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Objective:To investigate the effects of long non-coding RNA (lncRNA) RP11-1212A22.4 on the cell viability and invasive ability of esophageal cancer cell lines by targeting miRNA-483-5p (miR-483-5p).Methods:The expression of RP11-1212A22.4 in esophageal cancer tissues was analyzed by using GEPIA online database. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of RP11-1212A22.4 in human esophageal cancer cell lines EC9706, KYSE30, TE-13, Eca109 and normal esophageal epithelial cell line HET-1A. The lowest expression level of EC9706 cell line in RP11-1212A22.4 was divided into RP11-1212A22.4 group (transfected with pcDNA-RP11-1212A22.4 plasmid) and the control group (transfected with pcDNA-NC plasmid). The cell viability of EC9706 cell was analyzed by using methyl thiazolyl tetrazolium (MTT) method, and the invasion ability of EC9706 cell was detected by using Transwell assay. The targeting relationship between RP11-1212A22.4 and miR-483-5p was verified by using StarBase database prediction and dual luciferase reporter assay. The relative expression level of miR-483-5p of EC9706 cell in two groups was detected by using qRT-PCR. Western blot was used to detect the expressions of cyclin-dependent kinase 6 (CDK6), matrix metalloproteinase 2 (MMP-2), cyclin-dependent kinase 4 (CDK4), and matrix metalloproteinase 9 (MMP-9) proteins in two groups.Results:In GEPIA online database, compared with adjacent tissues, the relative expression level of RP11-1212A22.4 in esophageal cancer tissues was decreased, and the difference was statistically significant ( P < 0.001). The relative expression levels of RP11-1212A22.4 in esophageal cancer cell lines EC9706, KYSE30, TE-13, Eca109 and normal esophageal mucosal epithelial cell line HET-1A were 0.11±0.08, 0.32±0.09, 0.72±0.09, 0.59±0.13 and 0.97±0.12, and the difference was statistically significant ( F = 40.42, P < 0.001). The relative expression levels of RP11-1212A22.4 in EC9706 cells of RP11-1212A22.4 group and the control group were 11.9±2.4 and 1.0±0.3, respectively, and the difference was statistically significant ( t = 8.89, P < 0.001). Compared with the control group, the cell viability of EC9706 cell in RP11-1212A22.4 group was decreased (all P < 0.05). The number of invasive cells in RP11-1212A22.4 group was lower than that in the control group (48±12 vs. 106±22, t = 4.63, P < 0.001). StarBase database prediction and dual luciferase reporter assay both showed that RP11-1212A22.4 targeted miR-483-5p. The relative expression level of miR-483-5p in RP11-1212A22.4 group was lower than that in the control group (0.24±0.11 vs. 1.02±0.23, t = 5.98, P = 0.001). Compared with the control group, the expressions of CDK6, MMP-2, CDK4 and MMP-9 proteins in the RP11-1212A22.4 group were decreased. Conclusions:RP11-1212A22.4 is lowly expressed in esophageal cancer tissues and cell lines, and it inhibits the cell viability and invasive ability of esophageal cancer cells by targeting miR-483-5p.
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Objective To investigate the expression of TMS1/ASC gene induced by gemcitabine(GEM) in pancreatic carcinoma cell line PANC-1.To study the relationship between cysteine aspartase(caspase-1),nuclear factor-κB(NF-κB) and the expression of TMS1/ASC.Methods The pancreatic carcinoma cell line PANC-1 was cultured in Dnlbecco's modification of Eagle's medium(DMEM).Methyl thiazolyl tetrazolium (MTT)method was used to measure the effect of GEM at different time points(24,48 h)at different concentrations(1,2,4,8,16 μg/ml) on growth of PANC-l.RT-PCR was used to detect the expression of TMS1/ASC mRNA stimulated by medium alone and by GEM(4.27μg/ml)for 24 h and 48 h.Western blot analysis was performed with inhibitory protein of NF-κB multiclonal antibody,caspase-1 multiclonal antibody and β-actin monoclonal antibody to observe the expression of β-actin,caspase-1 and IκBα in GEM group and in the control group.The activation state of caspase-1 and NF-κB was examined.Results GEM inhibited the growth of PANC-1 cells in a concentrationand-time-dependent manners and its half maximal inhibitory concentration(IC50)was 4.27 μg/ml on 24 h.The expression of TMS1/ASC was 0.3 ±0.004 and 0.63 ±0.007 respectively in GEM group while it was 0.1 ±0.001 and 0.21 ± 0.006 in the control group on 24h and 48h.The difference between the 2 groups at the same time point had statistical significance (P ＜ 0.01).Western blot showed that GEM caused the activation of caspase-1.The expression of IκBα had no obvious differencebetween the 2 groups.GEM couldn't induce the activation of NF-κB.Conclusions GEM can inhibit proliferation of PANC-1 cells and induce their apoptosis.The drug sensitivity decreased with prolongation of exposure time.GEM might induce and increase the expression of TMS1/ASC,which might influence the apoptosis of the cells later.The apoptosis of PANC-1 cells induced by GEM is dependent of caspase-1 signaling pathway and independent of NF-κB signaling pathway.
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Objective To set up the method of estimation of age for Chinese girls at the age of 11-16 years according to their characteristic of radiographic changes in articulations of bone. Methods The samples of the X-ray photograph were come out of 6 articulations of humeri, cubiti, manus, coaxe, genus and ankle from 150 girls, ranging in age from 11-16 years old, in Henan province, North of China. The radiographic characteristics of osteoepiphysis at these joints were observed carefully and classified. The data were analyzed with the SPSS package. Result Regression equation was made for radiographic determine of age for Chinese girls at the age of 11-16 years. Conclusion The equation can be used for forensic practice about the determine of age.
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Objective:To investigate the roles of apoptosis-associated speck-like protein containing CARD(ASC),one of differentially expressed proteins in neutrophil death induced by ONO-AE-248.Methods:Human neutrophils were cultured in vitro with or without ONO-AE-248(5?10-5 mol/L).The expression of ASC mRNA was detected by RT-PCR.PMN proteins were extracted at 12 h and then separated by 2D electrophoresis.20 differentially expressed proteins were selected and determined by PDQest 2-DE software.Results:ONO-AE-248 decreased the expression of ASC mRNA strikingly in 2 hours;ASC was one of the important differentially expressed proteins between spontaneous apoptosis and non-apoptosis programmed cell death induced by ONO-AE-248 in 12 hour and ASC protein point was weaker in ONO-AE-248 group.Conclusion:ASC might act as double switch that leads to apoptosis of neutrophils in the high expression and removes the forbiddance of pathway of ONO-AE-248 signals in the low expression,resulting in non-apoptosis programmed cell death of neutrophils.
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Objective:To investigate the structural alterations of mitochondria and its role in neutrophil death induced by ONO-AE-248.Methods:Human neutrophils were cultured in vitro with ONO-AE-248(5?10-5 mol/L)and medium alone. Transmission electron microscopy(TEM) was used to detect the structural alterations of mitochondria and the level of mitochondria membrane potential by flow cytometry using mitocapture dying.Results:ONO-AE-248 resulted in extremely swollen mitochondria within 6 hours. Meanwhile, a rapid loss of mitochondrial membrane potential of neutrophils occurred, especially in 3 hours and 6 hours. There were obviously differences between spontaneous apoptosis and non-apoptosis programmed cell death induced by ONO-AE-248.Conclusion:The experiment results suggest that changes of mitochondrial structure and function be typically morphological, physiological and biochemical features in this unique form of neutrophil death, and that the mitochondrial pathway might play a more important role in ONO-AE-248-induced death of neutrophils.