OBJECTIVE:The purpose of this study was to investigate the relationship between the expression of Cdc25B and IER5 proteins in irradiated HeLa cells. METHODS:HeLa cells and IER5-knockdown Hela cells were irradiated with 4 Gy of γ rays and were harvested at 0,0.5,2,4,8,12,16,24,and 36 h after irradiation. Expression of IER5 and Cdc25B proteins was examined by Western blot. G2 cell cycle arrest was examined by flow cytometry. Expression level of Cdc25B mRNA was examined by real-time quantitative PCR. Unexposed cells were used as controls. RESULTS:The qPCR results show that within 0.5-24 h after irradiation of the normal HeLa cells the Cdc25B mRNA was lower from 0.5 h the time-pointand then increased after 2 h,the difference was statistically significant (P < 0.05);In IER5 knockdown Hela cells,Cdc25B mRNA decreased first and then increased after 0.5 h,the difference was statistically significant (all P < 0.05,except 2 h). The Western blot results show that within 0.5-24 h after irradiation of normal HeLa cells,the Cdc25B protein began to rise from the 2 h time-point,then began to decrease at 8 h,the difference was statistically significant (all P < 0.05,except 0.5 h). In IER5 knockdown Hela cells,Cdc25B began to decrease from the 0.5 h time-point,then began to rise at 8 h gradually,the difference was statistically significant (all P < 0.05,except 24 h). Compared with the control group, IER5 protein was upregulated in two cells types after their irradiation and during the timepoints mentioned above. After radiation,the level of IER5 in HeLa began to increase significantly from the 8 h time-point while IER5 knockdown Hela cells from the 0 h. The differences were statistically significant (all P < 0.05). The level of Cdc25B in HeLa began to decline significantly from 8 h,while IER5 knockdown Hela cells from 0 h. The differences were statistically significant (P < 0.05). The protein expressions of Cdc25B and IER5 were negatively correlated at the 8 h and 0 h ime-points of radiation (R₁=-0.740,R₂=-0.669,P < 0.01,respectively),while the expression of Cdc25B was decreased. The ratio of G2 cell cycle arrest was increased in the two cells lines (P < 0.05). CONCLUSION:Expression of Cdc25B protein in HeLa cells was induced by Cdc25B mRNA changes,and the expression of Cdc25B was negatively related with the expression of IER5 protein.

OBJECTIVE:To investigate changes of ROS and mitochondria on epithelial-mesenchymal transition. METHODS:Human lung cancer A549 cells were divided into control and treated groups. The latter were treated with different concentrations of TGF-β1 (0,2.5,5,10,20,40 ng/mL) and morphological changes were observed after 48 hours. In addition,cell viability was detected by using the CCK-8 kit and cell apoptosis by flow cytometry. DCFH fluorescent probe,MitoSOX fluorescent probe and Rhodamine-123 staining were used to detect ROS and changes in mitochondrial membrane potential, respectively. The content of ATP was detect by using the enhanced ATP kit. The epithelial-mesenchymal transition-related markers and mitochondrial-related proteins were detected by Western blot. RESULTS:The treated groups showed major differences from the control group:cell viability increased gradually and showed a dose-effect relationship with increased concentrations of TGF-β1 (r=0.941,P < 0.05);there was a significant difference on cell apoptosis after treatment with 5-20 ng/mL TGF-β1;mesenchymal cells were observed after treatment with TGF-β1 for 48 hours, indicating the appearance of epithelial mesenchymal transition;Western blot showed that the expression of epithelial marker E-cadherin decreased gradually and the expression of mesenchymal marker α-SMA increased gradually (P < 0.05);the detection of intracellular reactive oxygen showed that treatment with 5-20 ng/mL TGF-β1 induced generation of ROS and mitochondrial ROS (P < 0.05);mitochondrial membrane potential declined as indicated by fluorescence intensity (P < 0.05);the content of ATP also decreased (P < 0.05);and expression of mitochondrial-related proteins were downregulated (P < 0.05). CONCLUSION:TGF-β1 caused increase of reactive oxygen species which led to the occurrence of mitochondrial dysfunction and promotion of EMT.

