Effect Of UCP2 Inhibitor Genipin On Radiosensitivity Of Human Cervical Cancer Cells And Related Mechanism Research Of Mitochondria

Cervical cancer is the most common cancer of the Cervix and the second leading cause of cancer death in women worldwide.Premature sexual activity,multiple sexual partners,low condom use,multiple pregnancies and other factors contribute to a rising trend in morbidity,affecting the health of many women in developing countries.Most patients with cervical cancer will choose radiotherapy,because of the existence of factors such as hypoxia-resistant cells,the tumor cells have developed radiation resistance,and some tumor cells can not be eliminated during radiotherapy,therefore,it is very important to improve the radiation sensitivity of tumor cells.Mitochondrial Uncoupling protein 2（UCP2）is located in the Inner mitochondrial membrane and can influence tumor growth by changing the Ros content in the cell.Genipin,a specific blocker of UCP2,is a cyclic iridoid glucoside with the molecular formula C11H14O5,the active component isolated from the fruit of Gardenia rubiaceae is a kind of non-toxic edible dye.In recent years,it has been found that it has therapeutic effect on many kinds of tumors by inhibiting the expression of UCP2,thus affecting the generation of reactive oxygen species（ROS）,and further inducing the apoptosis of tumor cells by changing the function of Mitochondria.In this study,Siha and Me180 cells were used to investigate the role of Genipin,an inhibitor of UCP2,in radiosensitivity and its mitochondrial mechanism.Objective:An ideal radiosensitizer with low toxicity and high efficiency was found for clinical treatment.In this study,we investigated the radiosensitization of Genipin on human cervical cancer Siha and Me180 cells and its mechanism.To investigate whether Genipin inhibits the proliferation of cervical cancer cells and whether it affects the radiosensitivity of cervical cancer cells.To investigate whether Genipin plays its role by inhibiting the expression of Mitochondrial UCP2 and the specific molecular mechanism.Methods:1.CCK-8 method:by detecting the effect of Genipin on the activity of Siha cells and Me180 cells,the best drug concentration,action time and best radiation dose can be obtained;2.Clone formation method:by detecting the colony-forming ability of Genipin on cervical cancer cells,explore the effect of Genipin on the radiation sensitivity of cervical cancer cells;3.Cell scratch test method:to detect the effect of Genipin on the migration ability of cervical cancer cells induced by radiation;4.Q-PCR method:detect Genipin’s changes in the expression of UCP2 m RNA in cervical cancer cells induced by radiation;5.Western Blot method:detect Genipin’s radiation-induced apoptosis,cycle-related protein and UCP2 protein level changes in cervical cancer cells;6.Use DCFH-DA probe to detect the effect of Genipin on radiation-induced ROS in cervical cancer cells;7.Use Rhodamine 123（Rhodamine123）staining kit to detect Genipin’s radiation-induced changes in mitochondrial membrane potential of cervical cancer cells;8.Flow cytometry:detect the changes of Genipin’s radiation-induced apoptosis rate and cycle distribution of cervical cancer cells;9.Statistical analysis:All data were analyzed with SPSS 24.0 software,and graphs were drawn with Graph Pad Prism 6.0.The data are expressed in the form of mean±standard deviation（（?）±s）.The independent sample T test was used to analyze the comparison between different groups;the one-way analysis of variance was used for the comparison between multiple groups,and P<0.05 was considered to be statistically different.Results:1.Genipin’s effect on the proliferation and survival of cervical cancer cellsThe inhibition degree of Siha and Me180 cervical cancer cells increases with the increase of Genipin concentration,the extension of Genipin action time and the increase of radiation dose;the inhibition rate of Genipin to Siha and Me180 cervical cancer cells is close,and the IC₅₀ calculation shows that Genipin is The optimal concentration of the two cervical cancer cells were 145μmol/L and 142μmol/L,respectively;the optimal duration of action was 48 h;the optimal radiation dose was 4Gy;the proliferation and viability of Siha and Me180 cells after Genipin Compared with the negative control group,there was a greater reduction（P<0.05）;and after 4Gy irradiation,the proliferation and survival ability of Siha and Me180 cells after Genipin treatment decreased more significantly（P<0.05）;2.The effect of Genipin on the radiosensitivity of cervical cancer cellsGenipin’s effect on Me180 cells after 2,4,6,8 Gy irradiated,the colony forming ability is greatly reduced compared with the negative control group,and after 4 Gy irradiated,Genipin’s cervical cancer cells The ability of proliferation and survival of the drug is significantly reduced;3.The effect of Genipin on the migration ability of cervical cancer cells induced by radiationThe migration ability of Siha and Me180 cells treated with Genipin was significantly reduced compared with the negative control group（P<0.05）;and after 4Gy irradiation,the migration ability of Siha and Me180 cells treated with Genipin was obvious Decrease（P<0.05）;4.The effect of Genipin on UCP2 in cervical cancer cellsCompared with the negative control group,cervical cancer cells treated with Genipin had significantly down-regulated UCP2 m RNA and protein expression levels（P<0.05）;5.The effect of Genipin on the apoptosis of cervical cancer cells induced by radiationCompared with the negative control group,Genipin can enhance the apoptotic rate of Siha and Me180 cervical cancer（P<0.05）.After irradiation,the apoptosis rate of Siha and Me180 cervical cancer cells after Genipin’s action increased significantly,and Genipin enhanced radiation The induced apoptosis of Siha and Me180 cells（P<0.05）promoted the increase of apoptotic body formation related proteins and the expression of pro-apoptosis related proteins,and the decrease of the expression of anti-apoptotic proteins;6.Genipin’s effect on the cycle of cervical cancer cells induced by radiationWe have tested the cell cycle changes of Siha and Me180 cervical cancer cells by flow cytometry.Siha and Me180 cells will have obvious G₂/M phase block after 4 Gy irradiation.The blocking effect of Genipin group was more obvious（P<0.05）;Western blot experiment found that after irradiation,the expression of Cyclin B and CDC2 protein in Genipin group decreased compared with the negative control group;7.Genipin’s effect on mitochondrial function and ROS of cervical cancer cells induced by radiationAfter Genipin treatment of Siha and Me180 cells,the membrane potential and ROS increased compared with the negative control group.After irradiation,the membrane potential and ROS of the Genipin group increased compared with the negative control group（P<0.05）.Conclusion:1.Genipin’s inhibition of human cervical cancer Siha and Me180 cells is proportional to Genipin’s concentration,action time and radiation dose;Genipin’s inhibition of human cervical cancer Siha and Me180 cells is close;2.Genipin has an inhibitory effect on the proliferation activity and migration ability of human cervical cancer Siha and Me180 cells induced by radiation;3.Genipin can inhibit the production of UCP2 in human cervical cancer Siha and Me180 cells;4.Genipin can promote the increase of the inner membrane potential of cervical cancer Siha and Me180 cells and the generation of ROS,combined with ionizing radiation,the increase of membrane potential and the generation of ROS are more significant;5.Genipin can promote the apoptosis and G₂ phase block of cervical cancer Siha and Me180 cells,and the radiation-induced apoptosis and G₂ phase block of cervical cancer cells are more obvious;In general,Genipin promotes mitochondrial ROS generation by inhibiting UCP2,which further affects the activation of mitochondrial-induced apoptosis pathways.