| Lung cancer is the first place in the global incidence of malignant tumor, the global annual new lung cancer 1.2 million there were 600000 people die of lung cancer every year in our country. In the cities in China because of the death of the tumor and the population, lung cancer ranks the first.Histone deacetylase (Histone deacetylases, HDACs) is one of the key enzymes in the maintenance of chromosomal basic composition unit nucleosomal histone acetylation balance.Its function is to catalyze the deacetylation of histones, which has a close relationship with the gene transcription and inhibition of complex process, involving gene silencing.It is a popular target effect on antitumor drug design. Histone deacetylase 2 (HDAC2) is one of the members of the histone deacetylase (HDACs) family.It is widely expressed in embryos and mammalian cells, and almost involves in all cellular functions, including cell proliferation, apoptosis, differentiation. At present it is founded in many tumors with high expression of HDAC2, and the mechanism of action in different tumors is inconsistent,the role in lung cancer is not yet clear and unified report. Our research studied the biological effects and mechanism in A549 cells knockdown of HDAC2 by RNA interference in vitro by use cellular and molecular biology techniques. Detected the expression of HDAC2 in Lung adenocarcinoma and paratumorous tissues.Including the following three parts:PART I:EXPRESSION OF HISTONE DEACETYLASE 2 IN LUNG ADENOCARCINOMAABSTRACTObjectives:The expression of HDAC2mRNA and HDAC2 protein in lung adenocarcinoma tissues and paratumorous tissues and then investigated the relationship between them.Methods:1,30 cases of lung adenocarcinoma tissues and paratumorous tissues had been collected and all were diagnosed by the Department of Pathology of the first affiliated hospital of Guangxi medical university. The expression of HDAC2 protein was detected in all the tissues by Western blot and Immunofluorescence staining. The expression of HDAC2mRNA was detected in all the tissues by RT-PCR.Results:1,RT-PCR results show that the relative expression of HDAC2mRNA in 30 cases of lung adenocarcinoma were lower than paratumorous tissuesues, the difference was statistically significant (P=0.0461).2,Western blot results show that the lung adenocarcinoma tissues group HDAC2 was down regulation to the paratumorous tissuesues group, the difference was statistically significant (p=0.0451).3,Immunofluorescence staining results show that HDAC2 were expressed in the cytoplasm and nucleus showing green fluorescence in human lung adenocarcinoma tissue slices and paratumorous tissues slices.The latter is stronger than the former.The difference was statistically significant (p=0.000).Conclusions:1, HDAC2 was down regulation in the lung adenocarcinoma tissues group suggesting that HDAC2 is closely associated with lung adenocarcinoma.3,Immunofluorescence staining results show that HDAC2 were expressed in the cytoplasm and nucleus showing green fluorescence in human lung adenocarcinoma tissue slices and paratumorous tissues slices.The latter is stronger than the former.The difference was statistically significant (p=0.000).Conclusions:1, HDAC2 was down regulation in the lung adenocarcinoma tissues group suggesting that HDAC2 is closely associated with lung adenocarcinoma.PART II:STUDY OF ERYTHROMYCIN ON HISTONE DEACETYLASE 2 IN A549 CELLSABSTRACTObjectives:s:Lung cancer is one of the most common malignant tumor in the world at present, this paper discusses the erythromycin (erythromycin, EM) of cigarette smoke extract (cigarette smoke extract, CSE) stimulation of lung adenocarcinoma cells (A549) of the histone deacetylase 2 (HDAC2) function.This paper studies EM to cisplatin (Cisplatin, CDDP) of A549 cells induced by apoptosis, and its influence on the cell cycle of A549 cells.Methods:The A549 cells were used to the research as object and exposed to CSE,EM and CDDP.(1) In order to determine the drug concentration and action time,the concentration of CSE was set as 0.5%,1%,2.5%,5%, EM concentration was set as 10,20,40,80μg/ml.The experimental groups just like this:blank control group (only 10% inactivated fetal bovine serum FBS RPMI-1640 cells cultured in 0.5%CSE group); EM group; 0.5%CSE+10μg/ml.This groups were treated with different concentrations of CSE,EM and CDDP with different concentration and then to observe the morphology of A549 cells by optical microscope; CCK-8 was used to detect the effects of different concentrations of CSE and EM on the activity of A549 cells in different time.Enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay ELISA) method was used to detect A549 cells just like IL-6, IL-8, TNF-a after treatment with different concentration of CSE,EM and CDDP. The Western blotting (Western blot,WB) was used to detect the expression of A549 cell HDAC2; RT-PCR was used to detect the expression of HDAC2mRNA, IL-6mRNA cells, IL-8mRNA, TNF-alpha mRNA.(2) The A549 cells as the object was used to study, the preparation of CDDP concentration of 0.25mg/L,0.5mg/L, lmg/L,2mg/L,4mg/L,8mg/L Different concentrations of CDDP stimulated A549 cells 48h cells activity of A549 cells treated with different concentrations of CDDP after the test by CCK-8 method, and calculated the CDDP of their half inhibition concentration (50% inhibiting concentration, IC50). The experimental group, the blank control group (only 10% inactivation of FBS RPMI-1640 cells cultured in EM group); IC50-CDDP+10μg/mlEM. The cells were cultured 48h, apoptosis, cell cycle percentage of cells in each group was detected by flow cytometry.Results:(1)The activity of A549 cells were detected by CCK-8. Compared with the blank control group, the cells treated with 0.5%CSE 24,48 hours and the cells treated with 1% CSE for 24 hours,10μg/ml cells treated with EM 24, 48 hours, the cell activity was inhibited obviously, the difference was not statistically significant (P>0.05). While the other CSE, EM cell activity were inhibited to varying degrees, the difference was statistically significant (P< 0.05), in a dose and time dependent manner and in a certain range, the greater the drug dose, training time is longer, the more serious the degree of cell growth inhibition.