OBJECTIVE: This study aimed at identifying common genomic DNA alterations in esophageal squamous cell carcinoma (ESCC) which can be used as molecular markers for the detection and prognostic determination of the disease. METHODS: Genomic DNAs were extracted from tumors and morphologically normal operative margins of 112 ESCCs. Polymerase chain reactions (PCR) and polyacrylamide gel electrophoresis (PAGE) were performed to detect alterations of six microsatellite sites on chromosomes 3p and 13q. Loss of heterozygosity (LOH) of the microsatellites was further analyzed in combination with the frequently mutated genes that had been reported in our previous study. RESULTS: Among the detected microsatellite sites,D3S1768 had the highest LOH frequency (48.9%),while D3S2452 had the lowest (28.8%). When conjointly analyzed with the frequent mutations,we discovered an optimal five-marker combination including LOH of D3S1768,D13S171,D13S1493,and mutations of TP53 and TTN. Alteration of any two markers arising together in this combination had a frequency of 75.6% which was much higher than that of any single marker. The results of survival analysis showed that there was no statistical correlation between LOH of the microsatellites in this group of ESCC and survival of the patients. However,the mutations of PBRM1 and SYNE2 mainly presented in the case of short-survival. When mutations of these two genes were combined with lymph node metastasis,the overall survival time of ESCC patients with at least two positive indicators was significantly shorter than those with only one positive index or negative ones (P=0.027). CONCLUSION: A high LOH frequency was detected with microsatellite D3S1768 in ESCC. A five-marker combination,including TP53,increased the sensitivity of detecting ESCC. PBRM1 and SYNE2 mutations combined with lymph node metastasis might become an index of ESCC prognosis.

OBJECTIVE: To investigate the effect of Lactobacillus casei on 7,12-dimethylbenz(a)anthracene (DMBA) induction of breast cancer in rats. METHODS: Female Spraque-Dawley rats were randomly divided into control group (C),model group (M),low dose group of Lactobacillus casei (L1) and high dose group of Lactobacillus casei (L2). The rats were subcutaneously injected with 100 mg/kg DMBA to establish breast cancer rat model. 4 and 8 mL/kg Lactobacillus casei were administered to L1 and L2 by gavage,respectively,1 time/day,for 16 weeks in total. The latency of breast cancer in rats and Lactobacillus casei's tumor suppression rate were determined. The percentages of T lymphocytes and natural killer cells were quantified in peripheral blood of rat by flow cytometry. In addition,thymus index,spleen index and the level of interleukin 4(IL-4),IL-6,IL-10,IL-12,interferon γ (IFN-γ),and tumor necrosis factor α (TNF-α) in serum were evaluated. RESULTS: No tumor was detected in group C,whereas tumors were formed in the remaining three groups. Compared with the model group,the L2 group had extended latency of tumors,reduced tumor incidence and tumor weights (P < 0.05). In addition,the tumor inhibition rate in the same group reached 41.2%,the thymus index was increased significantly (P < 0.05),the percentage of TCRαβ⁺ CD161a⁺ NK cells and the CD3⁺CD8⁺ T cells were both increased significantly (P < 0.05),while the proportion of CD3⁺Foxp3⁺ T cells was decreased significantly (P < 0.05). ELISA results showed that,compared with the control group,the concentration of IL-4 was decreased in the model group while the concentrations of IL-6,IL-12 and TNF-α were increased (P < 0.05). In the L2 group,the concentration of IL-4 and IL-10 were significantly higher than those in the model group while the concentrations of IL-6 and IL-12 were decreased respectively (P < 0.05). In the L1 and L2 groups,TNF-α concentrations were decreased significantly compared with the model group (P < 0.05). CONCLUSION: Lactobacillus casei supplementation can inhibit the induction of tumors by DMBA in rats. A possible mechanism may involve immune-regulatory effects on the breast cancer rat model via improving the levels of inflammation-associated cytokines and changing the distribution of CD3⁺CD4⁺,CD3⁺CD8⁺ T cells,NK cells and regulatory T cells.

