| Objective:Glioblastoma multiforme(GBM) is the most common one of tumors in head and neck. Surgery is traditional treatment, with radiation and chemotherapy as supplement. The treatment and prognosis of glioma may be affected by many factors: firstly, the chemotherapy drugs that cannot pass the blood-brain barrier will have a negative effect on chemotherapeutic afficacy; secondly, effective chemotherapy drugs for central nervous system neoplasms is not yet absolutely sure, so it is difficult to select the suitable drugs; lastly, glioma is resistant to to chemotherapy drugs and failed to select sensitive drugs for different tumor. TGF-β family affect proliferation, differentiation and play an important role in both normal and cancer cells. As a member of TGF-β, GDF15 has a significant effect on the proliferation of tumors. Some papers reported GDF15 can affect proliferation and invasiveness of tumor, while there is no research about the mechanism of GDF15 in drug resistance. In this study, glioma U373 and U87 cells are infected by constructing shRNA lentiviral vector and packaging lentivirus, and establish a stable low expression of GDF15 in glioma cell lines. Then observe whether a low expression of GDF15 could affect the chemotherapy sensitivity of glioma cells, eventually discuss the relevant mechanism about it. Methods:1、The construction of GDF15 shRNA lentivirus vector, establish a stable low expression of GDF15 in U373 and U87 cell strainsFirstly, two target sequences of RNA interference about GDF15 genetic sequence were designed, then synthesis the single-stranded DNA oligo, finally the double-stranded DNA oligo were formed by annealing. The RNAi lentivirus was constructed by connecting with vector GV248, transforming, identifying of positive clones and packaging lentivirus.The lentivirus GV248-GDF15-RNAi(1)/(2) were used to infect U373 cells, and observe the green fluorescent through microscope from 24 to 72 hours. The expression of GDF15 was assessed by Western Blot.Screen out the sequence with high efficiency, then infect U373 and U87 cells with the lentivirus. Finally, screen out a stable low expression of GDF15 in U373 and U87 cell strains by puromycin.2、Detecting the effect of silencing GDF15 in U373 cells on drug sensitivityTo discuss the effect of silencing GDF15 in U373 cells on drug sensitivity, the experimental group of U373-GDF15-RNAi and negative control group of U373-NC-RNAi were treated with two antitumor drugs: VM-26(teniposide) and DDP(cis-dichlorodiamineplatinum). The final concentration of VM-26 was 0.1、0.5、2.5 and 12.5 μg/mL; the final concentration of DDP was 0.08、0.4、2 and 10 μg/m L. After treating 48 hours with the drugs, MTT assay was arranged to detect the cytotoxic effects of drugs, detected the morphological changes in apoptosis by Hoechst/PI double staining, assessed expression of Bcl-2 、 Bcl-x L 、 p53 and Caspase-3 by Western Blot.3、Detecting the effect of silencing GDF15 in U87 cells on drug sensitivityTo discuss the effect of silencing GDF15 in U87 cells on drug sensitivity, the experimental group of U87-GDF15-RNAi and the negative control group of U87-NC-RNAi were treated with two antitumor drugs: VM-26(teniposide) and DDP(cis-dichlorodiamineplatinum). The final concentration of VM-26 was 0.1、0.5、2.5 and 12.5 μg/mL; the final concentration of DDP was 0.08、0.4、2 and 10 μg/m L. After treating 48 hours with the drugs, MTT assay was arranged to detect the cytotoxic effects of drugs, detected the morphological changes in apoptosis by Hoechst/PI double staining, assessed expression of Bcl-2 、 Bcl-x L 、 p53 and Caspase-3 by Western Blot. Results:1. The GDF15 shRNA lentivirus and a stable low expression of GDF15 in U373 and U87 cell strains were constructed successfullyThe target sequences of shRNA lentivirus about GDF15 were designed, then the high suppression efficiency one will infect U373 and U87 cells. According to the results of western blot, GDF15 expression decreased obviously. A stable low expression of GDF15 in U373 and U87 cell strains were constructed successfully.2、The influence of down-regulation GDF15 on U373 drug sensibilityAfter treated with VM-26 and DDP for 48 hours, the cell viabilities decreased significantly with the increase of drug concentration, while the cell viabilities of U373-GDF15-RNAi cells were higher than U373-NC-RNAi cells at concentration. The results of Hoechst/PI double dye suggest that nucleus condensation and apoptotic appear with drug treatment. But the proportion of apoptotic in experimental group was obviously less than that in negative control group. Western blot showed a significant decrease Bcl-2 and Bcl-xL and a significant increase in the p53 and Caspase-3 with the increased concentrations of VM-26 or DDP in the same cell strains. But in every concentration of each drug, Bcl-2 and Bcl-x L level was obviously up-regulated while p53 and Caspase-3 level was obviously down-regulated in U373-GDF15-RNAi cells.3、The influence of down-regulation GDF15 on U87 cells drug sensibilityAfter treated with VM-26 and DDP for 48 hours, the cell viabilities decreased significantly with the increase of drug concentration, while the cell viabilities of U87-GDF15-RNAi cells were higher than U87-NC-RNAi cells at concentration. The results of Hoechst/PI double dye suggest that nucleus condensation and apoptotic appear with drug treatment. But the proportion of apoptotic in experimental group was obviously less than that in negative control group. Western blot showed a significant decrease Bcl-2 and Bcl-x L and a significant increase in the p53 and Caspase-3 with the increased concentrations of VM-26 or DDP in the same cell strains. But in every concentration of each drug, Bcl-2 and Bcl-x L level was obviously up-regulated while p53 and Caspase-3 level was obviously down-regulated in U87-GDF15-RNAi cells. Conclusion:1、GDF15 shRNA lentiviral vectors were constructed successfully, and establish down-regulation GDF15 U373 and U87 cell lines.2 、 Down-regulation GDF15 glioma can decrease the sensitivity to the chemotherapy drugs, and improve the survival rate of cells.3 、 Glioma cells morphological changes and nucleus pycnosis due to chemotherapy drugs, down-regulation GDF15 could reduce the cell apoptosis.4、After treated with the chemotherapy drugs, the Bcl-2 and Bcl-x L level increased, p53 and Caspase-3 expression decreased in U373/U87-GDF15-RNAi cells,which indicated the down-regulation GDF15 induce drug resistance induction of glioma cells was mediated by up-regulation of Bcl-2 and Bcl-x L expression, while down-regulation of p53 and Caspase-3. |
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