(2) The A549 cells were detected by ELISA method of IL-6, IL-8 and TNF-were. Compared with the blank control group,0.5%CSE in A549 cells treated with 24,48 hours, cells treated with 1%CSE for 24 hours, the secretion of IL-6, IL-8 and TNF-were increased, the differences were statistically significant (P< 0.05). The secretion of the cells of CSE group IL-6, IL-8 and TNF-were decreased, and with the increasing of CSE concentration and the extension of the culture time, the more serious the secretion of inflammatory factors decreased, the differences were statistically significant (P< 0.05). Compared with the blank control group, A549 cells were treated with different concentrations of EM at different time, inflammatory cytokines secretion were inhibited, and in a dose and time dependent manner, the larger dose of EM, culture time is longer, the secretion of inflammatory factors from the more serious the degree of inhibition, the differences were statistically significant (P< 0.05).(3) Compared with the blank control group, A549 cells after 0.5%CSE treatment, expression of HDAC2mRNA decreased, the difference was statistically significant (P< 0.05). And compared with the 0.5%CSE group,10 μg/ml EM expression in A549 cells can upregulate the expression of HDAC2mRNA of tobacco smoke exposure, the difference was statistically significant (P< 0.05).(4) Compared with the blank control group,the treatment of A549 cells with 0.5%CSE, IL-6mRNA, IL-8mRNA and TNF-mRNA expression were increased, the differences were statistically significant (P<0.05); compared with 0.5%CSE group, the expression of 10μg/ml EM can reduce tobacco smoke exposure of A549 cells to IL-8mRNA and TNF-mRNA, the differences were statistically significant (P<0.05).(5) CCK-8 results showed that different concentrations of CDDP A549 cell 48h, cell growth was inhibited, and the inhibition was concentration dependent, and the higher the concentration, the cell growth inhibitory effect is larger, compared with the control group, the differences were statistically significant (P< 0.05). IC50-CDDP 3.449 mg/L(6) The effect of IC50-CDDP on the A549 cells 48h after EM pretreatment, compared with the blank control group, early apoptosis cells increased, the difference was statistically significant (P<0.05). The results of cell cycle showed that the G0/G1 phase of the cell cycle increased, the difference was statistically significant (P< 0.05).(7) Compared with the blank control group,10μg/ml after EM stimulation of A549 cells, the cell cycle of G0/G1 phase was increased, the difference was statistically significant (P< 0.05). But no significant increase in apoptosis (p>0.05).Conclusions:(1)0.5%CSE can make A549 cells to produce inflammatory reaction, and EM can inhibit the inflammatory reaction of tobacco smoke exposure in A549 cells, and its mechanism may be through up regulation of tobacco smoke exposure of A549 cells to HDAC2 activity and effect.(2)The EM can enhance the blocking effect of CDDP cells on A549 cells and its mechanism may be related to regulation of EM, the increased expression of HDAC2 induced CyclinD1 activity decreased.PART Ⅲ SILENCING OF HDAC2 BY RNAi INFLUENCES BIOLOGICAL BEHAVIOR OF HUMAN LUNG ADENOCARCINOMA A549 CELLSABSTRACTObjectives:The expression of lung adenocarcinoma and paratumorous tissues HDAC2 mRNA and protein in lung adenocarcinoma patients preoperative and postoperative blood HDAC2 mRNA expression analysis of the relationship between them.Methods:1,30 cases of lung adenocarcinoma and paratumorous tissues had been collected and all were diagnosed by the Department of Pathology of the first affiliated hospital of Guangxi medical university. The expression of HDAC2 protein was detected in all the tissues by Western blot. And then investigated the relationship between the expression of Sp3,β-catenin and clinicopathologic features.2,30 cases of lung adenocarcinoma and 20 cases of non-lung cancer patients had been collected and all were diagnosed by the Department of Pathology of the first affiliated hospital of Guangxi medical university. These blood samples were extracted RNA, lung cancer patients one day before surgery and after three days of blood samples, HDAC2 mRNA expression levels were detected. Also compared with and without HDAC2 mRNA expression of burning fumes exposure history of lung cancer in patients with long-term biofuels, analyze their relevance.Results:1, RT-PCR was used to detect HDAC2 of A549 and RNAi-A59 with the difference was statistically significant (t=3.0176, P=0.041).2, RT-PCR assay results showed that A549 relative expression of HDAC2 mRNA was higher than three days after surgery, the difference was statistically significant.Conclusions:1, HDAC2 in human lung adenocarcinoma patients with blood and cancer tissue expression, suggesting that HDAC2 is closely associated with lung adenocarcinoma.2, RNAi-A549 cell HDAC2 mRNA was lower than A549 cell with CSE exposure, HDAC2 levels may be associated with HDAC2 RNAi related. |
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