OBJECTIVE: To explore the effects of cistanche phenylethanoid glycosides (CPhGs),acteoside and echinacoside on anti-proliferation and induction of apoptosis in hepatic stellate cells (HSC-T6) in vitro. METHODS: HSC-T6 cells were treated with different concentrations of CPhGs (25,50,100 μg/mL),acteoside (1.5,3,6.0 μg/mL) or echinacoside (125,250,500 μg/mL) for 48 h. The MTT methods was used to measure the anti-proliferation effect and half the inhibition rate (IC₅₀) of HSC-T6. Proportions of apoptosis were assayed by flow cytometry. Western blot was used to detect the effects of the expression of Bcl-2 and Bax. RESULTS: With increasing concentrations of the three chemicals,the rate of inhibition of proliferation increased gradually. IC₅₀ were 119.1,7.0 and 520.3 μg/mL for the three chemicals,respectively. The proportions of apoptosis for the three chemicals were significantly different from that of the untreated controls (P < 0.01). The highest apoptosis rates for CPhGs (at 100 μg/mL),acteoside (6.0 μg/mL) and echinacoside (500 μg/mL) were 24.40%±3.16%,34.73%±1.16% and 21.13%±0.98%,respectively. Except for the 1.5 μg/mL acteoside group,expression of Bcl-2/Bax was significantly decreased (P < 0.05) compared to the normal control group. CONCLUSION: CPhGs,echinacoside and acteoside caused proliferation inhibition and apoptosis. The sequence of the hepato-protective effects of the three chemicals was shown to be acteoside > CPhGs > echinacoside.

OBJECTIVE: To investigate the mechanism of cytotoxicity of CdSe/ZnS quantum dots (CdSe/ZnS QDs) on HepG2 cells. METHODS: Morphological images of CdSe/ZnS QDs were obtained with a transmission electron microscope (TEM) and the fluorescence spectra of the QDs were determined by a spectrophotometer. After treatment with 0,0.5 and 5 nmol/L CdSe/ZnS QDs for 4 hours, the HepG2 cells were imaged under a laser scanning confocal microscope (LSCM) and the uptake efficiency of the QDs was evaluated quantitatively using a flow cytometry machine. After treatment for 24 and 48 hours, the effect on cell proliferation was measured using the MTT assay. The Western blot assay was used to evaluate the effect on apoptosis-related proteins, Casepase-9 and Casepase-3. RESULTS: CdSe/ZnS QDs had a particle size of around 10 nm and the nanocrystals were highly mono-dispersed. The QDs exhibited a broad absorption spectrum from 250 nm to 500 nm and a narrow emission spectrum from 600 nm to 700 nm. The QDs entered cells and remained mostly in the cytoplasm within 4 hours. The uptake efficiency of the QDs was up to 99%. Compared with controls, cells treated with 0.5 and 5 nmol/L QDs for 24 hours exhibited no obvious suppression on cell proliferation but showed highly suppressed cell proliferation 48 hours later (P < 0.05). Compared with controls, the expression levels of Caspase-9 and Caspase-3 were increased in QDs-treated cells. CONCLUSION: The results suggest that CdSe/ZnS QDs which were taken in by HepG2 cells inhibited cell proliferation. The inhibition could be related to the induction of Caspase-9 and Caspase-3 expression.

OBJECTIVE: To identify the relationship among gene copy number and their expression with survival of glioma. METHODS: Based on the gene copy number variation data and mRNA expression information of 95 glioma patients,we organized a new expression profile. Samples were divided into two groups by the copy number variation and by using t test differential genes. The data were annotated into 300 pathways from KEGG pathway database. Applying with the Subpathway-GM method,we identified risky subpathways. Kaplan-Meier method and Log-rank test were used to predict genes which were highly related to the survival of glioma. RESULTS: 2 219 differential genes were obtained by using t test,as well as 48 risky subpathways by Subpathway-GM method. Studying the relathion between the 14 genes precticted by using survival method and the top 10 risky subpathways,we found that MDH2,YWHAE,SHB2B, CACNG4 and CACNA1G could be regard as survival markers of glioma. CONCLUSION: By combining glioma patients' copy number variation of gene,their expression information and the topological structure of the pathways,we identified some survival markers for the diagnosis and treatment of glioma.

