| Cervical cancer is one of the most common malignant tumors and occupies more than half of total gynecologic malignant tumors. Radiotherapy is one of the main methods of cervical cancer clinical treatments. But the effectiveness of radiotherapy is still limited according to the clinical data. The existence of radioresistant cancer cells is the main cause of recurrence and metastasis after radiotherapy for cervical cancer patients. Therefore, revealing the key molecular and mechanism responsible for cervical cancer radioresistance has a great significance for improving the effectiveness of radiotherapy. Multiple signal pathways,such as NF-κB,PI3-K/Akt and TGFβ pathways and some key moleculars, like HIF-1, p53 and p21 have been reported to participate in regulating the radiosensitivity of cervical cancer cells. The mechanism of cervical cancer radiosensitivity is a complex biological process involving various factors and procedures, has not been elucidated and remains to be studied further.EMT(epithelial mesenchymal transition) plays a crucial role in tumor metastasis and it is considered as one of the key mechanisms of tumor cells obtaining the abilities of metastasis and invasion. The abilities of invasion and migration of tumor cells are enhanced dramaticlly through EMT. Therefore, understanding the key mechanism and regulating factors, and intervening in the key signal pathway, can effectively inhibit the metastasis of tumor theoretically.micro RNAs(mi RNAs) is a kind of novel regulatory factors participating in regulating multiple biological processes and have an important role in regulating growth, development, disease and tumorgenesis. Researches found that mi RNAs also play a significant role in regulating the radioresistance and metastasis of cervical cancer. This article aims to find the key mi RNA which participates in regulating the radioresistance and metastasis of the cervical cancer and make it as the potential therapeutic target for improving the efficiency of radiotherapy. Aims:1. To establish the radioresistant cervical cancer cell lines and the differentially expressed mi RNAs were screened.2. The effect of mi R-630 expression on radiosensitivity of cervical cancer Hela cells was examined.3. To clarify the effects of mi R-630 on EMT of cervical cancer Hela cells. Methods:1. The radioresistant cervical cancer cell variants were established by repeated selection with irradiation.The mi RNA profiles of radioresistant cells and their corresponding controls were analyzed and compared using microarray.2. q RT-PCR were used to detect the expression level of mi R-630 in Hela cells after irradiation of γ-ray.3. Lentiviral vectors which overexpress mi R-630 and a lentiviral vector expressing scrambled RNA were used to establish Hela cells with stable expression of mi R-630 or mi R-630-inhibition. After puromycin selection, levels of mi R-630 were quantified using q RT–PCR.4. Cell viability was analyzed using MTT assay. Radiosensitivity of cervical cancer cells were determined using colony-forming assay. The spontaneous apoptosis and radiation-induced apoptosis were analysised using Flow Cytometry. The apoptosis-related protein levels were determined by Western blotting in Hela cells infected with mi R-630, mi R-630 inhibiton, or the corre-sponding negative control.5. Transplanted tumor assay was uesd to observe the tumorigenicity of Hela cells with diferrent expression level of mi R-630.6. The changes associated with EMT, including morphology, EMT markers, migration and invasion were observed by microscope, western blotting, imunofluroscence, scratch assay and transwell chamber assay respectively.7. Dual-luciferase reporter gene assay was used to verify the target gene of mi R-630.8. q RT-PCR was applied to detect the expression level of mi R-630 in Hela cells treated with hypoxia. Results:1. The radioresistant cervical cancer cell lines Hela-R11 and Siha-R15 were uccessfully established. mi R-630 was significantly upregulated in radioresistant cervical cancer cell lines.2. Radiation induced the expression of mi R-630 in Hela cell in a time and dose dependent manner.3. Hela cells with stable expression of mi R-630 or mi R-630-inhibition were successfully established.4. mi R-630 decreased the radiosensitivity of Hela cells by inhibiting spontaneous apoptosis and radiation-induced apoptosis. Overexpression of mi R-630 increased the expression of anti-apoptotic protein Bcl-2 and decreased the expression of pro-apoptotic protein Bax, Caspase 3, Caspase 7 and Caspase 9 in cervical cancer Hela cells.5. mi R-630 enhanced the tumorigenicity of cervical cancer Hela cells.6. mi R-630 promoted EMT and enhanced the migration and invasion in Hela cells.7. ZNF-451 and IGF1 R were not the target gene of mi R-630.8. Hypoxia induced the expression of mi R-630 fastly and long lasting in Hela cells. Conclusions:Radiation and hypoxia upregulated mi R-630 expression in cervical cancer Hela cells. mi R-630 enhanced the radioresistance of cervical cancer Hela cells by inhibiting the radiation-induced apoptosis. mi R-630 promoted the EMT and the metastasis of cervical cancer Hela cells. |
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