OBJECTIVE:Previous studies demonstrated that the EB-8m variant of Epstein-Barr virus (EBV)-encoded small RNA 2 (EBER2) might be associated with nasopharyngeal carcinoma (NPC). The aim of this study was to investigate whether the variant EBER2 would confer resistance to apoptotic by enhancing inhibition of RNA-activated protein kinase (PKR) in NPC cell lines. METHODS:The cell anti-apoptosis observation induced by 10 000 IU/mL interferon-α(IFN-α) or 2μg/mL cisplatin was conducted in EBV-negative NPC cell lines (CNE1 and HONE1) with stable expression of wild type or EB-8m variant EBER2 gene under the control of lentivirus vector transfection group. Western blotting was performed to determine phosphorylation of PKR, eIF2α and c-Jun (which are down streams of PKR activation),expression of Bcl-2 in IFN-α-treated cells with stable expression of EBER2 and control cells. RNA-binding protein immunoprecipitation against PKR was assayed in cells with stable expression of EBER2 to detect the fold enrichment of EBER2 with PKR. RESULTS:EBER2 -expressing NPC cells enhanced anti-apoptosis ability in contrast to the control cells (P < 0.05 or P < 0.01),and the cells expressing variant EBER2 showed higher anti-apoptosis ability compared with those expressing wild type EBER2 (P < 0.05). Compared with the control cells,EBER2-expressing cells showed lower level of phosphorylation of PKR, eIF2α and c-Jun and elevation of Bcl-2 (P < 0.05). The levels of phosphorylated PKR in variant EBER2 transfected HONE1 and CNE1 cells were significantly lower than that in wild type EBER2 transfected cells (P < 0.05). That of phosphorylated eIF2α in variant EBER2 transfected HONE1 cells was also significantly lower than that in wild type EBER2 transfected cells (P < 0.05). EBER2 could be detected in immunoprecipitation against PKR, and the variant EBER2 had higher fold enrichment than wild type EBER2 (P < 0.05). CONCLUSION:The EB-8m variant EBER2 demonstrated its inhibition of PKR,and of phosphorylation of eIF2α and c-Jun,and up-regulated Bcl-2 expression,thus enhanced the anti-apoptosis ability of NPC cell lines.

OBJECTIVE:To construct miR-106a promoter reporting plasmids and to explore their regulatory effects on the migration of human gastric cancer cells with transcription factor Krüppel-like factor 4. METHODS:Using UCSC genomic database to predict the promoter region of miR-106a,and to insert it into pGL3-basic reporter vector for construction of the wild-type reporter plasmid,pmiR-106a-WT-luc,and the point mutation reporter plasmid,pmiR-106a-MUT-luc. Using transcription factor database JASPAR to predict and to screen the transcription factor KLF4 that regulates the promoter region of miR-106a,and to clone it into pcDNA3.0 vector for construction of the over-expression plasmid KLF4-pcDNA3.0. KLF4^+/- -pcDNA3.0 and pmiR-106a-WT/MUT-luc were co-transferred into HEK293T cells and their fluorescence activities were detected by dual luciferase assay. 30 pairs of gastric cancer and adjacent non-tumor tissues were collected and qPCR was used to detect the expression level of miR-106a and KLF4. Transwell assay was used to detect the migratory capacity of human gastric cancer SGC-7901 cells. RESULTS:Both miR-106a reporter and KLF over-expression plasmids were constructed correctly through enzyme digestion,PCR amplification,sequencing and Blast comparison. KLF4-pcDNA3.0 significantly inhibited the luciferase activity of pmiR-106a-WT-luc (P=0.000);whereas, the effect on pmiR-106a-MUT-luc was little (P=0.553). The relative expression of KLF4 was 0.716±0.624 corresponding to 3.367±2.165 for miR-106a in gastric cancer tissues. KLF4-pcDNA3.0 partially blocked the stimulative effect of miR-106a on the migration of gastric cancer SGC-7901 cells (P=0.038). CONCLUSION:Our data show that there was a direct binding site between transcription factor KLF4 and the promoter region of miR-106a. In addition,KLF4 negatively regulated the expression of mature miR-106a at upstream transcription level. Thereby,it played a role in inhibiting the migration of gastric cancer cells induced by miR-106a.

