OBJECTIVE: To establish a pollutant-responsive gene-specific pyrosequencing assay for quantitative measurement of DNA methylation in peripheral blood lymphocytes of coke-oven workers. METHODS: We firstly analyzed DNA methylation levels of RAS-associated domain family 1 (RASSF1A) in 69 coke-oven workers and 46 controls using bisulfate sequencing method. The hyper-methylated hotspots in the exposure group were recognized as the target region for design of specific primers of pyrosequencing. As a pilot study,we examined the methylation status of RASSF1A in 11 paired coke-oven workers and controls. RESULTS: Pyrosequencing was the optimized assay for DNA methylation detection. Compared to BSP assay,pyrosequencing exhibited advantages in efficiency,simplicity,accuracy and economical performances. We were able to distinguish occupational exposed workers from controls effectively using this method. The methylation level of RASSF1A in coke-oven workers was increased by 87.15% compared to that in the controls (9.32%±3.82% and 4.98%±2.28%,respectively,P < 0.01). CONCLUSION: Compared to the BSP assay, gene-specific pyrosequencing method exhibited strength in specificity and accuracy of methylation detection of environmental pollutant-responsive gene in PAHs-exposed workers.

OBJECTIVE: To investigate expression status of KIAA1522 in colorectal cancer (CRC),and its correlation with clinicopathological features and prognosis. METHODS: Expression of KIAA1522 protein was determined with tissue microarrays-immunohistochemistry (TMA-IHC) technique in surgically-resected colorectal cancer tissues and adjacent normal epithelia from 96 patients. The relationship between KIAA1522 expression and clinicopathological characteristics and prognosis of the patients were analyzed. RESULTS: KIAA1522 protein was mainly localized in the cytoplasm of colorectal epithelial cells. While only 12.5% (12/96) of normal colorectal epithelia displayed positive staining,81.3% (78/96) of CRC specimens showed over-expression of KIAA1522. The difference between them was highly significant (P < 0.001). Kaplan-Meier survival analysis revealed that high level of KIAA1522 expression was correlated with lower 3-year disease free survival (DFS) rate of CRC patients (P=0.017). Further more,KIAA1522 up-regulation was significantly correlated with distant metastasis (P=0.012). More importantly,Cox regression analysis indicated that KIAA1522 protein expression was an independent prognosis factor in CRC (P=0.020). CONCLUSION: KIAA1522 was frequently up-regulated in CRC,and its over-expression was significantly correlated with distant metastasis and poor prognostic,thus KIAA1522 over-expression may served as a new prognostic marker in colorectal cancer.

OBJECTIVE: The influence of proanthocyanidins on oxidative stress,secretion of amyloid protein Aβ_1-42 and soluble amyloid precursor protein sAPPα which were induced by amyloid protein fragment Aβ_25-35 in SH-SY5Y cells was investigated. METHODS: SH-SY5Y cells were treated with Aβ_25-35 of different concentrations (0.1,1.0,10.0 and 20.0 μmol/L) for 24 hours. Cell viability was measured by MTT assay. Cell injury models were built with 1.0 μmol/L Aβ_25-35,with proanthocyanidin of different concentrations added to cell cultures and incubated for 24 h. Concentrations of Aβ_1-42 and sAPPα in cell supernatant were determined by using ELISA. Intracellular content MDA and total SOD were determined by the method of TBA test and WST-8,respectively. RESULTS: Compared with the control group,cells treated with Aβ_25-35 (1 μmol/L) had significantly declined cell viability (P < 0.01),increased Aβ_1-42 level (P < 0.01),declined sAPPα concentration (P < 0.01),increased intracellular MDA content (P < 0.01),and decreased total SOD vitality (P < 0.01). Compared with the Aβ_25-35 injury cell models,proanthocyanidin (0.1,0.5,1.0 and 5.0 μg/mL) significantly improved cell viability (P < 0.05),decreased the secretion of Aβ_1-42 (P < 0.01). Proanthocyanidin (0.5,1.0 and 5.0 μg/mL) increased sAPPα concentration (P < 0.05),decreased MDA content (P < 0.05),and strengthened total SOD vitality (P < 0.05). CONCLUSION: Proanthocyanidin alleviated the Aβ burden and oxidative stress in SH-SY5Y cells which were induced by Aβ_25-35.

