OBJECTIVE:To study the effect of TGF-β1 on expression of miRNA and the inflammatory protein,cyclooxygenase-2 (COX-2),in human keratinocytes (HaCaT) after UVB irradiation. METHODS:10 ng/μL of TGF-β1 was added to cultured HaCaT cells for 48 h and then irradiated with UVB:0,10,20,30 mJ/cm₂ UVB and different time (4,12 and 24 h) after 30 mJ/cm₂ UVB irradiation. Real-time fluorescent quantitative PCR was used to detected expression levels of miRNA-23a and let-7c 6 h after irradiation. Expression levels of COX-2 and caspase-3 were detected by Western blot at different time (0,2,4,8,12,24 and 48 h) after 30 mJ/cm₂ UVB irradiation. RESULTS:Expression of miR-23a was increased and let-7c was generally down-regulated. Expression of COX-2 was significantly increased (P < 0.05),and cleaved-caspase3 decreased gradually at 12-48 h after irradiation. CONCLUSION:The data indicate that TGF-β could regulate the expression levels of miRNA and COX-2 which have been shown to play a role in cell proliferation,infiltration,inflammation and anti-apoptosis.

OBJECTIVE:The role of EGFR in autophagy activity in lung adenocarcinoma A549 cells was investigated using small interference RNA to silence EGFR expression. METHODS:A549 cells were divided into four groups:untreated,EGFR siRNA-1,EGFR siRNA-2 and EGFR siRNA-3 groups. Real-time RT-PCR and Western blot were used to detect degradation of EGFR after transfection with EGFR siRNA. In addition,A549 cells were treated with different EGF concentrations:0,10,25 and 50 ng/mL. Protein expression of EGFR was detected using Western blot. Furthermore,A549 cells were divided into four groups:EGFR siRNA,negative control siRNA,transfection reagent (only add equivalent transfection reagent) and untreated groups. All four groups also received 50 ng/mL EGF. Western blot was used to detect expression of LC3Ⅱ/LC3Ⅰ which was observed as the effect on silencing EGFR expression on autophagy activity. A549 cells were divided into EGFR siRNA transfection and negative control siRNA groups,and autophagosomes were observed by transmission electron microscope. RESULTS:After treatment of A549 cells with the three different EGFR siRNAs,the mRNA and protein expression of EGFR were significant reduced. Western blot indicates that increased LC3-Ⅱ/LC3-Ⅰ ratios were induced by down-regulation of EGFR. Transmission electron microscopy shows that EGFR siRNA treatment,compared with control siRNA treatment,induced marked formation of autophagsomes. CONCLUSION:Reducing the expression of EGFR increased autophagy activities of lung adenocarcinoma A549 cells.

OBJECTIVE:The glioma-associated long non-coding RNA (LncRNA) MEG3 was preliminarily selected and detected in glioma tissues. Its expression in glioma cells together with proliferation,apoptosis,invasion and transformation of glioma cells were investigated after HHV-6A infection. METHODS:Expression of LncRNA MEG3 in 37 glioma,4 para-tumor tissues and 18 normal brain tissues,or HHV-6A infected glioma cells were measured by Real-time quantitative PCR. The D(450) value of HHV-6A infected glioma cells were assayed by using cell counting kit-8 method. The frequencies of apoptotic cells were detected by flow cytometry. The cell healing ability was monitored by the wound-healing assay. The epithelial-mesenchymal transition (EMT)-related proteins were evaluated by Western blot. RESULTS:Expression of LncRNA MEG3 was significantly lower in glioma tissues than in para-tumor tissues (P < 0.05). Expression of LncRNA MEG3 showed a down-regulation in glioma or normal glial cells after HHV-6A infection (P < 0.05). The apoptotic rates of HHV-6A infected glioma cells were significantly lower than that in the control group[(6.05±0.40)% and (8.23±0.75)%,respectively,P < 0.05]. The wound healing ability of glioma cells after HHV-6A infection was significantly better than that of the control group[(72.33±6.90)% and (43.67±6.30)%,respectively,P < 0.05]. Compared with the control group,expression of Snail and Vimentin were increased,while expression of Ecadherin was decreased in glioma cells after HHV-6A infection (P < 0.05). CONCLUSION:HHV-6A infection down-regulated the expression of LncRNA MEG3 in glial cells and glioma cells. The expression was associated with proliferation,invasion,epithelial-mesenchymal transition of glioma cells and inhibition of apoptosis.