OBJECTIVE:To study regulatory effects of Survivin on c-myc in esophageal ECA109 cells. METHODS:ECA109 esophageal squamous carcinoma cells were divided into Survivin shRNA interference,control shRNA and control groups (without any treatment). ECA109 cells were transfected with 2μg Survivin shRNA and 2μg control shRNA plasmids,respectively. After 48 h,expressions of Survivin,c-myc mRNA were detected by the RT-PCT method,proteins of Survivin,c-myc and p-ERK were detected by Western blot. ERK,p38,JNK,JAK/STAT3, PI3K/Akt signal pathway inhibitors were used to detect mRNA expression of c-myc by half quantitative RT-PCR and Western blot. RESULTS:Compared with the negative plasmid control group and the blank cell group,the expression of c-myc mRNA and protein was decreased after the interference of the Survivin shRNA group (P=0.022,P=0.000). c-myc mRNA and protein expressions were reduced in the ERK signal channel blockers group (P < 0.05). The expression of ERK phosphorylation protein was reduced after Survivin silence in the Survivin shRNA interference group. CONCLUSION:Survivin was effective in regulation of ERK activation state in upstream signal transduction. The role of Survivin in regulation of c-myc expression was related to the ERK signaling pathway.

OBJECTIVE:To investigate protective effect against cell death in DNA damage-induced breast 2 cancer cell line MDA-MB-231. METHODS:MDA-MB-231 cells were irradiated with 5 J/m² UVC and were used to establish our DNA damage cell model. Western blot was used to detect DNA damage marker phospho-H2AX;cell death related proteins:p53, p21, and PARP; and nuclear factor NF-90 which regulates p53 and p21 expression. 2 RESULTS:At 0.5 h after 5 J/m² UVC irradiation of MDA-MB-231 cells,phospho-H2AX expression was induced (P < 0.05),p21 was degraded (P < 0.05),and phosphor-p53 (p-p53) was induced (P < 0.05). At 8 hours after irradiation, PARP was cleaved (P < 0.05). There were not any significant changes on p53 and NF-90. CONCLUSION:From our cell model of DNA damage in MDA-MB-231 cell line,p21-PARP pathway was involved in induction of cell death,but phosphor-p53 was resistant. These imply that phosphor-p53 showed a protective role on this model.

OBJECTIVE:To analyze secretory proteins from three different kinds of cervical cancer cell lines (Siha, Caski and C33) and to identify specific proteins as biomarkers for earlier detection and for treatment. METHODS:Label-free quantitative proteomics technology processes were performed successively:protein cleavage, protein quantification and proteolysis. Then,the secretory proteins were analyzed and were picked out by LC-MS/MS and unlabeled Maxquant analyses. RESULTS:21 609 kinds of peptides and 2 895 different kinds of proteins were found. Meanwhile,collective proteins made up 29.2% of total with 729 different types. The numbers of proteins for Siha,Caski and C33 were 128,143 and 859,respectively. Among the collective proteins,there were 4 main protein species:heat shock proteins (HSPs)(14 proteins), heterogeneous nuclear ribonucleoprotein (HNRP)(8 proteins), laminin (LN)(4 proteins) and Aldo-keto reductase family 1(AKR1) (2 proteins). In addition,the lysosomal signaling pathway accounted for the largest proportion (39 proteins). CONCLUSION:There were 729 kinds of collective proteins among the three cervical cancer cell lines which belonged to the HPV16 type. Among the collective proteins,4 main protein species were concluded which have already been confirmed to play a significant role in the cervical cancer process. They may also belong to the lysosomal signaling pathway,The results may provide certain research directions for tumor molecular markers in the future.