OBJECTIVE:To investigate the effects of microcystin-LR on viability and apoptosis of human normal esophageal epithelial cells,and on expression of Caspase-3 and Caspase-9. METHODS:Human normal esophageal epithelial cells were treated with microcystin-LR at different concentrations (1,3.75,7.5,15,30,50 μg/mL) for 24 h,and cell viability was detected by the MTT assay. The data were used to calculate the half inhibitory concentration (IC₅₀) which was used to determine the MC-LR concentrations for subsequent experiments. Accordingly,cells were treated with 6,12 and 24 μg/mL MC-LR,and apoptosis was detected by PI-Annexin V double staining. Expression levels of Caspase-3 and Caspase-9 were detected by Western blot. RESULTS:Compared with the control group,microcystin-LR of 3.75-50 μg/mL decreased the viability of normal esophageal epithelial cells (P < 0.01);the 6-24 μg/mL concentrations increased the apoptosis rate (P < 0.05 or P < 0.01),and the expression of Caspase-3 and Caspase-9 (P < 0.01). CONCLUSION:Induction of apoptosis in human normal esophageal epithelial cells by microcystin-LR may be related to its activation of Caspase-3 and Caspase-9.

OBJECTIVE:To investigate the expression of long non-coding RNA EMX2OS (LncRNA EMX2OS) during glycidyl methacrylate (GMA)-induced malignant transformation of 16HBE cells. METHODS:Malignantly transformed 16HBE cells (induced by exposure to 8 μg/mL GMA) and solvent control cells (exposed to DMSO) at around the 30th generation were harvested. High throughput LncRNA microarrays were used to detect differences in expression profile of LncRNAs between the two groups of cells. Changes in LncRNAs were screened through strategies such as fold changes and neighboring genes analyses. Real-time fluorescence quantitative PCR (qPCR) and gene chip were applied to measure the expression levels of LncRNA EMX2OS and EMX2,respectively. RESULTS:Based on the result of LncRNA microarrays,expressions of LncRNA EMX2OS was up-regulated by 6.01 fold in the transformed cells campared to the control cells. In addition, qPCR analyses showed that LncRNA EMX2OS increased 3.37 fold and EMX2 increased 1.88 fold change (P < 0.05). The trends of increase in EMX2 and EMX2OS trends were identical. CONCLUSION:GMA induced significant increase in expression of LncRNA EMX2OS of 16HBE cells and the expression can be considered as a specific biomarker which is involved in GMA-induced malignant transformation 16HBE cells.

OBJECTIVE:To investigate the expression of TM4SF1 and its prognostic value in hepatocellular carcinoma (HCC) patients. METHODS:Immunohistochemistry was performed to determine the expression levels of the TM4SF1 protein in 120 cases of HCC cancerous tissues and their adjacent noncancerous tissues,as well as 30 normal liver tissues from patients without malignant tumors. Associations between TM4SF1 and clinicopathological features as well as prognosis of HCC patients were analyzed. RESULTS:Expression of TM4SF1 in HCC cancerous tissues was significantly higher than noncancerous tissues(P < 0.01). Expression was higher in the high level AFP,lymph node metastasis,PVTT,stages Ⅲ-Ⅳ,poorly differentiated,recurrence,and distant metastasis groups than the low level AFP,without lymph node metastasis,without PVTT,stages Ⅰ-Ⅱ,high differentiated,no recurrence,and no distant metastasis groups (P < 0.05). The high expression levels of TM4SF1 protein were associated with shorter DFS and OS after surgery (P < 0.01). High TM4SF1 level,PVTT,stage Ⅲ-Ⅳ were independent predictors for DFS and OS (P < 0.05). CONCLUSION:TM4SF1 was highly expressed in HCC tissues therefore it may play an important role in HCC carcinogenesis. High TM4SF1 is potentially valuable for prognosis of HCC.