OBJECTIVE:To assess health risk of drinking water in the Longgang district of Shenzhen,China. METHODS:Health risks associated with 15 indexes in drinking water were assessed using the US EPA health risk assessment model. RESULTS:The ranking of carcinogenic risk was Cr⁶⁺ > As > Cd > trichlormeth-ane > perchlormethane,the ranking of non-carcinogenic risk was fluoride > nitrate nitrogen > Hg > Cu > Pb > Se > Zn > ammonia nitrogen > Mn > Fe. The total health risk level was 4.113×10^-5,from 3.906×10^-5 to 9.457×10^-5. CONCLUSION:The total health risk level of drinking water was closed to that of ICRP recommended level (5×10^-5),and lower than that of US EPA recommended level (1×10^-4).

OBJECTIVE:To investigate the relationship between microRNA-95 (miR-95) expression and chemoresistance of gastric cancer cells to lobaplatin. METHODS:Expression of miR-95 in the cancer and paracancerous tissues from 30 patients with gastric cancer was detected using real time PCR. CCK-8 was used to detect sensitivity of gastric cancer tissues to lobaplatin in vitro. Anti-miR-95 and miR-95 mimics were transfected into SGC7901 to determine changes in inhibition rates of lobaplatin. Real-time quantitative PCR and Western blot were used to detect expression of the multidrug resistant genes:MDR1,GST-π,LRP,Survivin,xIAP and Bad. RESULTS:Levels of miR-95 in gastric cancers were higher than that in paracancerous tissues (P < 0.05). The cancer tissues with higher miR-95 expression levels showed higher activities after lobaplatin treatment (P < 0.05). After transfection with anti-miR-95 cells,sensitivity to lobaplatin increased significantly (P < 0.01). The mRNA and protein expressions of MDR1,Survivin and xIAP decreased significantly after the transfection (P < 0.05),while the expression of Bad mRNA and protein increased significantly (P < 0.05). After the transfection with miR-95 mimics,sensitivity of cells to lobaplatin decreased significantly (P < 0.01). The mRNA and protein expressions of MDR1,Survivin and xIAP significantly after the transfection (P < 0.05),while the expression of Bad mRNA and protein decreased significantly(P < 0.05). CONCLUSION:miR-95 was involved with sensitivity of gastric cancer cells to lobaplatin via regulation of some resistance-related genes.

OBJECTIVE:Expression of β1 integrin in breast cancer patients with pathological nipple discharge was investigated and its effect on prognosis of patients was evaluated. METHODS:From January 1st,2006 to December 31,2013,112 breast cancer patients with pathological nipple discharge in the People's Hospital of Gansu Province were brought into the study. Another 112 breast cancer patients with breast lumps which were detected by physical examination or not were selected as a control. Immuno-histochemistry was used to detect the difference in expression of β1 integrin and Her-2 in cancer tissues. Survival analysis was used to study the effect of β1 integrin on prognosis of the patients. RESULTS:Among patients with pathological nipple discharge,14.29% (16/112) showed positive expression of β1 integrin which was significantly lower than that in the control group (25%) (χ²=4.073,P=0.044). Survival analyses show that the control group had significantly lower survival than that of the pathological nipple discharge group (HR=1.773,P=0.012). The overall survival with higher expression of β1 integrin was significantly lower than that of lower expression in the nipple discharge group (HR=2.573,P=0.023). The expression of β1 integrin and Her-2 had a positive correlation in the nipple discharge group (r=0.281,P=0.001),Cox proportional hazards model test found that histological-grade,lymph node metastasis,and high expression of β1 integrin were three independent risk factors affecting the overall survival of patients with pathological nipple discharge (P < 0.01). CONCLUSION:High expression of β1 integrin in breast cancer tissues significantly reduced the overall survival of breast cancer patients. Furthermore,breast cancer patients with integrin had lower overall survival,High expression of β1 integrin was positively correlated with poorer prognosis among breast cancer patients with pathological nipple discharge and was positively related to worse pathologic features of breast cancer.