OBJECTIVE: To investigate the effects of cinobufacini on proliferation,invasion and chemotherapy sensitivity of human esophageal carcinoma,and to analyze its impact on epithelial-mesenchymal transition and its mechanism. METHODS: The effects of cinobufacini on proliferation and chemotherapy sensitivity of human esophagus cancer ECA109 cells were measured by the methyl thiazolyl tetrazolium (MTT) assay. After treatment with cinobufacini,cell apoptosis was analyzed and chemotherapy sensitivity by flow cytometry (FCW). In addition,transwell assay was employed to examine viability,migration and invasion. The effects of cinobufacini on invasion,epithelial-mesenchymal transition of ECA109 and multidrug resistance (MDR)-related proteins were examined by Western blot assay. RESULTS: Cinobufacini treatment significantly inhibited ECA109 cell proliferation in both time- and dose-dependent ways (r=0.985,P=0.000;r=0.988,P=0.000),and enhanced chemotherapy sensitivity compared to that in the control group (P=0.000). Cell migration and invasion were significantly suppressed (both P=0.000). In addition,the treatment also down-regulated the expression levels of Snail,Twist,MMP2,Vimentin and P-gp,but up-regulated the expression of E-cadherin. CONCLUSION: Cinobufacini effectively inhibited the proliferation and invasion of human esophageal carcinoma ECA109 cell,and enhance its chemotherapy sensitivity in culture. The mechanism might be partly related to the inhibition epithelial-mesenchymal transition by cinobufacini.

OBJECTIVE: The aim of this study was to detect the expression of Syntenin in non-small cell lung cancer (NSCLC) tissues and to explore its clinical significance. METHODS: We detected expression of Syntenin in 147 NSCLC tissues and 147 para-carcinoma tissues by tissue microarray and immunohistochemical staining method,and analyzed the relationship of Syntenin expression with clinicopathological parameters and prognosis of NSCLC patients. RESULTS: Expression level of Syntenin (positive rate 49.7%) in NSCLC tissues was significantly higher than that in adjacent lung tissues (positive rate 4.1%,P < 0.01). Over-expression of Syntenin was associated with lymph node metastasis,clinical stage and pathological type in NSCLC patients (P=0.039,P=0.038 and P=0.024),but not with age or gender. The Kaplan-Meier survival analysis revealed that Syntenin expression in primary tumors was significantly associated with patients' overall survival (P=0.028). CONCLUSION: Syntenin protein was over-expressed in NSCLC tissues,and was associated with poor prognosis of patients.

OBJECTIVE: To investigate expression of Sox17,β-catenin and cyclin D1 proteins in gastric cardia adenocarcinoma(GCA),and to establish their relationship with pathogenesis of GCA. METHODS: Immunohistochemistry method was applied to examine the expression of Sox17,β-catenin and cyclin D1 proteins in 78 samples of GCA and the corresponding adjacent non-cancerous tissues．RESULTS: In GCA tissues,the frequency of Sox17,β-catenin and cyclin D1 expression was 47.4% (37/78),83.3% (65/78) and 70.5%(55/78),respectively,which was significantly lower than that in corresponding adjacent non-cancerous tissues:69.2%(54/78),62.8%(49/78) and 44.9%(35/78),(P < 0.01). The frequency of β-catenin ectopic expression was 83.3%(65/78) in GCA tissues which was significantly higher than that in corresponding adjacent non-cancerous tissues of 62.8%(49/78) (P < 0.01). Sox17 expression was negatively correlated with ectopic expression of β-catenin and cyclin D1 (r=-0.264,P < 0.05;r=-0.287,P < 0.05). Sox17 were significantly correlated with tumor stage (P=0.045). β-catenin and cyclin D1 were significantly correlated with lymph node metastasis(P < 0.05). CONCLUSION: Low level expression of Sox17 protein may play an important role in the development of GCA via aberrantly activating the canonical Wnt/ β-catenin signaling pathway through influencing the expression of β-catenin and cyclin D1.

