| BACKGROUND AND OBJECTCervical cancer is one of the most common genital cancers in women worldwide. It is the second most prevalent and the fifth most deadly malignancy. There are approximately500,000new cervical cancer cases in the world each year, and more than60%of these cases die annually. And incidence of cervical cancer in our country ranks second in the world. Currently, many therapy methods are available for cervical cancer, such as surgery, radiotherapy and chemotherapy. According to the NCCN Clinical Practice Guidelines in Oncology—Gervical Cancer Guideline2010, radiotherapy is one of the important means for the treatment of cervical cancer. Radiotherapy is effective for almost all the stages of cervical cancer, particularly for patients with advanced cervical cancers or who can not be cured surgically. However the response to radiotherapy varies among patients because of the heterogeneity of individual conditions and tumors. Radiation resistance has become one of the most reasons of failure of cure and recurrence and metastasis. Therefore to clarify the mechanisms of radiation resistance may provide theoretical basis for individual treatment and also provide new therapeutic targets to reverse the radiation resistance of cervical cancer. The response of tumor cells to radiotherapy was affected by many factors in tumor microenvironment. Radiation ionizing activates genes which are related to cell apoptosis, DNA damage and repair, cell adhesion and angiogenesis. In return these genes regulate cell response to radiation. Epithelial-mesenchymal transition (EMT) is a process that epithelial cells lose their cell-cell adhesion and obtain mesenchymal features. Including redistribution or lost of specific epithelial connecting protein, E-cadherin, and mesenchymal markers, including N-cadherin and vimentin, were activated. Morphology of cell changes to cambiform phenotype and the ability of migration is enhanced. EMT is a biological process that epithelial cells transited to cells with mesenchymal characters in some physiological and pathological conditions. And it also occurs in the development of tumors. In recent years, the functions of EMT have been paid more attention. It has been reported that EMT played important role in development and progression, invasion and migration of tumor. And many factors regulated EMT.MicroRNAs (miRNAs) are small endogenous single-stranded,22-24nucleotide non-coding RNAs. Maps of miRNAs are different between tumors and normal tissues. Therefore these maps can be used as new methods of disease classification. MiRNAs influence differentiation, proliferation, metabolism, apoptosis of cells and development of diseases by regulating key target mRNAs. And in regulation of EMT, miRNAs also play important role. As a result it is very important to clarify the molecular mechanisms of occurrence of EMT and the functions of EMT in development of tumors. And it is also very meaningful to explore the diagnostic methods which are based on EMT.NF-κB is a key transcription factor in regulation of cell apoptosis and tumor progression. It is essential in induction and maintain of EMT. MiRNAs can regulate NF-κB and consequently the expression of its downstream genes were affected. The signal strength and lasting time of NF-κB are controlled by multiple negative feedback mechanisms. Thus NF-κB can keep its functions of oncogene in tumor cells.This study aimed to clarify the mechanisms of radiation resistance of cervical cancer and provide theoretic basis for individual treatment of cervical cancer. In this project three parts were studied. Part1was to establish radiation resistance cervical cancer cells. Part2was to study the molecular mechanisms of low-dose radiation induced EMT. And Part3was the screening of miRNAs which are resistant to radiation of cervical cancer and analysis of their functions.METHODSFractionated X-ray irradiation was utilized to generate radioresistant (FIR) cell model according to methods reported and NCCN Clinical Practice Guidelines in Oncology—Cervical Cancer Guideline2010. Cervical cancer cells, SiHa and C-33A, were irradiated with2GY of low-dose fractionated X-ray irradiation per day on five consecutive days per week, until the total concentration reached75GY. There was a two-week interval between the last fractionated irradiation and the experiments. Clonogenic assay, cell cycle and apoptotic assays were conducted to test the radiresistance of cells.During this process, morphological changes of FIR cells, which corresponded with EMT, were observed comparing of parent cells. To confirm the existence of EMT, we detected the expression of E-cadherin and N-cadherin, the marker proteins of EMT. After that, other