OBJECTIVE: To investigate effects and mechanism of miR-195-5p on radiosensitivity of esophageal squamous cell carcinoma cells in vitro. METHODS: KYSE510 and KYSE150 cells were exposed to an 8 Gy of X-rays,and non-irradiated cells were used as control. mRNA expression levels of miR-195-5p and of survivin were detected by qPCR. Protein expression levels of survivin were detected by Western blot. KYSE510 cells were transfected with different oligos which gave rise to the miR-195-5p mimic,antagomir and negative control groups. After KYSE510 cells were irradiated with X-rays,cell proliferation was measured by the Incucyte real-time dynamic living cell monitoring and EdU incorporation assays. Cell colony survival and apoptosis rates were measured by colony formation assay and flow cytometry analysis,respectively. Dual-luciferase reporter assay was used to verify the luciferase activity of KYSE150 cells in each group. RESULTS: Growth of KYSE510 and KYSE150 cells were significantly inhibited after irradiation. Compared with the non-irradiation group,expression of miR-195-5p in the irradiation group was decreased,and expression of survivin protein was increased (P<0.05 or 0.01). After X-ray irradiation,the survival rate of the miR-195-5p mimic group was lower than that of the negative control group (P<0.01). The rates for EdU positive cells and clonal survival were lower than that of the negative control group (P<0.05 or 0.01),and the apoptosis rate was significantly higher than that of the negative control group (P<0.01). However,the rates for EdU positive cells and clonal survival of the antagomir group was higher than that of the negative control group (P<0.01). The expressions of survivin mRNA and protein in the miR-195-5p mimic group was lower than that of the negative control group. In contrast,the expressions of survivin mRNA and protein in the miR-195-5p antagomir group was higher than that of the negative control group. Over-expression of miR-195-5p in KYSE150 cells inhibited luciferase activity of the BIRC5 3' UTR reporter gene (P<0.05). CONCLUSION: MiR-195-5p could enhance radiosensitivity of esophageal squamous cell carcinoma cells in vitro by targeting survivin gene.

OBJECTIVE: To investigate the effects of X-ray irradiation on pyroptosis of human umbilical vein endothelial cells (HUVEC). METHODS: Pyroptosis is an inflammatory programmed cell death process. Cells were irradiated with a single dose of 10 Gy X-ray. In order to investigate the occurrence of X-ray induced pyroptosis,levels of IL-1 β,IL-6,TNF-α and IL-18 which were secreted by HUVEC cells from 0 to 72 h after the radiation were determined using the ELISA assay. Caspase-1 which is cleaved to the activated state is a key step of pyroptosis. Therefore,the activated levels of Caspase-1 were detected using Western blot at 6-48 h after the single dose of 10 Gy X-ray irradiation. Morphological changes of HUVECs were detected using transmission electron microscopy (TEM) at 72 h after the irradiation. Flow cytometry (FCM) was used to detect the changes of pyroptosis after a single dose and a small dose of multiple exposures (2.5 Gy/d×4 d). RESULTS: Compared with the control group,the levels of IL-1 β,IL-6,TNF-α and IL-18 in HUVEC cells 0-72 h after the radiation were significantly higher (P<0.05) and the levels of activated Caspase-1 at 6-48 h after the radiation was increased (P<0.05). The cell volumes were increased with swelling. The percentages of pyroptoti cells after the single and multiple exposures were significantly higher than that of the control group (P<0.01). In addition,the pyroptotic death rates after the single dose was twice as high as that after the multiple doses (P<0.01). CONCLUSION: X-ray exposure induced pyroptosis in HUVEC cells. Under the same total dose,the damage levels from the single dose exposure were significantly higher than that from the multiple exposures.

OBJECTIVE: To investigate effects and mechanisms of isorhamnetin (ISO),an important component of sea-buckthorn,on aging. METHODS: In this study,the model of D-galactose (D-Gal)-induced aging was employed. Human umbilical vein epithelial cells (HUVEC) in their 20th-23th generations were divided into 4 groups:control,D-Gal,D-Gal+5 μmol/L ISO and D-Gal+10 μmol/L ISO groups. The D-Gal concentration was 10 g/L and the intervention time was 72 hours. CCK-8 kit was used to detect cell viability in each group. Senescence was determined by the Senescence-associated β-Galactosidase (SA-β-Gal) staining kit. Levels of total reactive oxygen species (ROS),malondialdehyde (MDA),superoxide dismutase (SOD) activities and catalase (CAT) activities were detected by their respective kits. Western Blot was used to detect expression levels of aging markers,p21 and p27,as well as nuclear factor,erythroid-2 related factor 2 (Nrf2),and several downstream molecules. RESULTS: Isorhamnetin with concentration ≤ 10 μmol/L had no obvious toxic effects on HUVEC. Compared with the control group,D-Gal treatment increased the activity of SA-β-Gal,the protein levels of p21 and p27,and the total ROS level and MDA content. At the same time,D-Gal treatment inhibited the activities of total SOD and CAT,the protein levels of Nrf2 and the downstream molecules (P<0.05). Compared with the D-Gal group,isorhamnetin treatment effectively inhibited the activity of SA-β-Gal,the protein levels of p21 and p27,and the total ROS level and MDA content. Moreover,isorhamnetin treatment improved the activities of total SOD and CAT,and enhanced the protein levels of Nrf2 and its downstream molecules. CONCLUSION: Isorhamnetin attenuated D-Gal-induced HUVEC senescence by reducing oxidative stress and activation of Nrf2 pathway might be an important mechanism.