OBJECTIVE: We used the Japanese big-ear rabbits to study the distribution and accumulation of strontium in their different organs. METHODS: 24 Japanese big-ear rabbits were randomly allocated into one blank group and five experimental groups. Rabbits of the blank group (group 1) were fed with deionized water while the experimental group were fed with deionized water plus the addition of strontium chloride hexahydrate every other day. The concentration of strontium chloride was 0.002 g/mL. The volume of water with strontium that was given to the rabbits from groups 2 to 6 were 1,2,3,4 and 5 mL respectively. After 30 days of oral treatment,samples of blood,liver and back leg tibialis were taken. All samples were dried,mashed and decompounded by nitric acid. The concentration of strontium was analyzed by inductively coupled plasma atomic emission spectrometry (ICP-AES). RESULTS: Plasma strontium concentrations in rabbits positively related to oral treatment dose with strontium chloride. R2 of linear fitting method was 0.82. Liver strontium concentrations in rabbits showed no significant correlation with oral treatment dose. Back leg tibialis strontium concentrations in rabbits positively related to oral treatment dose with strontium chloride. R2 of linear fitting method was 0.98. Plasma strontium concentrations positively related to back leg tibialis strontium concentrations. R2 of linear fitting method was 0.76. CONCLUSION: Strontium was absorbed by the body from the gut,transferred through plasma and concentrated in bone.

OBJECTIVE: To explore the relationship between the human leukocyte antigen (HLA) DRB1,DPA1 low resolution type alleles and susceptibility of Uighurs to develop Hodgkin's lymphomas (HL). METHODS: Using a case-control study design,DNA direct sequencing classification method (SBT) was used to detect alleles in HLA-DRB1 and expression of HLA-DPA1 gene among 40 cases of Uighur patients and 80 healthy controls. The HLA-DRB1 and DPA1 allele frequencies between the two groups were compared. The correlation between them with the pathogenesis of HL was analyzed. RESULTS: HLA-DRB1 and DPA1 low resolution alleles were detected 12,3 types in the case group and 13,4 types in the control group,the frequency distribution of which met the Hardy-Weninberg genetic balance test (P > 0.05). The results show that HLA-DRB1*15,DPA1*03 and DPA1*02-DRB1*13 gene frequencies were significantly more frequent in the Uighur patients than the controls (P=0.016,OR=3.476;P=0.002,OR=4.248;P=0.037,OR=3.028,respectively);HLA-DRB1*07 gene frequencies between the two groups were statistically significant (P=0.025,OR=0.314). CONCLUSION: HLA-DRB1,DPA1 gene polymorphism may be closely associated with the onset of HL among Uighurs and with pathogenesis of HL.

OBJECTIVE: To investigate the expression and clinical significance of long non-coding RNA (LncRNA) XIST and miR-101-3p in cervical cancer. METHODS: Cervical cancer tissues and matched non-tumor para-neoplastic tissues were collected from 58 patients. Quantitative real-time PCR was used to examine the expression of LncRNA XIST and miR-101-3p,the correlation between them and their correlation with clinicopathological features were analyzed. RESULTS: LncRNA XIST expression was significantly up-regulated in cervical cancer tissues than in adjacent non-tumor tissues (P < 0.05). The expression of miR-101-3p was significantly down-regulated in cervical cancer tissues as compared with the adjacent tissues (P < 0.05).The expression of them in nucleic acid levels were negatively correlated(r=-0.68,P=0.000) and correlated significantly with FIGO stage,lymph node metastasis,and tissue size of tumor (P < 0.05). CONCLUSION: Our findings indicate that LncRNA XIST and miR-101-3p may be potential prognostic markers and therapeutic targets for cervical cancer. The expression of LncRNA XIST and miR-101-3p was negatively correlated.

