AIM: To clarify the role of high in normal-1 (HIN-1) gene promoter methylation during gastric cancer development. METHODS: Gastric cancer cell lines and tissue specimens were analyzed for expression of HIN-1 mRNA and ...AIM: To clarify the role of high in normal-1 (HIN-1) gene promoter methylation during gastric cancer development. METHODS: Gastric cancer cell lines and tissue specimens were analyzed for expression of HIN-1 mRNA and protein using the semi-quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The methylation of the HIN-1 gene promoter was detected in gastric carcinoma cells and tissues using methylation-specific polymerase chain reaction. The 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay and flow cytometry were used to assess the changes in behaviors of gastric cancer cells with or without 5-aza-2’-deoxycytidine treatment. RESULTS: HIN-1 was not expressed in 4 of 5 gastric cancer cell lines. The demethylation reagent 5-aza-2’-deoxycytidine was able to induce or upregulate HIN-1 expression in gastric cancer cell lines, which is associated with reduction of tumor cell viability. Furthermore, methylation of the HIN-1 gene promoter was shown in 57.8% (26/45) of the primary gastric cancer and 42.1% (17/38) of adjacent tissue samples, but was not shown in normal gastric mucosa (0/10). From the clinicopathological data of the patients, methylation of the HIN-1 gene promoter was found to be associated with tumor differentiation (P = 0.000). CONCLUSION: High methylation of HIN-1 gene promoter results in silence of HIN-1 expression in gastric cancer. 5-aza-2’-deoxycytidine reverses HIN-1 methylation and reduces viability of gastric cancer cells.展开更多
In order to effectively reduce the chlorophyll content in flue-cured tobacco, improve the overall quality of tobacco leaves, chlorophyllase gene was cloned from Arabidopsis thaliana. After the expression of the expres...In order to effectively reduce the chlorophyll content in flue-cured tobacco, improve the overall quality of tobacco leaves, chlorophyllase gene was cloned from Arabidopsis thaliana. After the expression of the expression vector in E. coil, the recombinant engineering strain was obtained. Afterwards, IPTG （isopropy-β-D-thiogalactopyranoside）was used to induce the goal protein, and the chlorophyllase activity of the recombinant engineering strain was measured, so as to investigate its degradation effect on the chlorophyll in the extracts of tobacco leaves. The results were as follows： （1） the amplified chlorophyllase gene At- CLH1 constructed the expression vector pET28a-AtCLH1 successfully, obtaining the recombinant engineering strain; （2） induced under 30 ℃ for 22 h, the strain could well express the recombinant protein AtCLH1 with 0.5 mmol/L IPTG, and the molecular weight was about 35 kDa; （3） the strain showed good chlorophyllase producing capability, and the activity of the produced chlorophyllase could reach up to 24.9 U/mL, which could degrade the chlorophyll in tobacco extract and had a good application prospect in improving the quality of low quality tobacco; （4） based on the results of orthogonal test, the enzyme extract from the strain was added to the tobacco leaf surface, which could make the degradation rate of chlorophyll in the tobacco leaf reach 17.06% under the temperature of 37 ℃ at the humidity of 75% for 48 h; （5） after treated by the enzyme liquid, the test tobacco showed increase in the content of aromatic substances, enhancement of tobacco fragrance quality and amount, significant decrease of offensive odor and irritation, significant improvement of agreeable aftertaste, making the overall sensory quality of the tobacco leaf significantly improved.展开更多
基金Supported by National Basic Research Program (973 Program No. 2010CB912802)the Postdoctoral Fund of China, No. 20080441314
文摘AIM: To clarify the role of high in normal-1 (HIN-1) gene promoter methylation during gastric cancer development. METHODS: Gastric cancer cell lines and tissue specimens were analyzed for expression of HIN-1 mRNA and protein using the semi-quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The methylation of the HIN-1 gene promoter was detected in gastric carcinoma cells and tissues using methylation-specific polymerase chain reaction. The 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay and flow cytometry were used to assess the changes in behaviors of gastric cancer cells with or without 5-aza-2’-deoxycytidine treatment. RESULTS: HIN-1 was not expressed in 4 of 5 gastric cancer cell lines. The demethylation reagent 5-aza-2’-deoxycytidine was able to induce or upregulate HIN-1 expression in gastric cancer cell lines, which is associated with reduction of tumor cell viability. Furthermore, methylation of the HIN-1 gene promoter was shown in 57.8% (26/45) of the primary gastric cancer and 42.1% (17/38) of adjacent tissue samples, but was not shown in normal gastric mucosa (0/10). From the clinicopathological data of the patients, methylation of the HIN-1 gene promoter was found to be associated with tumor differentiation (P = 0.000). CONCLUSION: High methylation of HIN-1 gene promoter results in silence of HIN-1 expression in gastric cancer. 5-aza-2’-deoxycytidine reverses HIN-1 methylation and reduces viability of gastric cancer cells.
基金Supported by the Planning Project for the Scientific Research and Technological Development of China Tobacco Henan Industrial Co.,Ltd.(ZW201435)
文摘In order to effectively reduce the chlorophyll content in flue-cured tobacco, improve the overall quality of tobacco leaves, chlorophyllase gene was cloned from Arabidopsis thaliana. After the expression of the expression vector in E. coil, the recombinant engineering strain was obtained. Afterwards, IPTG （isopropy-β-D-thiogalactopyranoside）was used to induce the goal protein, and the chlorophyllase activity of the recombinant engineering strain was measured, so as to investigate its degradation effect on the chlorophyll in the extracts of tobacco leaves. The results were as follows： （1） the amplified chlorophyllase gene At- CLH1 constructed the expression vector pET28a-AtCLH1 successfully, obtaining the recombinant engineering strain; （2） induced under 30 ℃ for 22 h, the strain could well express the recombinant protein AtCLH1 with 0.5 mmol/L IPTG, and the molecular weight was about 35 kDa; （3） the strain showed good chlorophyllase producing capability, and the activity of the produced chlorophyllase could reach up to 24.9 U/mL, which could degrade the chlorophyll in tobacco extract and had a good application prospect in improving the quality of low quality tobacco; （4） based on the results of orthogonal test, the enzyme extract from the strain was added to the tobacco leaf surface, which could make the degradation rate of chlorophyll in the tobacco leaf reach 17.06% under the temperature of 37 ℃ at the humidity of 75% for 48 h; （5） after treated by the enzyme liquid, the test tobacco showed increase in the content of aromatic substances, enhancement of tobacco fragrance quality and amount, significant decrease of offensive odor and irritation, significant improvement of agreeable aftertaste, making the overall sensory quality of the tobacco leaf significantly improved.