OBJECTIVE: To investigate the role of protein kinase C (PKC) and c-Jun N-terminal kinase (JNK) in modulating lysophosphatidic acid (LPA)-induced monocyte chemo-attractant protein-1 (MCP-1) expression in human fetal lung fibroblasts (HLF-1). METHODS: Cultured human lung fibroblasts (HLF-1) were incubated with LPA(0,1,3 and 10 μmol/L) for 0.5,6,12 and 24 h. ELISA was used to detect MCP-1 protein levels in the supernatants,and quantitative real-time PCR (qPCR) for MCP-1 mRNA levels in the cell lysate. In addition,cells were pre-incubated with PKC inhibitor bisindolylmaleimide I (0,0.1,1 and 10 μmol/L) or JNK inhibitor SP600125 (0,0.1,1 and 10 μmol/L) for 30 min,and then treated with LPA (10 μmol/L) for 2 or 6 h,and ELISA and qPCR assays were conducted. Cells were also pre-incubated with PKC inhibitor bisindolylmaleimide I (1 μmol/L) for 30 min,and then treated with LPA (10 μmol/L) for 30 min. C-Jun N-terminal phosphorylation levels in the cell lysate were measured by Western blot. RESULTS: LPA stimulated MCP-1 protein expression in dose- and time-dependent manners. The MCP-1 protein production in the LPA (10 μmol/L) treated group was 2.4 times higher than that in the untreated group (P < 0.01). MCP-1 protein production in the LPA (10 μmol/L) treated group for 24 h was 1 time higher than that in the untreated group (P < 0.01). LPA stimulated MCP-1 mRNA expression in a time-dependent manner. The MCP-1 mRNA expression in the LPA (10 μmol/L) treated group for 2 h was 5.3 times higher than that in the untreated group (P < 0.01). PKC inhibitors bisindolylmaleimide I and JNK SP600125 clearly inhibited LPA-induced MCP-l mRNA and protein expressions. The LPA (10 μmol/L)-induced MCP-1 protein production was reduced by 60% with bisindolylmaleimide I(1 μmol/L) compared with that in the control group (P < 0.05). With 3 μmol/L of bisindolylmaleimide I,the induced MCP-1 mRNA expression was reduced by 40% (P < 0.05). The LPA (10 μmol/L)-induced MCP-1 protein production was reduced by 78% with SP600125 (1 μmol/L) (P < 0.05). The 10 μmol/L LPA-induced MCP-1 mRNA expression was reduced by 40% with SP600125 (1 μmol/L) (P < 0.05). The activity of JNK was enhanced by LPA in HLF-1 while PKC inhibitors bisindolylmaleimide I (1 μmol/L) suppressed the activity of JNK which was induced by LPA(10 μmol/L). CONCLUSION: PKC and JNK mediated LPA-induced MCP-1 expression in HLF-1.

OBJECTIVE: To study the anti-tumor effect of cisplatin by chrysin (ChR) in vitro. METHODS: Cultured cells from human colorectal cancer (HCT-116),gastric cancer (BGC-823),nasopharyngeal carcinoma (CNE1) and liver cancer (HepG2) were treated by chrysin (40 μmol/L),cisplatin (5.0 μg/mL),and chrysin (40 μmol/L)+cisplatin (5.0 μg/mL) separately for 24 hours. The inhibitory rate of tumor cells was determined by MTT assay. The apoptosis rate of tumor cells was determined by Hoechst 33342 fluorescence staining. The sensitive cell line was identified by the inhibition rate and apoptosis index. The L₉(3³) orthogonal design of three levels of chrysin (10,20,40 μmol/L),three levels of cisplatin (1.3,2.5,5.0 μg/mL),and three time-points (12,24,36 h) was used to screen for the best combination effect of chrysin and cisplatin in the sensitive cells. RESULTS: Among all cell lines,the MTT assay showed that the inhibition rates from exposure to the combination of chrysin and cisplatin were significantly higher than that of the single treatment groups of chrysin and cisplatin (P < 0.01). Similarly,Hoechst 33342 staining showed that the apoptosis rates from the combined treatment were significantly higher than those from the single treatment groups (P < 0.01). From the combined treatment,the inhibitory rate in BGC-823 was (78.0±2.0)% and the apoptosis index was (72.3±6.5)%,indicating that this was the most sensitive among all tested cell lines. The results of in vitro orthogonal experiment showed that the most effective conditions were:40 μmol/L chrysin,5.0 μg/mL cisplatin,and 24 hours of treatment time. CONCLUSION: Chrysin enhanced the inhibitory effect of cisplatin via inhibition of cell proliferation and promotion of apoptosis in human tumor cell lines. Among the tested cell lines,the human gastric cancer cell line BGC-823 was the sensitive one to the combined effect of chrysin and cisplatin.