EMT markers and detailed molecular signaling pathways were detected by real-time PCR on mRNA level and by western blot on protein level. SiRNA was used to interfere the expression of NF-κB p65to study the role of NF-κB p65in low-dose radiation induced EMT involved in cervical cancer radioresistance.Recently,microRNAs (miRNAs) were reported to play important role in regulation of EMT, cell proliferation, cell differentiation, apoptosis and the development of diseases. For this reason, we used miRNA microarray analysis to detect the FIR cells and parent cells to find the miRNAs which were related to radioresistance. The primers of relative miRNAs were synthetized and real-time PCR was conducted to further confirm the results of microarray and the functions of miRNAs which work in radioresistance of cervical cancers were studied. Mimics of miRNAs which were down-regulated in FIR cells were synthetized and transfected to detect the change of EMT markers. At the same time, the expressions of NF-κB p65 were detected by western blot. SiRNA was used to silence the expression of NF-κB p65to make sure the expression change of miRNAs by real-time PCR.RESULTSClonogenic assays revealed that the ability of FIR cells to form clones were enhanced comparing with parent cells after2GY of X-ray irradiation. When accepting different dose of X-ray irradiation, the apoptosis rate of FIR cells decreased obviously. This indicated that FIR cells were resistant to radiation and cervical cancer cell model of radioresistance were generated successfully.During the processes of generating radioresistant cervical cancer cells, FIR cells were observed obvious morphological changes which corresponded with EMT. And the abilities of migration and invasion of FIR cells also enhanced.To clarify the existence of EMT, we detected the expression of E-cadherin and N-cadherin which were marker proteins of EMT and found that E-cadherin had low expression and N-cadherin had high expression in FIR cells. This indicated that FIR cells developed EMT. Further studies, including real-time PCR on mRNA level and western blot on protein level, revealed that another epithelial marker, CK-18, was down-regulated and mesenchymal marker, vimentin, was up-regulated. After silencing of p65by siRNA in FIR cells, E-cadherin was up-regulated and N-cadherin was down-regulated. These results showed that NF-κB p65worked in regulation of low-dose radiation induced EMT in cervical cancer.MiRNAs expression profile is a powerful tool to study miRNAs. After analyzing the miRNAs expression profiles of radiation resistance cervical cancer cells and parent cells, miRNAs which are related to radiation resistance can be screened out. And this will provide new therapeutic targets to reverse the radiation resistance of cervical cancer patients. The results of miRNA microassay analysis showed that20in total1198miRNAs varied in FIR cells compared with parent cells. And real-time PCR confirmed these results. Mimics of miRNAs which were down-regulated in FIR cells were synthesized and transfected into FIR cells to detect the changes of EMT markers. Results revealed that after transfection of mimic miR-1236, the expression of E-cadherin was enhanced and N-cadherin was decreased obviously in FIR cells. It indicated that miR-1236play role in low-dose radiation induced EMT involved in cervical cancer radioresistance.Further detailed studies found that after transfection of miR-1236, the expression of NF-κB p65were up-regulated. And when using siRNA to interfere the expression of NF-κB p65, miR-1236was elevated compared with control group. This illustrated that the mutual regulation between miR-1236and NF-κB p65regulated the low-dose radiation induced EMT in cervical cancer.CONCLUSION1. low-dose radiation could induce EMT in cervical cancer to develop the radioresistance.2. NF-κB p65played role in regulation of low-dose radiation induced EMT in cervical cancer radioresistance.3. MiR-1236worked in regulation of low-dose radiation induced EMT in cervical cancer radioresistance.4. Mutual regulation between miR-1236and NF-κB p65regulated the low-dose radiation induced EMT involved in cervical cancer radioresistance.SIGNIFICANCE1. Clarifying the mechanisms of radioresistance in cervical cancer could provide the theoretical basis for individual treatment of cervical cancer patients.2. NF-kB p65and miRNAs which were found relative with cervical cancer could provide new therapeutic targets for cervical cancer to reverse the radioresistance.3. The.radioresistance cervical cancer cells which we established in this study could be used in other molecular research related to radioresistance. |
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