OBJECTIVE:To investigate genomic alterations in STR loci on autosomal and sex chromosomes among human papillary thyroid cancers. METHODS:Tumor and stromal cells were separated from papillary thyroid cancer tissues from 43 unrelated patients using laser capture microdissection. DNA of tumor cells,stromal cells and adjacent normal tissues were extracted using Chelex-100,amplified using Goldeneye^® DNA 22NC,Goldeneye^® DNA 27YB and Goldeneye^® DNA 17X PCR amplification kit and analyzed using ABI 3500 Genetic Analyzer. RESULTS:The genotypes of stromal cells from different cancer tissues were consistent with that from corresponding adjacent normal tissues. Genomic alterations were detected in 11 out of 43 tumor cell specimens. Eleven alterations were detected on 37 STR loci within autosomal and X chromosomes,including additional alleles:3 times,partial loss of heterozygosity:6 times,and new alleles:2 times. Moreover,the alternations might have occur simultaneously at more than one loci in the same tumor cells. There were no genomic alterations detected on STR loci of the Y chromosome. With respect to clinical data,there was no significant association between STR variations and staging of the cancers,nor patient's gender. However,there were significant differences between age of the patients and STR loci alterations (P < 0.05). CONCLUSION:There were genomic alterations in the STR loci among autosomal and sex chromosomes in human papillary thyroid cancer cells. In addition,these alterations occurred more often in older patients. Stromal cells could be separated accurately from tumor tissues by microdissection which would provide more opportunity for investigations.

OBJECTIVE:To investigate the effect of acid sphingomyelinase (ASMase) and ceramide (Cer) on the death receptor pathway and on oxidative stress in vitamin E succinate (VES)-induced apoptosis in gastric cancer cells. METHODS:Human gastric cancer cells in cultures were treated with medium,desipramine (DES,12.5 μmol/L),VES (20 μg/mL) or VES+DES (cells were pretreated with DES at 12.5 μmol/L for 2 h,then with VES at 20 μg/mL). ASMase activity was assessed using the ELISA assay and cellular expression of Cer was detected by immunostaining at different time points. Apoptosis was evaluated using DAPI staining at 24 h. Western blotting was used to detect the expression of proteins in the death receptor pathway,including Fas,death receptor 5 (DR5),caspase-8,caspase-9 and PARP. 2',7'-dichlorofluorescein-diacetate was used to quantitate the levels of reactive oxygen species (ROS). RESULTS:VES increased the activity of ASMase and accumulation of Cer in cellular membrane at 1.5 h and reaching the peak at 3 h. The ratio of apoptosis was (41.00±1.00)% at 24 h. The inhibition of ASMase activities was significantly associated with blockage of VES-induced apoptosis (20.00±2.00)%,via perturbing the levels of Fas,DR5,caspase-8/9 and PARP cleavage,and oxidative stress (all P < 0.01). CONCLUSION:In the VES-induced apoptosis in the SGC-7901 human gastric cancer cells,our results demonstrate that ASMase and Cer were upstream events which were followed via the death receptor pathway and expression of oxidative stress.

OBJECTIVE:The aim of this study was to investigate the toxicological activityity of total flavonoids from Xinjiang semen Nigellae,the unique national drug resources of Xinjiang,in human cervical cancer cell lines to investigate the mechanism by using. METHODS:Two humna cervical carcinoma cell lines,SiHa and HeLa,were treated with different concentrations of the total flavonoids from the semen nigellae(2.5,5,10,15,20,25,50 μg/mL). Their toxicological activities were detected using the MTT assay which determined the effective concentrations and IC₅₀. Anti-migration effects were detected by cell scratch assay. Apoptosis rates were detected by flow cytometry. RESULTS:Compared with the control group,proliferation of the treated cervical cancer cells was inhibited in a concentration-dependent manner. The IC₅₀ of total flavonoids was 16.935 μg/mL for SiHa and 18.366 μg/mL for HeLa. By comparing the scratch widths before and after the treatments,the relative migration distance of tumor cells in the SiHa cell line control group was (223.333±13.868) px,and that in the treated group was (56.333±10.970) px. The relative migration distance of the HeLa cell control group was (360.667±15.308) px,and that in the treated group was (13.000±3.606) px. Total flavonoids also induced apoptosis in both cervical cancer cell lines. The apoptosis rates in the 10,15 and 25 μg/mL treatment groups were (8.5±0.5)%,(35.9±1.3)% and (85.7±1.6)% for SiHa,and were (5.3±0.4)%,(11.4±0.8)% and (85.9±1.9)% for HeLa. The differences were significantly different from that of the control (P < 0.05). CONCLUSION:The total flavonoids from Xinjiang semen nigellae demonstrated anti-proliferation anti-migration activities in vitro and induced apoptosis in human cervical cancer cell lines.