OBJCTIVE: To investigate expression and role of SERPINB5 in esophageal squamous cell carcinomas (ESCC). METHODS: Expression of SERPINB5 mRNA in ESCC tissues was analyzed using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. ESCC specimens and their matched morphologically normal operative margins were used to detect SERPINB5 protein expressions using the Western blot technique. Relationship between SERPINB5 expressions and the clinicopathological parameters of ESCC patients were analyzed. Furthermore,ESCC cells were transfected with an SERPINB5 over-expression vector,the empty vector or without any vectors (parental). These cells were used to detect proliferation and clony formation to determine the roles of SERPINB5. Effects of SERPINB5 on tumorigenesis were detected using the nude mouse xenograft model in vivo. Western blot was used to analyze changes in downstream pathways from over-expression of SERPINB5. Finally,potentials of SERPINB5 in treatment of ESCC through targeted inhibition were investigated. RESULTS: Expression levels of both the SERPINB5 mRNA and protein were down regulated in ESCC compared with the normal tissues (P<0.01). In addition,lower SERPINB5 mRNA levels were correlated with lower degrees of tumor differentiation (P=0.004) but higher clinical stages (P=0.037). From our in vitro and in vivo studies,our results show that over-expression of SERPINB5 significantly inhibited the proliferation and colony formation of ESCC cells,as well as the growth of tumor xenografts (P<0.05). At the molecular level,over-expression of SERPINB5 decreased the phosphorylation levels of AKT and mTOR. Data from targeted inhibition experiments show that the over expression of SERPINB5 significantly increased the sensitivities of ESCC cells to Alisertib (Aurora A inhibitor) or Everolimus (mTOR inhibitor) (P<0.01). CONCLUSION: SERPINB5 acted as a tumor suppressor in ESCC. Elevating the expression of SERPINB5 in tumor cells might be a potential strategy for the treatment of ESCC.

OBJECTIVE: To explore the differential expression and activities of lncRNAs and mRNAs in lymphocytes from acute myeloid leukemia patients compared with that from normal controls. METHODS: Microarrays were used to detect discrepant lncRNAs and mRNAs in peripheral lymphocytes from 6 acute myeloid leukemia patients compared with 6 normal controls. GO and KEGG were used to analyze the primary biological functions of differentially expressed genes. RESULTS: In lymphocytes from the two groups of individuals,there were 3 256 differentially expressed lncRNAs between patients and controls. Among these lncRNAs,1 161 lncRNAs were upregulated and 2 059 were downregulated. In addition,there were 3 544 differentially expressed mRNAs between patients and controls;1 950 mRNA were upregulated and 1 594 were downregulated. GO analyses show that functional annotation of the differential expression genes mainly included detection of chemical stimulus involved in sensory perception of smell,G1/S transition of mitotic cell cycle and inflammatory response. The graft versus host disease and cell cycle,HTLV-I infection and other pathways were shown by KEGG. CONCLUSION: These findings extended the current knowledge and provided some novel mechanisms on pathogenesis of leukemia.

