Effect Of UCP2 On Radiosensitivity Of Human Cervical Cancer Cells And Mitochondrial Related Mechanism

Cervical cancer is one of the most common malignant tumors in gynecology,and it is also one of the main causes of cancer death among women all over the world.Under the influence of early marriage,childbirth,human papillomavirus(HPV)infection and other factors,the prevalence rate of cervical cancer in women is often higher.Due to the rapid development of the disease,most women are in the middle and late stage at the time of first diagnosis.In clinic,there are many treatments for cervical cancer,radiotherapy is one of the main treatment methods,but due to the existence of radiation resistance,it seriously affects the prognosis of patients with radiotherapy.However,the research on the mechanism of radiation resistance of cervical cancer is not clear at present.Therefore,in order to increase the efficiency of diagnosis and improve the effect of radiotherapy,it is particularly important to explore the mechanism of radiation resistance of cervical cancer.UCP2(uncoupling protein2,UCP2)is an anion carrier protein located in the intima of mitochondria,which mediates the proton leakage of mitochondrial inner membrane,thus affecting the production of intracellular reactive oxygen species,regulating the process of oxidative stress,and further changing mitochondrial autophagy,cell proliferation,cell invasion and apoptosis,tumor progression and chemotherapy resistance to affect the prognosis of tumor patients.In this study,Hela and Caski cells were taken as the main research objects,and UCP2 was used as the target gene to explore the role of UCP2 in radiosensitivity and mitochondrial-related mechanisms.Objective:The purpose of this study is to explore the mechanism of UCP2 in the radiosensitivity of cervical cancer cells by regulating cellular oxidative stress,so as toprovide a theoretical basis for optimizing individual treatment regimens for cervical cancer and making it a new target for diagnosis and treatment of cervical cancer.Methods:1.The background expression in different kinds of cervical cancer cells was detected by Q-PCR and WesternBlot.The cells used in the follow-up experiment were selected to detect the changes of UCP2 expression in Hela cells at different time points after 4Gy irradiation.2.The UCP2 silencing and overexpression models of Hela and Caski cells were constructed by transient transfection,and the transfection efficiency was detected by QPCR and WesternBlot,respectively.3.Clony formation and CCK-8 assay were used to detect the colony formation,proliferation and survival ability of the treated cells.4.Using DCFH-DA probe to detect the effect of UCP2 on ROS of cervical cancer cells induced by radiation.5.MitotrackerGreenFM detection of UCP2 was used to detect the changes of mitochondrial membrane integrity of two kinds of cervical cancer cells at 6 h,12 h,12 h and 24 h after irradiation.6.The changes of mitochondrial membrane potential of two kinds of cervical cancer cells at 6 h,12 h,12 h and 24 h after irradiation were detected with rhodamine123(Rhodamine123).7.Apoptosis and cell cycle of UCP2 silencing and overexpression models combined with X-ray irradiation were detected by flow cytometry.8.The expression of DNA damage related protein ?-H2 AX in Hela cells was detected by immunofluorescence.9.WesternBlot was used to detect the changes of apoptosis,cycle and DNA damage-related protein expression in cervical cancer cells,and to explore the molecular mechanism of UCP2 in cervical cancer cell apoptosis,cycle and DNA damage.10.The experimental data were expressed as average ±standard deviation(±s).Statistical analysis was carried out by SPSS24.0,histogram was drawn by software GraphPadPrism6.0,and comparison between groups was made by ANOVA.Results:1.The background expression of UCP2 was different in different cervical cancer cells,and irradiation could induce the expression of UCP2 in Hela cells.The background expression of UCP2 was higher in Hela,and Hela was used as the model of UCP2 silencing.The background expression of UCP2 in Caski cells was relatively low,and Caski cells were used as the model of UCP2 overexpression.The expression of UCP2 in Hela cells enhanced with the increase of time at 6 h,12 h,24 h after irradiation.2.Effect of UCP2 on proliferation and survival of cervical cancer cells.After UCP2 silencing,the colony forming ability of Hela cells after 2,4,6,8 and10 Gy irradiation was significantly lower than that of the negative control group,and after 4 Gy irradiation,the proliferation and survival ability of Hela cells silenced by UCP2 was significantly decreased,but the proliferation and survival ability of UCP2 overexpressed Caski cells irradiated by 8 Gy was not obvious compared with the negative control group,which was related to the transfection efficiency.3.Effect of UCP2 on ROS in radiation-induced cervical cancer cells.In the two kinds of cervical cancer cells,the production of ROS in the radiation group was higher than that in the non-irradiation group,especially in Hela cells.The ROS production of Hela cells silenced by UCP2 at 6 h and 12 h after irradiation was significantly higher than that of the negative control group,and there was no significant difference in ROS production at 24 h after irradiation,which was considered to be related to the dissipation of ROS,while at 6 h and 12 h after irradiation,the Caski cells in the over-expression