OBJECTIVE:To investigate correlations between gene polymorphisms and cancer,we studied polymorphisms in the XRCC3 double strand DNA repair gene in breast cancer. METHODS:Data on four gene loci (rs861534,rs861537,rs3212092 and rs861530) were combined with genotypes on breast cancer. High throughput chip detection assay was used for the polymorphism of XRCC3 in 517 cases of breast cancer and 1 008 disease-free controls which were collected in Gansu. RESULTS:With logistic regression analyses of the polymorphisms,rs861534 was found have significantly different distributions at the AG/AA genotype (P < 0.01,OR=0.36;P=0.013,OR=0.08) compared with the-GG-allele. The rs861530 AG+AA alleles-were significantly different from the controls (P=0.044). With respect to clinical indicators,the rs861537 GG/AG genotype was significant associated with positive ER protein (P=0.048,OR=1.50). In tumer tissues,the rs3212092 CT genotype was significantly associated with the Her-2⁺ protein (P=0.049),while the CC+CT genotype was significantly different (P=0.027,OR=2.06). CONCLUSION:Our results indicate that-XRCC3 rs861534 and rs861530-were significant associated with breast cancer risk. The GG/AG genotype with ER⁺ at XRCC3 rs861537 may impose a higher risk for breast cancer. Patients with the positive of ER protein and with the rs861537 locus may have increased risk from endocrine therapy. After adjusting for covariates,the rs3212092 homozygote TT-and heterozygote CT with Her-2⁺ expression were associated with the risk of recurrence and metastasis.

OBJECTIVE:The in vitro micronucleus test was applied to evaluate genotoxicity of the traditional Chinese medicine Panax notoginseng extracting solution. METHODS:The Chinese hamster lung cell line (CHL) were treated with Panax notoginseng extracting solution which was prepared by soaking with alcohol repeatedly at three concentrations (0.625,1.250,2.500 mg/mL). Cell toxicity was determined by the neutral red uptake test. Genotoxicity was determined with and without the S9 metabolic activation via the in vitro cytokinesis-block micronucleus test. Cyclophosphamide and mitomycin C were used as the positive controls,and MEM was used as the negative control. RESULTS:The neutral red uptake test showed that the 2.500 mg/mL concentration of Panax notoginseng extracting solution induced OD value decreased significantly compare with negative control (P < 0.05) which indicates that the cell survival rate was 80.3%. The frequencies of micronuclei in the three concentrations of 0.625,1.250,2.500 mg/mL of Panax notoginseng extracting solution were 17‰,16‰,16‰ in the presence of +S9. In the absence of S9,the frequencies were 17‰,15‰,16‰ which were not significantly different from the negative control (+S9 16‰、-S9 15‰,P > 0.05). CONCLUSION:Panax notoginseng extracting solution did not show genotoxicity under our study conditions.

OBJECTIVE:Electric mosquito repellents are widely used therefore their toxicologic effects should be better understood. The purpose of this study was to investigate genoxicity of the repellents in plant cells. METHODS:Plant cells were exposed to various concentrations (25%,50% and 75%) of 3 brands of electric mosquito repellents (BBN,JJ and YB electric mosquito liquid). The micronucleus test was used to monitor genotoxic effects. RESULTS:When the Vicia faba root tip cells were exposed to the three brands of electric mosquito repellents at the 75% concentration,the micronucleus rates were significantly higher than that in the untreated control group (P < 0.05). Under the similar experimental conditions using the garlic root tip cells,only the JJ and YB brands showed significant induction of micronuclei (P < 0.05). Under the similar conditions using the onion root tip cells,both the 50% and 75% concentrations induced significantly higher micronuclei frequencies (P < 0.05). The micronucleus rates in the treated onion root tip cells at the 75% concentration were significantly higher than those of Vicia faba (P < 0.05). The genotoxic effects from the three kinds of electric mosquito repellents showed no significance differences (P > 0.05). CONCLUSION:Three brands of electric mosquito repellents caused genotoxicity to root tip cells of Vicia faba,garlic and onion,and the damage to onion cells was significantly higher than that to Vicia faba. There was no statistical difference in the damage caused by three kinds of electric mosquito repellents.