OBJECTIVE:To explore the new miRNA target gene 3'untranslated region (3'UTR) single nucleotide polymorphism (SNPs) among digestive tumor associated genes, and their association with gastric carcinogenesis. METHODS:The 1:1 matched case-control study was conducted. SNP microarray was used to detect the SNP loci of 3'UTR region of digestive tract tumor-associated genes in 96 patients with gastric adenocarcinoma and 96 healthy controls in the high incidence area of gastric cancer in Xianyou County,Fujian Province. Time-of-flight mass spectrometry (TFMS) was used to detect the SNP loci in 622 patients with gastric cancer and 622 healthy individuals. RESULTS:SNP chip and bioinformatics analyses selected EGFR rs884225,EGFR rs10277413,FAS rs1468063,MSH2 rs17502941 and MSH2 rs11125144 as candidate genes. The SNP genotyping of 622 candidate genes in the control group showed no correlation between the above 5 loci and susceptibility to gastric cancer. Further stratification analyses showed that the genotype (CT+TT) with T allele in EGFR rs884225 was associated with cardiac cancer (OR=0.67,95% CI:0.46-0.99), while other loci were not associated with the risk of cardiac cancer and non-cardiac cancer (P > 0. 05). CONCLUSION:The rs884225 polymorphism of EGFR gene 3'untranslated region is associated with the occurrence and development of gastric cancer in Xianyou County,a high incidence area of gastric cancer in Fujian Province. T allele may be a genetic protective factor for gastric cancer.

OBJECTIVE:To investigate the mechanism of MDM2 mRNA and protein expression in the development of skin scar carcinoma. METHODS:Skin scar carcinoma and pathological scar were analyzed in comparison to normal skin.Expression of MDM2 protein was detected using immunohistochemical method and MDM2 mRNA using in situ hybridization.Their differential expressions (optical density and positive area) among the three groups of tissues were detected using computerized image-analysis.All data were input into computer and analyzed statistically using SPSS (17.0) software.RESULTS:Expressions of MDM2 mRNA and protein in skin scar carcinomas were positive or strongly positive,weakly positive or positive in skin pathological scar and negative or weakly positive in normal skin tissues.Differences between any two groups were statistically significant (P < 0.05). CONCLUSION:High expression of MDM2 mRNA and protein was significantly associated with development of skin scar carcinoma. Therefore, their expression may be useful for diagnosis of skin scar carcinoma in the earlier stages.

OBJECTIVE:To study expression of a costimulatory molecule, B7 homologue 4 (B7-H4), in precancerous lesions of human esophageal carcinoma and to understand its clinical significance in the formation of esophageal squamous cell carcinoma. METHODS:58 esophageal tissue specimens of endoscopic resection were collected. There were 16 cases of normal tissues,23 cases of low-grade intraepithelial neoplasia (LGIN) and 19 cases of high-grade intraepithelial neoplasia (HGIN). Hematoxylin eosin staining (HE) was used to evaluate their pathological changes. Expression of B7-H4 mRNA in esophageal tissues was detected by real-time fluorogenic quantitative-PCR (qPCR). Immunohistochemistry and Immunoblotting (Western blot) methods were used to detect B7-H4 protein expression in esophageal tissues. Expressions of IL-6, IL-10 and IFN-γ in esophagi were detected by Enzyme-linked Immunosorbent assay (ELISA). Additionally,correlations between expression of these cytokines and B7-H4 protein were analyzed. RESULTS:Immunohistochemistry and HE results indicate that B7-H4 protein expression was positively correlated with esophageal pathological grade (P < 0.01). Moreover,the qPCR results show that there was no significant difference in expression of B7-H4 mRNA between precancerous tissues and normal esophageal tissues (P > 0.05). Western blot data indicate that B7-H4 protein expression was increased significantly in precancerous esophagi compared with that of normal esophagi (P < 0.05). Furthermore,results of Western blot and ELISA tests show that B7-H4 expression was positively correlated with IL-6 concentration (P < 0.01). CONCLUSION:B7-H4 protein expression was gradually upregulated with the increase of precancerous grade of esophagi. B7-H4 may promote esophageal squamous cell carcinoma formation by interacting with IL-6.