OBJECTIVE: To investigate the role of DNA methylation as a possible mechanism of Benzo[a]pyrene (BaP) induction of malignant transformation in vitro. METHODS: Human bronchial epithelial cells (16HBE cell line) were treated with different concentrations BaP (0,10,20 and 40 μmol/L) for 1,9 and 15 weeks. Then,5-methylcytosine immunofluorescent assay was used to detect genomic DNA methylation level,Western blotting and RT-PCR were used to detect protein and mRNA levels of DNMT1,DNMT3a,DNMT3b and MBD2. RESULTS: BaP treatment decreased expression of 5-mC (methylation) in 16HBE cells in a dose- and time-dependent manner. The protein and mRNA levels of DNMT1 showed in a significant dose- and time-dependent reduction (P < 0.05),but there were no signi fi-cant changes in protein levels of DNMT3a,DNMT3b and MBD2(all P > 0.05). CONCLUSION: BaP treatment decreased the global DNA methylation levels in 16HBE cells and reduction of DNMT1 expression could play an important role.

OBJECTIVE: To observe the effect of RNAi silenced p53 gene on the expression of nuclear transcription factor STAT3 in cervical cancer HeLa cells,and on their proliferation and invasion. METHODS: 3 kinds of short hairpin RNA (shRNA) which target the p53 gene were constructed and were used for liposome-transfection into HeLa cells. Then,expression of p53 were detected by PCR and Western blot. After silencing the p53 gene,expression of STAT3 was detected by PCR and Western blot. Proliferation and invasion of HeLa cells were detected by the MTT and Transwell assays. RESULTS: The transfection efficiency of p53-shRNA3 group was 85.7%. After the transfection,the expression of p53 mRNA and protein were 0.23±0.05 and 0.41±0.11,respectively,significantly lower than the non-transfection,empty plasmid,p53-shRNA1 and p53-shRNA2 groups (P < 0.05). In addition,expression of STAT3 mRNA and protein of the p53-shRNA3 group were 1.09±0.21 and 1.46±0.25,respectively,significantly higher than non-transfection group and empty plasmid group (P < 0.05). MTT results showed that at 24,36,48 h and 72 h,cell proliferation ability of the p53-shRNA3 group was significantly higher than the non-transfected group and empty plasmid group (P < 0.05). Transwell results showed that the number of cells in the p53-shRNA3 group (0.05±59.36) was significantly higher than the non-transfection group (15.73±1.29) and the empty vector group (16.01±1.34) (P < 0.05). CONCLUSION: Silencing the p53 gene can significantly increase the expression of STAT3,and promote the proliferation and invasive ability of HeLa cells.

OBJECTIVE: To investigate the mechanism and effect of catechin on cisplatin (DDP)-induced oxidative damage in HEK293 cells. METHODS: Survival HEK293 cells in vitro after their exposure to catechin or DDP was determined. In addition,the effect of catechin pretreatment on DDP-induced cell growth inhibition was determined by the MTT method. Superoxide dismutase (SOD) activity was examined by the xanthine oxidase method. Malondialdehyde (MDA) content was measured by the thiobarbituric acid method. Glutathione (GSH) content was determined by the nitrobenzoic acid method. RESULTS: Cell survival rate decreased gradually as the DDP concentrations increased. Pretreatment with catechin caused increased cell survival initially but decreased gradually afterward. The initial increase was not significant (P > 0.05). However,catechin treatment significantly increased the intracellular MDA content,but decreased the SOD activity and GSH level compared with that in the DDP control group (P < 0.05). CONCLUSION: Although catechin pretreatment did not improve the survival rate of DDP-treated HEK293 cells,it alleviated the DDP-induced oxidative damage.

OBJECTIVE: To investigate the expression of miR-19a and miR-19b in osteosarcoma and adjacent non-cancerous tissues,and their correlation with clinicopathologic characteristics. METHODS: 32 cases of osteosar-coma tissues and paired adjacent non-cancerous tissues were collected. qPCR was used to detect the expressions of miR-19a and miR-19b in the two tissues. Correlation between expression of the miRNA and clinical pathologic changes was performed. RESULTS: Expression of miR-19a and miR-19b was significantly up-regulated in osteosarcoma tissues as compared with the adjacent non-cancerous tissues. Their expression nucleic acid levels was positively and significantly correlated with pathologic gradings (all P < 0.05). The expression of the miR-19a was also positive correlated with clinical stage of osteosarcoma and related to the prognosis (r=0.685,P < 0.05). Multivariate analyses suggested that miR-19a expression was an independent predictor for overall survival (P=0.037). CONCLUSION: Our study showed that miR-19a and miR-19b may play an important role in the tumorigenesis and progression of osteosarcoma. In particular,miR-19a may serve as a prognostic marker and therapeutic target for osteosacoma.