OBJECTIVE: To investigate toxic effects of various components in PM_2.5 and of their combined exposures on human umbilical vein endothelial cells (HUVECs),and to determine their interactive effects. METHODS: Immortalized HUVECs were exposed in culture to 0,5,10,20,40,80,160 μg/mL carbon black,0,2.5,5,10,20,40,80,160 μg/mL dust,0,6.25,12.5,25,50,100,200,400 μmol/L lead acetate,0,5,10,20,40,80,160 μmol/L sodium arsenite and cadmium chloride for 24 h. The CCK-8 assay was used to determine cell viability and IC₅₀ values were calculated. For the combined toxicity study,the doses at which cell viability was 80%,carbon black (or dust) was combined with lead,arsenic,and cadmium for 24 h,and the CCK-8 assay was used to determine cell viability. Additionally,according to the actual proportion of each fraction in PM_2.5,carbon black (or dust) was proportionally (m_lead:m_arsenic:m_cadmium:m_{carbon black/dust}=10:5:1:500) combined with lead,arsenic,and cadmium,respectively,for 24 h,and the CCK-8 assay was used to determine cell viability. RESULTS: The cell viability test shows a decreasing trend with increasing doses of carbon black,dust,lead acetate,sodium arsenite,and cadmium chloride (P<0.05),with IC₅₀ of 16 μg/mL,110 μg/mL,184 μmol/L,15 μmol/L,and 14 μmol/L,respectively. Cell viability was decreased by 17.8%,43.8%,41.2% when carbon black was combined with lead acetate,sodium arsenite,and cadmium chloride,respectively,and by 11.6%,27.8%,28.3% when dust was combined with lead acetate,sodium arsenite,and cadmium chloride,respectively. When carbon black and dust were combined with lead acetate,sodium arsenite,and cadmium chloride,the cell viability was significantly lower than when they were exposed individually (P<0.05). There were interactions between carbon black and sodium arsenite or cadmium chloride (all P<0.05). CONCLUSION: Different components in PM_2.5 can cause toxic effects to the vascular endothelial cells in vitro,the cytotoxic effect of carbon black was greater than dust particles. Upon combined exposure,dust was more cytotoxic upon simultaneous exposure to the other three metals. The order of toxic effect of lead,arsenic and cadmium was cadmium chloride > sodium arsenite > lead acetate,and the interactions between carbon black particles and sodium arsenite and cadmium chloride wa synergistic.

OBJECTIVE: To compare efficacies and acute toxicity between concurrent chemo-radiotherapy (CRT) and radiotherapy alone (RT) among elderly patients with locally advanced esophageal cancers. METHODS: Patients who were aged >70 years and who had locally advanced esophageal cancers were recruited from the Fourth Hospital of Hebei Medical University from 2010.1 to 2016.1. Patients' complete response rates (CRR),partial response rates (PRR),disease control rates (DCR),as well as progression-free survival (PFS),overall survival (OS) and acute adverse reactions were evaluated and followed by analysis of prognostic factors. RESULTS: 82 patients were recruited for our study. The CRR in the CRT group (33.3%) was significantly higher than that in the RT group (14.5%,P=0.049). There was no significant difference in PRR and DCR between the two groups. (P=0.058,0.064). The median PFS and the proportion of patients with PFS ≥ 1,2,and 3 years in the CRT group was significantly higher than that in the RT group(P=0.007). The median OS and the proportion of patients with OS ≥ 1,2,and 3 years in the CRT group was significantly higher than that in the RT group (P=0.012). Analysis of Univariants data show that different treatment options and CRR after treatment mainly affected PFS and OS. Multivariate analyses show that CRR was an independent prognostic factor for PFS,treatment plan and CRR were independent prognostic factor for OS (all P<0.05). Some acute adverse reactions in the CRT group:including nausea and vomiting,leukopenia,thrombocytopenia,were significantly more frequent than the RT group(all P<0.05). CONCLUSION: Chemo-radiotherapy can be used as a treatment option for elderly patients with locally advanced esophageal squamous cell carcinoma,and its efficacy was better than that of radiotherapy alone. During the treatment process,close attention should be paid to patients' adverse reactions.

OBJECTIVE: To investigate effects of p38MAPK gene on expression of oncogenes and apoptosis related genes in human kidney epithelial cells (HK-2 cells) which had been exposed to PM_2.5. METHODS: According to the mRNA sequence of p38MAPK gene provided by GenBank,interference sequences were designed and synthesized,and a recombinant lentiviral vector was constructed and was transfected into HK-2 cells to construct p38MAPK gene-silenced cells. Real-time fluorescent quantitative PCR (qPCR) and western blot were used to identifyeffects from p38MAPK gene silencing. HK-2 cells and p38MAPK gene-silenced cells were treated with 50 μg/mL PM_2.5 suspension for 24 h,and qPCR and western blot were used to detect their expression of oncogenes (c-myc,c-fos and p53) and apoptosis-related genes (Caspase-8,Caspase-9 and Bcl-2). RESULTS: p38MAPK mRNA expression levels in p38MAPK-silenced cells were inhibited by 58.50% and p38MAPK protein expression levels by 51.33% when compared with the normal HK-2 cells (P<0.01). These results indicate that the p38MAPK silenced cells were successfully constructed. The qPCR results showed that when compared with the HK-2 cells in the control group,the PM_2.5-exposed cells indicated the mRNA of c-myc,c-fos,Caspase-8 and Casepase-9 in expressions were significantly increased by 39.89%,15.12%,19.47% and 15.45%,respectively,and p53 and Bcl-2 mRNA expressions were significantly decreased by 21.54% and 31.77% respectively (P<0.05). When compared with the PM_2.5 exposed HK-2 cells,the PM_2.5-exposed p38MAPK-silenced cells showed that the mRNA of c-myc,c-fos,p53,Caspase-8 and Caspase-9 expressions were significantly decreased by 84.55%、63.55%、34.49%、37.19% and 54.97% respectively,(P<0.05). At the protein level,when compared with the HK-2 cells in the control group,the PM_2.5 exposed cells showed that the protein levels of c-myc,and Caspase-8 were increased and that of Bcl-2 were decreased. When compared with the PM_2.5-exposed HK-2 cells,the PM_2.5-exposed p38MAPK-silenced cells showed that the mRNA levels of c-myc,Caspase-8 and Bcl-2 were decreased. CONCLUSION: p38MAPK-silenced cells were successfully constructed in this study. Our data show that PM_2.5 promoted the expression of oncogenes and apoptotic-related genes in HK-2 cells,and the p38MAPK gene was involved in the cytotoxicity of PM_2.5.