group of UCP2 decreased to some extent compared with the negative control group,although the difference was not significant,but the difference was statistically significant.4.Effect of UCP2 on Mitochondrial structure and function of Cervical Cancer cells induced by radiation.After UCP2 silencing,Hela cells could significantly increase the mitochondrial membrane potential and promote the production of ROS after 6 hours and 12 hours after irradiation,and the mitochondrial membrane potential collapsed under the action of excessive ROS,that is,the membrane potential of Hela cells decreased significantly in 24 hours after irradiation,while the change of membrane potential of UCP2 overexpressed Caski cells after irradiation was always higher than that of negative control group.Under the influence of the changes of mitochondrial membrane potential and ROS,the integrity of mitochondrial membrane in two kinds of cells also changed.We observed that the mitochondrial membrane integrity of Hela cells after UCP2 silencing was further damaged by irradiation compared with the control group,and the destruction of mitochondrial membrane in Caski cells with UCP2 overexpression was relatively reduced by irradiation.5.Effect of UCP2 on radiation-induced apoptosis of cervical cancer cells.There was no significant difference in the apoptosis rate of cervical cancer cells in the non-irradiation group,but the apoptosis rate of cervical cancer cells increased significantly after irradiation.UCP2 silencing enhanced the radiation-induced apoptosis of Hela cells,and the level of Bcl-2,Bcl-xl were reduced,and the level of Cleaved-Caspase3,Cleaved-Caspase9 and Cyt-c which relating to the apoptosis pathway induced by mitochondria.The level of Apaf-1 increased further in UCP2 silent group.However,only the expected changes in the expression of Bcl-xl,Caspase-9 and Cyt-c were observed in Caski cells,which was related to the low transient transfection efficiency of UCP2 overexpression plasmid.6.Effect of UCP2 on radiation-induced celvical cancer cell cycles.The cell cycle changes of the two kinds of cells were detected by flow cytometry.There was no significant difference in cell cycle changes in the unirradiated group,but there was obvious G2 / M phase arrest in Hela and Caski cells irradiated by 4Gy and 8Gy,respectively.Compared with the negative control group,the arrest effect was enhanced by silencing UCP2 in irradiated Hela cells.In irradiated Caski cells,compared with the negative control group,the overexpression of UCP2 reduced the cell cycle arrest effect induced by irradiation to a certain extent.WesternBlot results also showed that cell cycle-related proteins CyclinB and CDC2 related to G2 phase cell cycle checkpoint also showed corresponding changes.7.Effect of UCP2 on radiation-induced DNA damage in cervical cancer cells.After UCP2 silencing in Hela cells,we used immunofluorescence to detect the number of ?-H2 AX focus formation at different time points after irradiation.It was found that at 6 h and 12 h after irradiation,compared with the non-radiation group,the?-H2 AX focus formation was significantly increased,but there was no significant difference within the irradiated groups,but at 24 h after irradiation,the ?-H2 AX focus formation in the UCP2 silent group was significantly higher than that in the negative control group,but at 24 h after irradiation,the ?-H2 AX focus formation in the UCP2 silent group was significantly higher than that in the negative control group.That is,UCP2 enhanced the damage of DNA double strand breaks in Hela cells induced by X-rays.The WesternBlot results also showed that the expression of Ku70,Rad51-proteins related to DNA damage and repair,decreased to a certain extent in the UCP2 silent group after irradiation,and it was also detected by WesternBlot that the ?-H2 AX protein in the UCP2 overexpression group decreased compared with the negative control group 12 hours after irradiation,and the UCP2 overexpression attenuated the DNA damage caused by ionizing radiation,while the Ku70 protein increased in the UCP2 overexpression group.Conclusion:1.Ionizing radiation can induce the expression of UCP2 in cervical cancer cells.2.UCP2 can affect ROS production in cervical cancer cells induced by ionizing radiation.Silencing UCP2 can promote radiation-induced ROS production in Hela cells,while overexpression of UCP2 can reduce radiation-induced ROS production in Caski cells to some extent.3.UCP2 can cause some structural and functional changes of mitochondria in cervical cancer cells induced by radiation,mainly mitochondrial membrane potential and membrane integrity,which further affect the activation of mitochondrial-induced apoptosis pathway.4.UCP2 can change the cell cycle of cervical cancer cells induced by ionizing radiation,and the arrest of G2 phase is the most significant,that is,UCP2 silencing can promote radiation-induced arrest of G2 phase of cervical cancer cells,while the overexpression of UCP2 can weaken the blocking effect accordingly.5.Most of the activation mechanisms of the above apoptotic pathways can be attributed to the changes of ROS production in cervical cancer cells induced by UCP2,and the production of ROS indirectly affects radiation-induced DNA damage and repair in cervical cancer cells.In short,UCP2 plays a very important role in the radiosensitivity of cervical cancer cells and is expected to become a new target for radiotherapy of cervical cancer.