The Mechanism Of Micrornas Involved In The Regulation Of Glioma Radiosensitivity

Part I Screening and Preliminarily Identification of radiosen- sitivity related microRNAs in human glioma cell lineObjective To screen and identify the miRNAs associated with the radiosensitivity of human glioma. Methods 1. Assess the different radiosensitivity of human glioma cell lines. U87MG, U251MG and SHG-44 cell lines were exposed to X-rays with different doses respectively and number of non-irradiated or irradiated (2, 5, 10 Gy) cells were assessed every 24 h during a period of 7 days. The apoptosis of three cell strains after exposed to 5, 10Gy doses irradiation were analyzed with flow cytometry. 2. The growth inhibiting effects of irradiation with different doses on the U87MG cell line were detected with MTT. The doses leading to 50% growth inhibition of U87MG cell was determined by the curve fitting analysis, which would be used in the following study. 3. Irradiation planning and delivery. External beam irradiation was delivered on the linear accelerator with a 6 MV photon beam. A 40x40 cm field size was utilized and the dishes with monolayer cells were placed 100 cm from the source. The radiation was given with conventional fractionation (2 Gy/ day, 5 d/wk, 400 cGy/ min) in one direction as the treatment given to a planning target volume of patients with malignant glioma. Cells were irradiated to the appropriate radiation doses, which produced 50% inhibition effect of growth in U87MG cells. 4. miRNA microarray was used to monitor the differentially expression profiles of miRNAs between U87MG and the cells after radiation treatment. 5. The results obtained by microarray profiling were validated using real-time RT-PCR analysis. Results Escalating single doses of irradiation (2, 5, 10 Gy) promoted a dose-dependent decrease in cell proliferation in these three human glioma cell lines. But it showed different growth inhibition effects responding to the same doses. The assays revealed a significant growth inhibition at 48 h post 10 Gy irradiation in all the three cell lines. This inhibition persisted during the period of the study (7 days) after irradiation. But at lower doses, the cell lines demonstrated different relative radiosensitivity. At doses of 2 or 5 Gy, the cell lines U251MG and SHG-44 were relatively radiosensitive comparing to U87MG. Flow cytometric analysis detected the apoptosis of three cell lines following 5, 10 Gy irradiation. The apoptosis peak of the three glioma cell lines: SHG-44 > U251MG > U87MG. The curve fitting analysis of the growth inhibition of U87MG indicated that 18.8Gy led to 50% growth inhibition. U87MG cell was exposed to 18.8Gy irradiation following the planning described above and the normal U87MG cell was used as control. miRNA microarray identified 21 miRNAs as differentially expressed: 13 miRNAs up-regulated (≥2.0-fold increase) and 8 down-regulated (≥2.0-fold decrease). The down-regulated miRNAs after irradiation in U87MG were hsa-miR-181a, hsa-miR-521, hsa-miR-302b*, hsa-miR-206, hsa-miR-582, hsa-miR-518b, hsa-miR-107, hsa-miR-620, and the up-regulated miRNAs were hsa-miR-22, hsa-miR-500, hsa-miR-601, hsa-miR-379, hsa-miR-641, hsa-miR-548d, hsa-miR-191, hsa-miR-335, hsa-let-7g, hsa-miR-646, hsa-miR-22_MM1, hsa-miR-9*_MM2, hsa-miR-451. Among them, miR-521 has been reported sensitizing prostate cancer to radiotherapy. Let-7 takes an important role in radiation modulation in lung cancer. miR-181a has relationship with tumor development. But none has been reported in the field of glioma radiosensitivity. The irradiation process was repeated and the RNA was extracted for the qRT-PCR. miR-181a and miR-521 were down-regulated (≥2.0-fold decrease), which matched the the result of the miRNA microarray.Conclusion 1. U87MG cell line is radioresistant. 2. There are some miRNAs differently expressed following the irradiation procedure in U87MG cells. These miRNAs may take great part in modulating the raiosensitivity. 3. Among the differently expressed miRNAs, the significant down-regulation of miR-181a and miR-521 was verified by qRT-PCR. And they are chosen as the targets of our future study.Part II The role of miR-181a and miR-521 in modulating the radiosensitivity of gliomaObjective To study the role of miR-181a and miR-521 in modulating the radiosensitivity of glioma. Methods 1. Chemically synthesized mimics of miR-181a, and miR-521 were used to transfect U87MG cells and the expression levels were verified by RT-PCR. 2. MTT assay was carried in U87MG cells to access its response to radiation before and after the transfection. 3. Soft agar colony formation assay was used to assess the influence of the mimics on the cellular anchorage-independent growth. Results The expression of miR-181a and miR-521 was up-regulated by transfection of the specific miRNA mimics. In the condition of no radiation, the up-regulation of both miR-181a and miR-521 had no significant influence on the cellular proliferation. After exposed to the radiation, the transfected cells showed a significant growth inhibition compared with cells transfected with control mimic. The influence appeared at 72 h, got to the peak at day 4 afte the treatment, and then decreased. The non-irradiated U87MG cells tansfected with miR-181a, miR-521 and control mimics respectively showed no significant differences in the ability of cellular anchorage-independent growth. But the radiation led to less colonies formation in the miR-181a and miR-521 up-regulated group. Conclusion Both miR-181a and miR-521 sensitize human glioma cell line U87MG to radiation.Part III Molecular mechanism of miR-181a and miR-521 in radiation modulatingObjective To investigate the possible molecular mechanisms by which miRNAs modulated radiation sensitivity in radiation response. Methods 1. Predict the target proteins of miR-181a and miR-521 by bioinformatics software on the website. 2. RT-PCR and western blot were used to assess the mRNA and protein level of the targets before and after the radiation. 3. Specific pre-miRNA eukaryotic expression vectors expressing pre-miR-181a and pre-miR-521 were constructed and transfected into U87MG cell to up-regulate mature miR-181a and miR-521. The mRNA and protein level of target genes were tested at several time points by RT-PCR and western blot respectively. 4. G418 was used to screen the transfected cells in order to select the cell colony stably expressing the eukaryotic expression vector. The apoptosis under 2Gy radiation was assessed by flow cytometry and the colony formation ability was assessed by the plate colony formation assay to compare to radiosensitivity of the miR-181a or miR-521 up-regulated U87MG cell to the control.Results 1. A comprehensive analysis indicates that Bcl-2 and CSA were probably the target genes of miR-181a and miR-521 respectively. 2. Irradiation led to the increase of Bcl-2 and CSA protein, but not significant increase of the mRNA. 3. The protein expression of Bcl-2 or CSA decreased significantly at 72 h after the transfection with pre-miR-181a or pre-miR-521. No decrease of the Bcl-2 or CSA mRNA had been detected at the time points 24 h, 48 h and 72 h after the transfection. 4. U87MG cells stably expressing the vector survived the G418 screening. The U87MG cell stably expressed pre-miR-181a or pre-miR-521 had less colony formation ability than the one expressing the control. Conclusion The miR-181a and miR-521 post-transcriptinally down-regulate the target proteins Bcl-2 and CSA respectively, which sensitizes the glioma cell line U87MG to radiation.