OBJECTIVE:To investigate the role of Notch signaling pathway in the development of cystitis glandularis (CG) and bladder urothelial cell carcinoma(BUC). METHODS:Expression of Notch1 and Hes1 protein were assessed by immunohistochemistry in bladder sections of 16 normal bladders and 26 CG samples from 50 patients with BUC. The relationship between CG and BUC,and the pathological grade and clinical stages of BUC were evaluated. RUSULTS:Notch1 was observed in normal bladder,CG samples and BUC samples,and the positive rates were 65.38% (17/26),70% (30/50) and 25% (4/16),respectively. The positive rates of Hes1 in CG,BUC and normal bladder tissues were 65.38% (17/26),76% (38/50) and 18.75% (3/13),respectively. Expressions of Notch1 and Hes1 in BUC and CG samples were significantly higher than that in normal bladder samples (P < 0.05) and increased with the pathological grades (P < 0.05). However,there was no significant difference (P > 0.05) in their expression in BUC and CG samples. CONCLUSION:Our study shows that Notch1 and Hes1 expressions were associated closely with the occurrence and pathological grade of BUC,as well as the occurrence and development of CG. Notch pathway might influence CG for the development of bladder cancer. Therefore,the expression may be used as a biomarker for diagnostic indices and for treatment option of CG.

OBJECTIVE:To explore the roles of miR-34a and its downstream genes plus iron overload in rats with nonalcoholic fatty liver disease. METHODS:Thirty-six male SD rats of 5 weeks old were randomly divided into control diet group (basal diet),high iron diet group (basal diet containing 1% FeSO₄),high fat diet group (high fat diet 35% energy supply from fat) and high fat plus high iron diet group (high fat diet containing 1% FeSO₄). These rats were sacrificed after 12 weeks. Body weights were measured and liver lipid accumulation was observed by oil red O staining. The contents of ALT and AST in rat serum were detected. Quantitative real-time PCR (qPCR) was used to detect mRNA levels of miR-34a, silent information regulator (SIRT1) and peroxisome proliferator-activated receptor α (PPARα). SIRT1 and PPARα protein levels were detected by Western blot. RESULTS:Compared with control diet group,the body weight,the serum ALT level and the level of miR-34a in high fat diet group and high fat plus high iron diet group were increased significantly (P < 0.05). The levels of mRNA and protein expression of SIRT1 and its downstream PPARα were decreased (P < 0.05). The degree of lipid accumulation in the liver was gradually aggravated. Compared with high fat diet group,the levels of mRNA and protein expression of SIRT1 and PPARα in high fat plus high iron diet group were decreased significantly (P < 0.05). CONCLUSION:The combined effect of high fat and high iron diets could activate the expression of miR-34a and then inhibit the expression of downstream genes, thus aggravating liver lipid disorder in NAFLD rats.

OBJECTIVE:To investigate the effects of flutamide exposure in pregnant mice on their uterus and ovarian development,and on oxidative stress among offspring,and to provide mechanistic understanding of reproductive system malformation. METHODS:40 eight-week-old conceived ICR females mice were randomly assigned into treatment and the control groups. Flutamide was orally administered to pregnant mice at 300 mg/(kg·d) during gestation days 12-18,while the control group was treated with equal volume of soybean oil. More than 7 weeks after birth,the appearance of female offspring was observed. The body weights, uterine weight, ovarian weight were collected to calculate the organ coefficient. The uterus and ovarian tissues were taken for sectioning,and the pathological changes were observed. Follicles of each level were counted and their pathological changes observed. Then 12 animals were randomly taken out for measuring the superoxide dismutase (SOD),glutathioneperoxidase (GSH-Px) and malonaldehyde (MDA) of the uterus and ovary in each dose group,and their abundance of Nox1,Sod1 and Gpx1 was detected by qPCR. RESULTS:Compared to the control group,there was no significant difference in the appearance of the female offspring in the treatment group,but the body weight was lower than control(P < 0.05),the visceral coefficients of uterus and ovary had no statistical difference from the control group(P > 0.05). The results of follicle count showed that the numbers of primordial follicles and mature follicles in the experimental group were less than control(P < 0.05). The SOD activity and GSH-Px level in the uterus of the treatment female mice were lower than those in the control group,and the MDA level was higher(P < 0.05). In the ovary of the treatment group,the SOD activity and GSH-Px level were lower(P < 0.05),and the MDA level had no significantly difference(P > 0.05). The mRNA abundance of Noxo1 in the uterus of the experimental group was increased,while the abundance of Sod1 mRNA was decreased (P < 0.05),and the abundance of Gpx1 mRNA was not statistically different from the control(P > 0.05). The mRNA abundance of Noxo1 was increased and the abundance of Gpx1 in the ovary of the treatment group were decreased (P < 0.05),and the mRNA abundance of Sod1 has no statistically difference from the control group(P > 0.05). CONCLUSION:Data from our study show that pregnant mice exposed to flutamide caused abnormal uterus and ovarian development in their offspring and demonstrated anti-oxidative stress.