OBJECTIVE: To investigate effects of CASIN,a specific cell division circle 42 (CDC42) inhibitor,on viability,migration and pseudopodia of esophageal cancer cells,and its mechanism of inducing apoptosis. METHODS: According to expression of the CDC42 protein,four groups were set up:endogenous CDC42 high/low expression groups and exogenous CDC42 high/low expression groups. Western blot was used to detect expression of endogenous CDC42 protein in 9 esophageal cancer cell lines. From the analyses,two endogenous high-expression and two low-expression esophageal cancer cell lines were selected. Cell viability assay was used to detect the effect of CASIN on cell viability. Wound healing assay was to detect cell invasion. Western blot was to detect protein expression levels of Caspases-3 and PARP. After constructed the Flag-CDC42 and GFP-CDC42 plasmid successfully,they were transfected into esophageal cancer cells to distinguish exogenous CDC42 high/low expression groups. Cell viability assay and wound healing assay were used as described above. Furthermore,the immunofluorescence experiment was used to detect effects of CASIN on pseudopodia formation. RESULTS: In the endogenous CDC42 high/low expression groups,our data show that CASIN at concentrations of 15-30 μmol/L significantly inhibited cell viability and cell invasion (P<0.05),and increased the expression of Caspase-3 and lyse PARP. The latter suggests that CASIN might induce cell apoptosis. Compared with the exogenous CDC42 low expression group,20 μmol/L CASIN significantly reduced expression in the exogenous CDC42 high expression group (P<0.05). In addition,data from the immunofluorescence experiments show that CASIN suppressed the effect of CDC42 in promoting the formation of pseudopodia. CONCLUSION: CASIN reduced the viability and migration of esophageal cancer cells,inhibit pseudopodia,and induce cell apoptosis in vitro. Therefore,CASIN can be developed further as a new targeted drug against esophageal cancer.

OBJECTIVE: To investigate the effects of total saponins from the seeds of Nigella glandulifera Freyn (TSNG) on proliferation,migration,apoptosis and autophagy of SiHa cells. METHODS: SiHa cells were divided into untreated control group,TSNG (0.8,0.9,1.0,1.1,1.2,1.4,1.6,1.8 mg/mL) treatment group and HCQ (10 μmol/L) intervention group. Growth inhibition was detected by using the MTT assay. Anti-migration effects were detected by the cell scratch assay. Apoptosis rates were determined using the flow cytometry assay with Annexin V-FITC/PI staining. Nuclear apoptosis was observed after Hoechst staining. The formation of autophagosomes was detected after MDC staining. Protein expressions of LC3-Ⅱ and p62 were detected by Western blotting. RESULTS: Compared with the control group,TSNG had an inhibitory effect on cell growth which shows a dose-dependent effect relationship (P<0.05 or 0.01). The relative migration rates from the 0.9,1.0,1.1mg/mL TSNG treatment groups were (20.40±2.49)%,(15.14±0.39)% and (5.05±1.04)%,which were lower than those of the control group (36.63±2.52)% (P<0.01). The apoptosis rates from the 1.0,1.2,1.4 mg/mL TSNG treatment groups were (39.10±0.22)%,(68.37±0.58)% and (80.93±0.12)%,respectively,which were higher than those of the control group (10.73±0.82)% (P<0.05 or 0.01). Compared with the control group,the cell nuclei from thef TSNG treated group showed more apoptotic changes, increased autophagosomes,and increased the LC3-Ⅱ and p62 protein expressions (P<0.05 or 0.01). Compared with the TSNG treatment group,LC3-Ⅱ protein expressions had no significant changes after HCQ pretreatment. CONCLUSION: TSNG exposure caused inhibition of proliferation and of migration,induced apoptosis and blocked autophagy in SiHa cells.