OBJECTIVE: To investigate changes in expression of the tumor stem cell marker LGR5 gene before and after neoadjuvant chemoradiotherapy and its predictive effect on neoadjuvant chemoradiation. METHODS: Ninety-four rectal cancer patients with preoperative chemoradiotherapy were enrolled in the Affiliated Tumor Hospital of Xinjiang Medical University between January 2014 and February 2016. Quantitative real-time PCR(qRT-PCR) method was used to determine the expression of LGR5 mRNA before and after neoadjuvant chemoradiotherapy. Overall survival(OS) rates were estimated by the Kaplan-Meier method and compared using the Log-rank test for univariate analysis. Cox regression analysis was performed to provide multivariate analysis. RESULTS: Tumor pathological regression reaction of rectal cancer after neoadjuvant chemoradiothepy was good. The efficiency was at 87.2%,and 18.1% patients achieved pathological complete response rate (pCR) after neoadjuvant chemoradiothepy. The expression level of LGR5 mRNA in rectal carcinoma tissues after chemoradiothepy (8.847±6.664) was significantly less than that before chemoradiothepy (14.396±9.924;P < 0.05). The expression level of LGR5 mRNA after chemoradiothepy was up-regulated in 27 patients and down-regulated in 67 patients. Among them,the down-regulated group had significantly better tumor regression degree and 1,2 year-survival rates than that of the up-regulated group. Univariate analysis showed that several factors such as the CA19-9 levels before chemoradiothepy, clinical stage,grade of TRG and expression changes of LGR5 mRNA were significant prognostic factor for OS. CA19-9 levels before chemoradiothepy and expression changes of LGR5 mRNA showed significant differences in multivariate analysis. CONCLUSION: Neoadjuvant chemoradiotherapy caused changes in the expression of LGR5 mRNA. Our study shows that LGR5 mRNA expression changes before and after neoadjuvant chemoradiation and levels before chemoradiothepy had the prognostic potential on the effect of neoadjuvant chemoradiation for locally advanced rectal cancer.

OBJECTIVE: To evaluate genotoxicities of chromium (tri- and hexavalent states) using a new yeast mutation SUP4-o assay system. METHODS: YCpMP2 plasmid was transferred into MKP-o (SJR576) to obtain SJR576-p strain by electroporation. After treatment of the yeast cell SJR576-p with the same concentrations of Cr³⁺ and Cr⁶⁺ for 24 h,the cells were coated on canavanine resistance screening plates in uracil synthetic medium (SC-Ura),and the number of clones were subsequently counted. RESULTS: Compared with the untreated controls,the mutation rates of the treated groups with potassium dichromate and chromium trichloride increased five times. The values were about 23 and 26 parts per million,respectively. While there was no significant difference between the groups treated with Cr³⁺ and Cr⁶⁺. CONCLUSION: Our data suggest that Cr³⁺ can cause DNA damage and genotoxicity. Additionally,the yeast SUP4-o mutation assay system is useful for detecting genotoxicity.

OBJECTIVE: The teratgenic and toxic effects of chlorella vulgaris tablets were evaluated in SD rats for health food safety concerns. METHODS: Pregnant rats were randomly assigned to chlorella vulgaris tablet groups (1 875,3 750,7 500 mg/kg),positive control group (300 mg/kg aspirin) and negative control group. Rats were treated on the 7^th to the 16^th day during the gestation period and were killed on the 20^th day. Indicators for teratogenicity were examined:weight of uterus with embryo,fetal weight and body length,proportion of male and female fetuses,number of implantation,live fetuses,dead fetuses and reabsorbed fetuses,the morphology of fetuses,and abnormalities in the fetal skeletons and internal organs. RESULTS: Aspirin dose of 300 mg/kg produced maternal toxicity,embryonic toxicity and substantial teratogenic effects. The groups treated with doses of 1 875,3 750 and 7 500 mg/kg of the tablets did not produce maternal toxicity,embryonic toxicity nor teratogenicity. None of the observed effects was significantly different from the negative control group (P > 0.05). CONCLUSION: Chlorella vulgaris tablet did not cause maternal toxicity,embryonic toxicity,or teratogenicity in pregnant rats.