| Malignant glioma is a common head and neck primary tumors, which is located in the brain and spinal cord. Chemotheraphy for gliomas after surgery resection is a kind of necessary method. However, because of the particularity position as it is,and the effect of blood brain barrier, drug could not reach the point of tumor on a effective concentration, and glioma resistance might be induced. This is the main cause of chemotherapy failure. To explore the mechanism of drug resistance and screen out targets associated with drug resistance could provide somevalue information of clinical treatment. HDAC1 belongs to the histone acetylation enzyme, which is closely related to pestablish HDAC1 overexpression and low expression cell lines to observe the drug resistance related function of HDAC1, and then, the mechanism will be researched briefly. Objiective:To explore the degree of drug resistance of malignant glioma cell line after overexpressed and lowexpressed HDAC1 and related mechanism. Methods: Part I:1. The establishment of HDAC1 overexpressed cell line U87MG-HDAC1.Total RNA was extracted from glioma U87 MG cells, after RT-PCR reaction, TA cloning, enzyme digestion and sequence analysis. The HDAC1 coading region was connected with vector p CDH-CMV-MCS-EF1-Puro. Then the purified lentiviral expression vector was cotransfected with other 3 packaging plasmids into 293 T cells for virus packaging, the packaged virus was infected U87 MG cell line and the infected cells were screened by puromycin and identified by Western blotting 3 days later.2. The construction of HDAC1 lowexpressed cell line U87MG-HDAC1-RNAi.According to the HDAC1 sequence,we synthesis oligo DNA in accordance with the requirements of the short hairpin RNA. By forming double strand after annealing reaction and ligating with GV-248, the purified lentiviral RNAi expression vector was cotransfected with other 2 packaging plasmids into 293 T cells for virus packaging.Then the virus was used to infect U87 MG cell line and the infected cells were screened by puromycin and identified by Western blotting 3 days later. Part II:Effects of HDAC1 on glioma chemotherapy resistance.The subjects were divided into HDAC1 overexpressed group(U87MG-HDAC1 & U87MG-Control) and HDAC1 lowexpressed group(U87MG-HDAC1-RNAi & U87MG-NC-RNAi).The two groups were treated with VM-26 and DDP, respectively. The final concentrations of VM-26 is 0.1?0.5?2.5?12.5 ?g/m L and that of DDP is 0.08?0.4?2?10 ?g/m L?After treated for 48 h, the two groups were detected by MTT,Hoechst / PI double staining and Western Blot. Results: Part I:1. The HDAC1 coading region was amplificated correctly and the lentiviral vector p CDH-CMV-MCS-HDAC1-EF1-Puro was packaged successfully. HDAC1 stable overexpressed cell line U87MG-HDAC1 was established. There is a significant increased HDAC1 protein expression in U87MG-HDAC1 compare to that of U87MG-Control(1.148 ± 0.024 vs 0.580 ± 0.003,P<0.01).2. The HDAC1 sh RNA oligo were synthesized correctly and the lentiviral vector GV248-HDAC1-RNAi was packaged successfully. HDAC1 stable lowexpressed cell line U87MG-HDAC1 was established. There is a significant decreased HDAC1 protein expression in U87MG-HDAC1-RNAi compare to the U87MG-NC-RNAi(0.705±0.009 vs0.352± 0.006,P<0.01).Part II:MTT showed the survival rate of the overexpressed group(U87MG-HDAC1 & U87MG-Control) was decreased in a dose-dependent manner. On the same concentration of VM-26 or DDP, the survival rate of U87MG-HDAC1 was significantly higher than that of U87MG-Control(P<0.05); the IC50 of U87MG-HDAC1 is 3.11 times that of U87MG-Control after treated with VM-26; while the IC50 value is 2.60 times after treated with DDP. Meanwhile, the lowexperessed group(U87MG-HDAC1-RNAi& U87MG-NC-RNAi) was decreased in a dose-dependent manner. On the same concentration of VM-26 or DDP, the survival rate of U87MG-HDAC1-RNAi was significantly lower than that of U87MG-NC-RNAi cell line(P<0.05); the IC50 of U87MG-HDAC1-RNAi is 0.49 times that of U87MG-NC-RNAi after treated with VM-26; while the IC50 value is 0.62 times after treated with DDP.Hoechst 33258 / PI double staining showed the apoptosis ratio of the overexpressed group increased with the increasing concentration. On the same concentration of VM-26 or DDP, the apoptosis ratio of U87MG-HDAC1 was lower than that of U87MG-Control significantly. Meanwhile, the apoptosis ratio of the lowexpressed group increased with the increasing concentration. On the same concentration of VM-26 or DDP, the apoptosis ratio of U87MG-HDAC1-RNAi was higher than that of U87MG-NC-RNAi significantly.Western Blot showed the overexpressed group(U87MG-HDAC1 & U87MG-Control), as the concentration increasing, Bax and Caspase-3 increased while the Bcl-2 decreased. On the same concentration of VM-26 or DDP, the Bcl-2/Bax ratio was significantly higher than that of U87MG-Control(P<0.05) and the Caspase3 expression was significantly lower than that of U87MG-Control(P<0.05). Meanwhile, as the concentration of the lowexpressed group(U87MG-HDAC1-RNAi & U87MG-NC-RNAi) increasing, Bax and Caspase-3 increased while the Bcl-2 decreased. On the same concentration of VM-26 or DDP, the Bcl-2/Bax ratio was significantly lower than that of U87MG-NC-RNAi(P<0.05) and the Caspase3 expression was significantly higher than that of U87MG-NC-RNAi(P<0.05). Conclusion:1. The HDAC1 overexpressed cell line U87MG-HDAC1 and its negative control U87MG-Control were successfully established.2. The HDAC1 downregulated cell line U87MG-HDAC1-RNAi and its negative control U87MG-NC-RNAi were successfully established.3. The overexpression and lowexpression of HDAC1 enhance or weaken the drug resistance after chemotherapy, which was closely related to the ratio of Bax/Bcl-2 and the expression of Caspase-3. |
PDF Full Text Request