The Role Of CIP2A In Radiotherapy Sensitivity Through The Regulation Of Autophagy

BackgroundCervical cancer, ranked the second place in the morbidity of female carcinoma, is a deadly disease dramatically affected women’s health.Radiotherapy is one of the important means for the treatment of cervical cancer. 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 common reasons responsible for the failure of 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. Autophagy is a very primitive biological phenomena in the course of life evolution, which exists widely in plants and other lower creatures. Autophagy’s function in cancer’s treatment is always in dispute, and vividly depicted as "doubleedge sword". Presently, autophagy’s role in radiotherapy tend to be described as a "sensitizer of radiotherapy". CIP2A is a newly identified oncoprotein that stabilizes c-Myc protein level by inhibiting the activity of protein phosphatase 2A(PP2A) dephosphorylating c-Myc in S62 in cancer cells. We and other researchers have reported that CIP2A is overexpressed in many carcinomas and demonstrated as a prognostic marker. Our research group have found that CIP2A is involved in the formation of multidrug resistance of cervical cancer via upregulation of P-gp protein expression.Purpose:1. To identify the relationship between CIP2A and radiotherapy resistance of cervical cancer 2. To identify the relationship between CIP2A and autophagy in cervical cancer 3. To identify the mechanism by which CIP2A regulates radiotherapy resistance.Methods and Results:1. Relationship between CIP2A and radiotherapy resistance of cervical cancer:we treated HeLa, SiHa cells with CIP2A siRNA and control siRNA,and then we radiated the cells with various doses of X ray. At last we tested the cell viability via cell counting, CCK8 assay and clone formation assay. The result shows that:when CIP2A was knocked down, the cells were more sensitive to the irradiation, displayed as less cell counts(p<0.01,p<0.05), lower cell growing rate, and smaller colonies(p<0.05). These results demonstrate that knockdown of CIP2A can enhance the radiotherapy sensitivity of HeLa and SiHa cells.2. Relationship between CIP2A and DNA damage in cervical cancer under radiation:DNA double strand break is the primary mechanism through which radiation regulates cell death. So we tested the expression of cleaved PARP1 by western blotting assay after depletion of CIP2A under irradiation, and then we observed the structure of cell nuclear through DAPI assay and Comet assay. The result shows that:after CIP2A depletion, the cleaved PARP1 protein expression is upregulated, more apoptotic body and tailed cells were formed. These results demonstrate that knockdown of CIP2A can aggravate DNA damage in HeLa and SiHa cells.3. Relationship between CIP2A and autophagy in cervical cancer under radiation:we tested the LC3B and P62 protein expression of HeLa and SiHa cells after knockdown of CIP2A under irradiation. And then we used the electron microscope to detect the autophagosome in the two cell groups. The result shows that the expression of LC3B expression was significantly increased, while the expression of P62 was significantly decreased in the siCIP2A group compared with the NC group. Under the electron microscope, we found more autophagosome in siCIP2A group compared with the NC group (p<0.05). These results demonstrate that knockdown of CIP2A can upregulate autophagy level of HeLa and SiHa cells under irradiation.4. Identify whether CIP2A regulates radiotherapy resistance through autophagy:we set two groups:the siBeclinl group as the experimental group and the siNC group as the control group. Both groups were conducted under the fundamental that the CIP2A was knocked down. And then we tested the cell viability of the two groups under irradiation using colonies formation assay. The result shows that the siBeclinl group reveals a better growing status and more colonies were formed(p<0.01). This indicates that inhibition of autophagy can partly reverse the effect of CIP2A in HeLa and SiHa cell.Conclusions:our study first demonstrate that CIP2A can regulate radiotherapy sensitivity of cervical cancer. And this regulation mainly functions through an autophagy dependent way. When we added the autophagy inhibition reagent, the enhancement radiotherapy sensitivity was partly reversed. So we believed that our results suggest that CIP2A, as a new target of gene therapy,has a promising future in multimodality therapy of cervical cancer.