OBJECTIVE: To develop new,safe and effective radioprotectors,the protective effects from different water extracts of Carthamus tinctorius were investigated. METHODS: Safflower water extract was prepared with distilled water at 60 ℃ and isolated by HP-20 macroporous resin. Water elution fraction,30%,50%,70% and 95% ethanol elution fractions were separated for the study. The free radical scavenging ability of the different extraction fractions were evaluated by electron spin resonance (ESR) spectra. CCK-8 assay was performed to evaluate the survival rate of human liver L02 cells in vitro. Flow cytometry was applied to study apoptosis,cell cycle and the ratio of Bcl-2/Bax of human lymphocyte AHH-1 after ⁶⁰Co ay irradiation. RESULTS: The ESR results showed that all the different effective parts of safflower water extract had the ability to scavenge free radicals,with IC50 at 1.7-79.7 mg/mL. The survival rates from the 30% and 50% ethanol elution were 128.3% and 130.9% by the CCK-8 assay. The survival rates were significantly higher than the irradiated group and suggested that the two parts had equal radioprotection effect to the water extract of safflower. The decreased apoptosis rate of AHH-1 cells:the 30% fraction (15.3%±1.6%) and the 50% fraction(15.2%±1.9%) were significantly lower than that of the irradiation group (24.7%±4.4%) (P < 0.01). The results of AHH-1 cell cycle analysis showed that the 30% and 50% ethanol elution fractions had significant improvements in the G2/M phase block that was induced by irradiation (P < 0.01). At the same time,the two fractions increased the Bcl-2/Bax ratio (P < 0.05) and inhibited the cell apoptosis. CONCLUSION: Our data show that the 30% and 50% ethanol elution fractions had good radioprotective effects for L02 and AHH-1 cells which were irradiated with ⁶⁰Co γrays. The mechanism for the protection may be related to the elimination of free radicals,the improvement of G2/M cell cycle block,and the regulation Bcl-2/Bax ratio in order to inhibit cell apoptosis. The two fractions should be further studied as potentially safe and effective radioprotectors.

OBJECTIVE: To determine effects of 1,4-BQ on mitochondrial dysfunction and apoptosis in human chronic myeloid leukemia cells (K562 cells). METHODS: K562 cells were treated with 0,10,and 20 μmol/L 1,4-BQ for 24 h. Cell viability was detected by CCK-8 assay,production of reactive oxygen species (ROS) was detected by flow cytometry;function of mitochondria was evaluated by measuring mitochondrial membrane potential and adenosine triphosphate (ATP);and caspase-3 enzyme activity was detected using spectrophotometry. RESULTS: Compared with the control,the relative growth rate of K562 cells in the 1,4-BQ 10 and 20 μmol/L treatment groups decreased with the increased concentration of 1,4-BQ (P < 0.05). Production of ROS and cell apoptosis rates were elevated while mitochondrial membrane potential and the amounts of ATP were reduced. Between the 1,4-BQ 20 μmol/L exposure groups and the control group,the difference was statistically significant (P < 0.05 or P < 0.01). The activity of caspase-3 was increased,and the difference was statistically significant (P < 0.01) between the control and both 1,4-BQ treatment groups. CONCLUSION: 1,4-BQ induced increase of ROS in K562 cells,inhibited their proliferation and resulted in mitochondrial dysfunction and expression of apoptosis. This indicates that mitochondrial dysfunction was involved with 1,4-BQ -induction of apoptosis in K562 cells.

OBJECTIVE: The aim of the study was to observe the effect of iron overload on fertility of sexually mature male rats. METHODS: According to bodyweights,40 healthy male Wistar rats were randomly divided into four groups:control,iron deficiency,low-dose iron overload and high-dose iron overload groups. All rats were injected intraperitoneally every other day. The content of the Fe2+ in these four groups were 0.9,0.3,9 and 18 mg,respectively. After 6 weeks,blood samples from these rats were collected to determine serum iron levels. Testes and epididymides were removed and weighed. Sperm numbers were counted. Sperm mobility and teratosperm rates were calculated. Iron concentration,catalase (CAT),total superoxide dismutase (T-SOD),glutathione peroxidase (GSH-Px) and malonaldehyde (MDA) levels in the testes were determined. RESULTS: Compared with the control group,the serum iron concentration of the iron deficiency group was significantly lower (P < 0.01). However,there was no significant difference in the iron concentrations of the testes (P > 0.05). The serum and testes iron concentrations in the low- and high-doses iron overload groups were both significantly higher than that in the control group (all P < 0.01). There was no significant difference in the sperm count,the sperm motility rate and the teratosperm rate between the iron deficiency group and the control group (all P > 0.05). Compared with the control group,the sperm motility rate in the low-dose iron overload group was significantly decreased (P < 0.01);the sperm count and the sperm motility rate in the high-dose iron overload group were all significantly lower than those of control group and low-dose iron overload group,but the teratosperm rate was significantly higher than that of control group and low-dose iron overload group (all P < 0.05). The rats in the iron deficiency group had significantly lower GSH-Px levels in the testis comparing with the control group. Moreover,compared with the control group,the GSH-Px levels in the testis of the low-dose iron overload group were significantly lower,but the MDA level was significantly higher (all P < 0.05). The CAT and GSH-Px levels in the testis of the high-dose iron overload group were all significantly lower than those of control group and low-dose iron overload group,but the MDA level was significantly higher than that of control group and low-dose iron overload group (all P < 0.05). CONCLUSION: Iron overload can affect the fertility of sexually mature male rats,including reduced sperm count,decreased sperm motility rate and increased teratosperm rate. These adverse effects may be related to the increased oxidative stress level caused by iron overload.

