[The effect of PM(2.5) on oncogene expression in HBE cells].

Objective: To study the effect of particulate matter 2.5 (PM(2.5)) on oncogene expression in human bronchial epithelial (HBE) cells. Methods: HBE cells were selected as the study subjects, and PM(2.5) treatment group (10 µg/ml and 50 µg/ml) , negative control group and positive control group (10 µmol/L Cr(6+)) were set. CCK8 assay was used to test the IC(50) value of PM(2.5). HBE cells were treated with PM(2.5) for 24 h at 10 µg/ml and 50 µg/ml, additionally, cells were treated with blank as negative control, 10 µmol/L Cr(6+) as a positive control for 24 h. After the treatment, mRNA expression of oncogenes including c-myc, c-fos, k-ras and p53 were detected by fluorescent quantitative RT-PCR, the protein expression of oncogenes were detected with western blot. Results: The IC(50) value of PM(2.5) in HBE cells is 70.12 µg/ml. The qRT-PCR data showed that compared with the control group, the expression level of c-myc gene increased by respectively 500.1%、780.7%、305.3% after exposure to 10、50 µg/ml PM(2.5) and positive control group; c-fos gene increased respectively 34.0%、76.7%、131.3% after exposure to 10、50 µg/ml PM(2.5) and positive control group; k-ras gene increased respectively 50.3%、107.0%、49.7% after exposure to 10、50 µg/ml PM(2.5) and positive control group; p53 gene decreased by 28.3%、28.7%、59.7% after exposure to 10、50 µg/ml PM(2.5) and positive control group. The western blot results showed that compared with the control group, c-myc protein increased respectively 29.7%、77.3% after exposure to 50 µg/ml PM(2.5) and positive control group; c-fos protein increased respectively 200.3%、137.0% after exposure to 50 µg/ml PM(2.5) and positive control group; k-ras protein increased respectively 106.3%、130.3%、116.7% after exposure to 10、50 µg/ml PM(2.5) and positive control group; p53 protein decreased by 43.7%、53.3%、52.1% after exposure to 10、50 µg/ml PM(2.5) and positive control group. Conclusion: PM(2.5) could promote the expression of oncogenes in HBE cells, the carcinogenicity of haze might be related to promotion of oncogenes expression induced by PM(2.5).

[Construction of 3ß-HSD gene silencing cell line and its effects on apoptosis induced by DEHP].

Objective: To construct 3ß-HSD gene shRNA lentivirus interference vecto, then transfect into human MCF-7 cells, and construct cell line with 3ß-HSD gene silencing, finally to study the effects of 3ß-HSD on apoptosis induced by di- (2-ethylhexyl) phthalate (DEHP) . Methods: According to the mRNA sequence of 3ß-HSD gene provided by GenBank, three interference sequences were designed and connected to PLVX-shRNA2-puro after annealing. The recombinant lentivirus vector was transfected into 293FT cells, the virus supernatants were collected and infected with MCF-7 cells. After puromycin screening, MCF-7 cells with 3ß-HSD gene silencing were constructed. The cells with 3ß-HSD gene silencing were identified by real-time quantitative PCR and western blot. Then the 3ß-HSD gene silencing cells and MCF-7 cells were treated at various doses of DEHP for 24 hours to detect the gene expression and protein expression of apoptosis genes including Bax, Caspase-3 and Caspase-8. Results: The interference sequence of 3ß-HSD gene inserted into lentivirus vector PLVX-shRNA2-puro is consistent with the designed sequence. 3ß-HSD gene expression level in MCF-7 cells with 3ß-HSD gene silencing was 77% lower than than that of control MCF-7 cells. 3ß-HSD protein level in MCF-7 cells with 3ß-HSD gene silencing was 74% lower than that of control MCF-7 cells. After DEHP treatment in MCF-7 cells with 3ß-HSD gene silencing and control MCF-7 cells, qRT-PCR results showed that Bax gene expression levels increased by 28%-54%, Caspase-3 gene increased by 13%-49%, Caspase-8 gene increased by 21%-70% in MCF-7 cells when compared with the control group. Additionally, in the 3ß-HSD gene silencing cells, Bax gene expression level decreased by 11%-28%, Caspase-3 gene expression decreased by 12%-23%, Caspase-8 gene expression decreased by 11%-34%, compared with the same treatment group of MCF-7 cells. Western blot results showed that Bax protein expression level increased by 28%-61%, Caspase-3 protein expression level increased by 40%-48%, Caspase-8 protein increased by 31%-84% in MCF-7 cells when compared with the control group. In 3ß-HSD gene silencing cells, Bax protein expression level increased by 11%-27%, Caspase-3 protein increased by 21%-40%, Caspase-8 protein increased by 12%-25%, compared with the same treatment group of MCF-7 cells. Conclusion: The stable 3ß-HSD gene silencing cell line are successfully constructed in this study. DEHP can induce increased expression of apoptotic gene and protein. Silencing of 3ß-HSD gene can inhibit the activation of apoptotic gene by DEHP in a certain degree.

[Effects of 3ß-HSD gene silence or overexpression on DEHP induced oxidative damage in MCF-7 cells].

Objective: To study the oxidative damage of di- (2-ethylhexyl) phthalate (DEHP) on MCF-7 cells, and to investigate the effects of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) gene silence or overexpression on DEHP-induced oxidative damage. Methods: MCF-7 cells, 3ß-HSD gene silencing cells and 3ß-HSD gene overexpression cells were treated with different doses of DEHP (0,0.05,0.1,0.2,0.4,0.8 mmol/L) for 24h, then intracellular oxidative damage index such as MDA, SOD, GSH, GSH-PX were detected, DNA repair gene hOGG1, hMTH1 mRNA expression were tested by Q-PCR, hOGG1, hMTH1 protein expression were detected by western blot. Results: After MCF-7 cells were treated by DEHP, MDA levels increased; SOD activity, GSH content, GSH-PX activity decreased, hOGG1 and hMTH1 mRNA expression levels increased, hOGG1 and hMTH1 protein expression levels increased, the differences were statistically significant when compared with control (P<0.05 or P<0.01) . In 3ß-HSD gene silencing cells which were treated by DEHP, when compared with the same dose group of MCF-7 cells, MDA content increased, SOD activity, GSH content, GSH-PX activity decreased, hOGG1 and hMTH1 mRNA expression levels decreased, hOGG1 and hMTH1 protein expression levels decreased, the difference were statistically significant (P<0.05 or P<0.01) . In 3ß-HSD gene overexpression cells which were treated by DEHP, when compared with the same dose group of MCF-7 cells, MDA content decreased; SOD activity, GSH content, GSH-PX activity increased, of hOGG1 and hMTH1 mRNA expression levels increased, hOGG1 and hMTH1 protein expression levels increased, the difference were statistically significant (P<0.05 or P<0.01) . Conclusion: DEHP could cause oxidative damage in MCF-7 cells, induce the changes of related genes and proteins, 3ß-HSD plays an antioxidant role in the process of DEHP ox-idative damage.