OBJECTIVE:We investigated the expression of CALCA and TFPI-2,and their association with expression of human papillomaviurs (HPV) 16 coding proteins in cervical carcinoma cells. METHODS:We designed and synthesized small-hairpin RNAs (shRNA) which were specific to the sequences of E6 or E7 genes of HPV16. In addition,we prepared a series of shRNA expression constructs using pRNAi plasmid that could simultaneously express shRNA and green fluorescent protein (GFP) reporter gene. After transient expression of shRNA vectors in HPV16-positive SiHa cervical carcinoma cells by lipid transfection,we analyzed transfection efficiency,inhibition of HPV-coding gene expression and its effect on candidate gene expression using fluorescent imaging and quantitative RT-PCR. RESULTS:Relatively high transfection efficiency was confirmed by using confocal microscopy on cell population based on their release of green fluorescence. According to quantitative RT-PCR analysis,E6 or E7 expressions were,to a very high extent,inhibited after transfection of shRNA vectors. Expressions of CDK4 and BCL-2 were significantly decreased after selective inhibition of E6 or E7 gene expression. These observations suggest that the inhibition of HPV16 coding gene expression might have inhibited cell proliferation and induced cell-cycle arrest. Expressions of CALCA and TFPI-2 were significantly upregulated after inhibition of E7 gene expression but not remarkable after inhibition of E6 gene expression. CONCLUSION:Results of this study together with previous reports suggest that HPV16 E7 protein may promote the downregulation of CALCA and TFPI-2 in cervical carcinoma cells. The mechanism may be involved with HPV-driven cervical carcinogenesis.

OBJECTIVE:To assess the quality of life of advanced esophageal cancer patients who received chemotherapy in the Yancheng area,China and to guide the choice of clinical chemotherapy regimens. METHODS:Data on demographic information,disease and treatment,quality of life scale and EORTC QOL-C30 scale were collected from 49 new primary esophageal cancer cases undergoing chemotherapy. Data were collected at one day before chemotherapy and one week,four weeks after chemotherapy. According to five functional areas and three semiotic areas including six single measurement items,and one general health area,scores of QOL were changed into standard measure to analyze demographic data,disease characteristic,patients' QOL before and after chemotherapy and factors influencing patients' QOL undergoing chemotherapy. Software 17.0 was applied to describe and to analyze data including demographic data,disease characteristic,patients' QOL before and after chemotherapy and factors influencing patients' QOL undergoing chemotherapy. RESULTS:Compared with one day before chemotherapy,function score improved one week after chemotherapy. But symptoms of vomit and nausea,pain,insomnia,diarrhea and financial difficulty got worse in general after chemotherapy(P < 0.05). Except social support function,function score increased,and somatic symptoms aggravated more significantly 4 weeks after chemotherapy than that of one day before chemotherapy(P < 0.05). Compared with one week after chemotherapy,physical,role and overall functions improved 4 weeks after chemotherapy(P < 0.05). But symptoms of vomit and nausea,loss of appetite and diarrhea got worse (P < 0.05). Quality of life was related to age,educational level,residence clinical stage,and different routes of metastasis. CONCLUSION:Patients' quality of life after undergoing chemotherapy was affected by duration of chemotherapy,age and educational level,which can be improved by selecting individual scheme and adopting comprehensive management.

OBJECTIVE:To study the effects of di-(2-ethylhexyl) phthalate (DEHP) on expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) in MCF-7 cells. METHODS:MCF-7 cells in cultured were exposed to DEHP in different doses (0.05,0.1,0.2,0.4,0.8 mmol/L) for 12,24,48 h,or to one dose (0.8 mmol/L) at different times (3,6,12,24,48 h). Expression of 3β-HSD mRNA was detected by real-time PCR,expression of 3β-HSD protein was detected by western blot. RESULTS:After treatment of MCF-7 cells with DEHP at 0.1-0.8 mmol/L for 12-48 h, expression levels of 3β-HSD mRNA were significantly higher than that of the control group (P < 0.01). Treatment with the lowest dose of DEHP treatment (0.05 mmol/L) for 48h also induced significant increase of 3β-HSD mRNA expression (P < 0.05). Our study also show a dose-effect relationship. Expression of 3β-HSD mRNA also showed time-dependent effects after treatment with 0.8 mmol/L DEHP for 6,12,24,48 h (P < 0.01). Expression of 3β-HSD protein was also higher after treatment with DEHP at doses of 0.2-0.8 mmol/L (P < 0.05 or P < 0.01). In the 0.4,0.8 mmol/L dose group,expression of 3β-HSD protein level increased gradually with the increasing doses (P < 0.05 or P < 0.01). CONCLUSION:DEHP induced up-regulation of 3β-HSD mRNA and protein. Our findings indicate that the mechanism of DEHP toxicity may be related to the induction of changes of 3β-HSD during steroid synthesis.

