by puromycin selection.Then the HTFs were treated with 0.10 g/L of hydroxycamptothecin for 5 minutes and consecutively cultivated for 24 hours,and the untreated cells were used as the control group.Western blot assay was used to detect the expressions of autophagy specific proteins in the cells,including autophagy related gene 5 (ATG-5),Beclin-1,light chain 3 (LC-3).Cyto-ID staining was used to identify the autophagosome in the cells.The experimental results were analyzed and compared between different treating groups.Results The gray scales for the expressions of Beclin 1,ATG-5,LC-3-Ⅰ and LC-3-Ⅱ proteins in HTFs were 0.365:±0.045,0.765 ±0.055,0.120±0.030 and 0.215 ±0.035 in the control group,and those in the hydroxycamptothecin treated group were 0.980±0.070,1.495±0.095,0.585±0.025 and 0.785±0.055,showing a significant decline in the hydroxycamptothecin treated group(P=0.018,0.022,0.007,0.013).The green fluorescence of the autophagosome was stronger in the hydroxycamptothecin treated group compared with the control group.Western blot revealed that the gray scale of PERK expression in the cells was 0.130±0.030 in the PERK-knockout group,with a significant reduce in comparison with 0.765 ±0.055 of the control group (P =0.010).However,no obvious distinctions were seen in the band intensities of the expressions of Beclin-1,ATG-5 and LC-3 proteins between the two groups.Western blot indicated that the grey scale of the PERK expression in the cells was 1.790± 0.060 in the 0.10 g/L hydroxycamptothecin group,which was significantly higher than 0.880 ± 0.070 of the control group (P =0.010).Expression levels (gray scales) of Beclin-1,ATG-5,LC-3-Ⅰand LC-3-Ⅱ in the PERK-knockout+ 0.10 g/L hydroxycamptothecin group were 0.475 ± 0.045,0.390 ± 0.040,0.055 ± 0.015 and 0.075 ± 0.025,which were significantly lowed in comparison with 0.955 ± 0.065,0.765 ± 0.055,0.155 ± 0.015 and 0.280 ± 0.030 of the control+ 0.10 g/L hydroxycamptothecin group (P =0.026,0.031,0.042,0.034).In addition,the fluorescence intensity of autophagosomes was weaker in the PERK-knockout+0.10 g/L hydroxycamptothecin group compared with the control+0.10 g/L hydroxycamptothecin group.Conclusions Hydroxycamptothecin induces the autophagy of HTFs by PERK signal pathway.
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Background The fibrosis of filtering area caused by proliferation of human Tenon fibroblasts (HTFs) is one of failure causes following glaucoma surgery.Researches revealed that hydroxycamptothecin can induce the apoptosis of HTFs,but its influence on autophagy of HTFs is unclear.Objective This study attempted to investigate whether hydroxycamptothecin can cause an alteration of autophagic activity in HTFs.Methods Human Tenon capsular tissue was obtained from 3 patients during strabismus correction surgery under the informed consent of patients and their parents for the primary culture and passaged of HTFs in DMEM containing 10％ fetal bovine serum.The generation 3 to 6 cells then were incubated with 0.0,0.5,1.0,4.0,10.0 mg/L hydroxycamptothecin for 24 hours,respectively.A cell counting kit-8 (CCK-8) was used to detect the cell viability in different treated groups.The autophagic activity of HTFs was evaluated by a Cyto-ID autophagy detection kit,and then the autophagic flux was evaluated by counting the Cyto-ID positive cells under a fluorescence microscope,and the green fluorescence intensity was determined by flow cytometry.Quantitative reverse transcriptase PCR (qRT-PCR) and Western blot analysis were employed to assay the relative expressions of autophagic-associated genes and their proteins in HTFs,including Beclin-1,autophagy related gene 5 (ATG-5) and light chain 3 (LC-3).Results The cell viability of HTFs in the 0.0,0.5,1.0,4.0 and 10.0 mg/L hydroxycamptothecin groups were (100.00 ± 6.44) ％,(91.70 ± 6.36) ％,(81.47 ± 6.00) ％,(68.43 ± 6.69) ％ and (59.97 ± 6.98) ％ respectively,showing a gradually declining trend with the increase of hydroxycamptothecin doses,with a significant difference among them (F=19.040,P＜0.001),and the viability of HTFs in the 1.0,4.0 and 10.0 mg/L hydroxycamptothecin groups were significantly decreased than the control group (P＜0.05,P＜0.01,P＜0.01).qRT-PCR analysis revealed that the relative expression levels of Beclin-1 mRNA,ATG-5 mRNA and LC-3 mRNA in 4.0 mg/L hydroxycamptothecin group were (3.225 ±0.346),(2.839 ±0.418) and (3.761±0.224) folds higher than those of the control group.The expressions of Beclin-1 and ATG-5 proteins were significantly increased in the 4.0 mg/L hydroxycamptothecin group in comparison with the control group,and the expression intensity ratio of LC-3-Ⅱ/Ⅰ was 0.965±0.159 in the hydroxycamptothecin group,which was significantly higher than 0.275 ±0.860 of the control group (P =0.003).Cyto-ID staining showed that the percentage of autophagic cells increased dramatically from (11.333±4.010) ％ to (55.000±9.013) ％ upon the exposure of HTFs to 4.0 mg/L hydroxycamptothecin (P=0.002).Flow cytometry analysis showed that the green fluorescence intensity in the 4.0 mg/L hydroxycamptothecin group was (3.037 ±0.513) fold relative to that in the control group,showing a significant difference between the two groups (P =0.003).Conclusions Hydroxycamptothecin can induce autophagy in HTFs in vitro.