OBJECTIVE:To investigate reproductive toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D) in juvenile male and female SD rats. METHODS:Male and female SD rats were exposed to different concentrations of 2, 4-D for 28 days (18.1,36.3 and 72.5 mg/kg),and to a placebo. The general behavior of animals,body weight changes and vaginal opening time were monitored during the period of exposure. After the exposure,the testis,epididymis, uterus,and ovary were weighed to calculate the wet weight and organ coefficient. In addition, blood serum follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), testosterone (T) and cholesterol levels were measured using an automatic biochemical analyzer. Pathological examinations were performed on hematoxylin-eosin (HE) stained rat testis,epididymis,ovary and uterus. RESULTS:Compared with the control group,the body weight gain of SD rats decreased significantly with increase of exposure doses (P < 0.05). The vaginal opening time of the SD rats in the 36.25 mg/kg group was significantly shortened while the vaginal opening time of the SD rats in the 72.5 mg/kg group was significantly prolonged (P < 0.05). The serum T levels from the 72.5 mg/kg group were decreased compared with the control group (P < 0.05). The cholesterol levels of female SD rats in the 72.5 mg/kg group were increased (P < 0.05). The serum E2 levels of female rats in the 36.25 mg/kg group increased significantly,while they were decreased in the 72.5 mg/kg group,and the differences were statistically significant (P < 0.05). The uterine cavity was larger and the uterine wall was thinner in the rats exposed to 72.5 mg/kg,and the number of spermatogenic cells in the epididymal tissues from the highdose group was reduced in the seminiferous tubules (layers). The uterine cavity was larger and the uterine wall was thinner in the rats exposed to 72.5 mg/kg,and the number of spermatogenic cells was reduced in the seminiferous tubules. CONCLUSION:Our study shows that 2,4-D interfered with development of reproductive system among young SD rats and the female rats were more affected than the males. A possible mechanism is that 2,4-D inhibited the synthesis of sex hormones by inhibiting the synthesis of testosterone from cholesterol,thereby affecting the development of reproductive systems.

OBJECTIVE:To ascertain the potential relationship between tea consumption and risk of premenopausal and post-menopausal breast cancer. METHODS:Web of Science,PubMed,Medline,CNKI,VIP and WanFang database were independently searched from January 1st, 1996 to August 31st, 2017 by using "tea OR catechin OR tea polyphenol" AND "breast OR mammary" AND "cancer OR tumor OR carcinoma" as key words. The combination of the effect values and the heterogeneity test were performed using Stata 11.0 software. Each study was scored using the NOS (New Castle Ottawa Scale),and the publication bias was calculated using the funnel and Egger regression. RESULTS:A total of 9 studies (5 case-control studies;4 cohort studies) were included. The studies showed that tea intake was insignificantly associated with risk of breast cancer (OR=1.01,95% CI:0.86-1.18),the highest tea intake was also insignificant for risk of premenopausal (OR=0.92,95% CI:0.82-1.04) and postmenopausal breast cancers (OR=1.11,95% CI:0.93-1.33). In addition, we did not identify any statistically significant association between tea consumption and breast cancer risk in all the subgroup analyses (P < 0.05). CONCLUSION:In conclusion,our findings indicate that menopausal status had no effect on tea consumption and breast cancer risk. But the current evidence is still relatively limited. Therefore, large sample,multi-center cohort or case-control studies should be conducted to further clarify the accuracy of this conclusion.

OBJECTIVE:To investigate embryo toxicity and teratogenicity of a silicone adsorbent for cold turbidity of beer on rats. METHODS:SD rats during pregnancy were given intragastric administration of silicon-based sorbent at 0.83,1.67 and 3.33 g/kg,respectively. After continuous administration of 10 d,growth of pregnant rats, embryonic development,fetal appearance,visceral deformities,and skeletal deformities were monitored. RESULTS:There was no significant difference in the observation indexes between pregnant rats and fetal rats in each dose group and the negative control group (all P > 0.05). CONCLUSION:The silicon sorbent did not show reproductive toxicity nor teratogenic effects in SD rats.