OBJECTIVE: To study the serum level and diagnostic value of Dickkopf-1(DKK1) in patients with adenocarcinoma at the esophago-gastric junction (AEGJ). METHODS: Serum levels of DKK1 in 79 patients with AEGJ and 101 normal controls were measured by enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristics (ROC) was used to calculate its diagnostic value. RESULTS: The serum levels of DKK1 were significantly higher in AEGJ than in normal controls (P=0.001). ROC curves showed that the optimum diagnostic cutoff for serum DKK1 was 2 615.9 pg/mL,providing an area under the curve (AUC) of 0.650 (95%CI:0.569-0.731),a sensitivity/specificity of 34.2%/95.0%. Moreover,serum DKK1 had early diagnostic value for AEGJ [AUC 0.674 (95%CI:0.521-0.827)],35.7% sensitivity and 96.0% specificity. Moreover,the positive rate of DKK1 was not significantly related to age,gender,size of tumor,depth of tumor invasion,lymph node status,or TNM stage (P > 0.05),but related to distant metastasis (P < 0.05). CONCLUSION: Serum levels of DKK1 was high in AEGJ patients which indicates that serum DKK1 may be a potential biomarker for early diagnosis of AEGJ.

OBJECTIVE: To study the clinical characteristics of thyroid cancer in Xinjiang. METHODS: We collected information of 907 cases from the archive and analyzed the difference among the morbidity trend,gender,age,and ethnic groups. RESULTS: The number of thyroid cancer patients had markedly increased year by year. Among the cancer,papillary thyroid cancer (90.96%) was the dominant type. Morbidity of thyroid cancer showed significant differences among various age ranges and gender (male to female ratio of 1/2.88)(P < 0.05). However,both genders had the same age peak (40-49). In this age group,cases increased in women over men. The types of thyroid cancer was different between Han and Uyghur (P < 0.05),with the ratio of papillary thyroid cancer higher in Han than in Uyghur (93.77% and 83%,respectively). CONCLUSION: The incidence of thyroid cancer had increased in the Xinjiang population over the last 10 years. The increase in female was higher than that in male. The peak age was 40-49. The main pathology type was papillary thyroid cancer which showed ethnic differences.

OBJECTIVE: To compare the expression of low molecular-weight proteins (LMP) in breast cancers between Uighur and Han women. METHODS: We collected 165 cases of paraffin-embedded breast fibro adenoma or breast cancer tissues from Uighur and Han women and analyzed the protein expression of LMP2,LMP7 and LMP10 by immunohistochemistry. RESULTS: The total loss rate of LMP2 and LMP7 was significantly increased in breast cancer (67%/46.1%),the normal expression level was reduced with a significant difference (P < 0.01),the alternation trend of the loss rate of LMP10 was common to breast cancer and breast fibro adenoma (breast fibro adenoma 48%,breast cancer 56.5%),and the difference between the two groups is not statistically significant (P > 0.05). The alteration trend of the expression of LMP2,LMP7 and LMP10 was common to women from both Uighur and Han ethnic groups,in Uighur groups (43.1%,43.1%,47.7%),in Han groups (46%,42%,42%) and the difference between two ethnic groups is not statistically significant (P > 0.05). However,LMP2,LMP7 or LMP10 were significantly correlated with tumor lymph node invasion and distant metastasis (P < 0.05),LMP10 was significantly correlated with tumor size (P < 0.01). CONCLUSION: The loss of expression of LMP protein may be an important marker for breast cancer and was common to women from both Uighur and Han ethnic groups. This suggest that the loss may be a molecular mechanism for breast cancer and for both ethnic background.