The Study Of MiR-221/222 Induced By Radiation Enhances The Radiation Resistance Of Glioblastoma

Objective: Glioma is the most common primary central nervous system tumors.Because of invasive growth, malignant gliomas can not be totally resected.Postoperative radiotherapy is important for patients with glioblastoma. However, the therapeutic effect of radiotherapy is not good enough on account of the radiation resistance of glioblastoma. It has been reported that the expression of miR-221 and miR-222 are obviously higher in glioblastoma tissue than normal tissue, and knocking down miR-221 and miR-222 in human glioblastoma will partly reverse malignant biological behavior of tumor. In this present study, the expression of miR-221 and miR-222 in cellular stress response induced by ionizing radiation was investigated by RT-PCR. In addition, we also detected sensitivity of tumor to radiation by clone formation and DDR-related proteins by western blot after knocking down miR-221 and miR-222 in glioma. We hope to learn about the mechanism that DDR and miRNA regulate each other, with period for deeper understanding of mechanism of radioresistance and seeking new treatment strategy of glioblastoma.Method :1. The expression of miR-221 and miR-222 was detected by RT-PCR at 3h and 6h after U251, U87 and LN229 glioma cell lines were irradiated with 2Gy X-ray. The transcription factor of miR-221 and miR-222 was also identified by Ch IP.2. The AS-miR-221/222 was transfected into U251, U87 and LN229 glioma cell lines by using liposome. Cells of each group were exposed to 0Gy, 2Gy, 4Gy, 6Gy, 8Gy and 10 Gy X-ray, then using Clonigenic assay to measure the radiosensitivity. The mechanism of miR-221 and miR-222 regulate glioma radiosensitivity was also preliminary investigated. The established U251 subcutaneous glioma model in nude mice was treated with AS-miR-221/222 and radiotherapy to further explore the effect of knocking down miR-221/222 on radiosensitivity. In addition,immunohistochemical stain was analyzed to observe the expression of radiosensitivity-ralated protein.Results :1. The expression of miR-221 and miR-222 in glioma cells increased dependent ofactivation of c-jun after radiation.2. Knocking down the expression of miR-221 and miR-222 in glioma cells can enhances the sensitivity of tumor to radiation. Mi R-221 and miR-222 can initiate Akt pathway to repair DNA damage, which leads to radioresistance of glioblastoma. The growth rate of established U251 subcutaneous glioma model in nude mice treated with AS-miR-221/222 and radiotherapy was slowed down obviously.Immunohistochemical stain showed the expression of p Akt and DNA-PKcs protein reduced with downregulation of miR-221/222.Conclusions:1. The expression levels of miR-221 and miR-222 in glioblastoma cells increased after radiation.2. C-jun is the transcription factors of miR-221 and miR-222 which can promotor gene expression.3. C-jun was activated by radiaton and induced the expression of miR-221 and miR-222.4. Knocking down the expression of miR-221 and miR-222 in glioma cells can enhances the sensitivity of tumor to radiation.Conversely, the glioma cells will be highly resistant to radiotherapy.5. Mi R-221 and miR-222 can enhance the expression of protein DNA-PKcs by depending on PI3K/Akt signaling pathway, which contributes to radiation resistance of gliomas cells.6. Mi R-221 and miR-222 can be the molecular marker to forecast the sensitivity to radiotherapy as well as to be the therapeutic target for increasing the sensitivity to radiotherapy of gliomas.