OBJECTIVE:To study the hepatotoxicity of aqueous extract of Polygoni Multiflori Radix with or without incubation with the rat liver microsome. METHODS:Aqueous extract of Polygoni Multiflori Radix was incubated with or without rat liver microsome in vitro and then dried by freeze drying. Liver L02 and HepG2 cells were incubated with different concentrations of the freeze-dried extract(1.5,3,6,12 mg/mL) for 24 h,48 h. Cell vitability was assayed by MTT method. In addition,contents of three main components (trans-2,3,5,4'-tetrahydroxystibene-2-O-β-D-glucopyranoside,emodin-8-O-β-glucopy-ranoside,emodin) from aqueous extract of Polygoni Multiflori Radix after incubation were quantitated by HPLC analysis. RESULTS:The aqueous extract without incubation with rat liver microsome showed more toxicity at the concentration of 12 mg/mL than that with incubation (P < 0.01). Contents of the three main components of aqueous extract incubated with rat liver microsome were lower than those without rat liver microsome. CONCLUSION:Hepatotoxicity of aqueous extract of Polygoni Multiflori Radix was decreased after their metabolism by rat liver microsome. The relationship between reduced contents of the main components of the aqueous extract and the reduction of hepatotoxicity need further study for clarification.

OBJECTIVE:To investigate the regulatory mechanism on proliferation of miR-202 in A549 lung cancer cells. METHODS:Through the lentivirus transfection into A549 cells,up-and down-expression of miR-202 were investigated. DIANA-TOOLs and KEGG database were used to analyze the bioinformatic of miR-202 expression and to explore the signaling pathways that might be involved with potential target genes. Then,MTT assay and flow cytometry technology were applied to detect the influence of miR-202 on cell proliferation,apoptosis and cycle. RT-qPCR and Western blot methods were applied to detect the miR-202 regulation for its predicted target gene on both mRNA and protein levels. RESULTS:Bioinformatics analysis show that,miR-202 was involved in non-small cell lung cancer and other tumor-related pathways,and possibly in regulation of PIK3CA. Functional studies show that,compared with the negative control group,up-regulation of miR-202 inhibited cell proliferation (P < 0.05),caused cell cycle G1/S phase arrest (P < 0.05),but did not affect cell apoptosis (P > 0.05). Target gene study show that,compared with the control group,up-regulation of miR-202 reduced the expression of PIK3CA protein (P < 0.05),but had no effect on the expression of PIK3CA mRNA(P > 0.05). CONCLUSION:Expression of miR-202 influenced the proliferation and cycle of A549 cells through the negative regulation of PIK3CA and the study provides a new clue to the role and mechanism of miRNA in lung cancer.

OBJECTIVE:To explore the influence of the high expression of DNMT1 on MNNG-induced malignant transformation of Kazakh esophageal epithelial cell and to provide theoretical basis for pathogenesis of esophageal cancer. METHODS:Normal esophageal cells in culture were derived from Kazakh esophagus epithelium. MTT and clonal assays were conducted to determine the malignant transformation doses of MNNG. DNMT1 overexpression cells and the normal epithelial cells were exposed to MNNG for one hour. MTT and soft agar colony forming experiment were performed to observe the formation of cell colony under different exposure conditions and different multiplying rate. Nude mouse tumorigenicity assay was conducted to detect malignantly transformed cells. RESULTS:After treatment with MNNG (1.50×10^-5 mol/L), cells with high expression of DNMT1 were able to form colonies in soft-agar medium after the 27th generation in vitro. These cells also formed local mass in nude mice but pathological evaluation showed that the local mass had disorderly structure and without squamous cell carcinoma morphology. On the other hand,innoculation of epithelial cells with high expression of transformed cells induced tumors in nude mice which showed squamous cell carcinoma morphology. CONCLUSION:A malignant transformation model of Kazakh esophageal epithelial cell with high DNMT1 expression was successful established. In addition,the model showed that DNMT1 high expression could promote induction of malignant transformation by MNNG.