The Effect Of RMP On Radiosensitivity Of Tumor Cells And Preliminary Study On Its Mechanism

Objective:In this study,we explore the differences in the radiation sensitivity of different tumor cells and investigate the effects of RMP on the radiosensitivity of tumor cells and explore the specific mechanism.Methods:1.The CCK-8 experiment detects the proliferation of different types of tumor cells after irradiation with different doses of ⁶⁰Coγ-rays,screens for radiation-sensitive cell lines and radiation-tolerant strains,and confirms the experimental irradiation dose of CCK-8 in subsequent experiments.2.Clone formation assays were performed to detect the formation of clones after irradiation with different doses of ⁶⁰Coγ-rays for CNE-1,HEp-2,and Hep G2.3.The comet assay was used to detect DNA damage repair after treatment with⁶⁰Coγ-rays.4.Flow cytometry was used to detect cell apoptosis of different sensitivity cell lines treated with ⁶⁰Coγ-rays for 24 h and 72 h.5.Western Blot was used to detect the expression of p-ATM,p65 and other repair related proteins before and after irradiation in different irradiation sensitivity cell lines.6.CCK-8 was used to detect the cell proliferation of 2μg RMP over-expression plasmids transiently transfected with different sensitivity cell lines after ⁶⁰Coγ-ray irradiation.7.Flow cytometry was used to detect apoptosis of MCF-7 cells after transient transfection of 2μg RMP with ⁶⁰Coγ-ray irradiation.8.The effects of RMP on the growth of xenografts before and after irradiation were detected by subcutaneous xenografts in nude mice.9.HE staining was used to detect the growth of transplanted tumors.Results:1.CCK-8 results showed that human nasopharyngeal carcinoma cell line CNE-1and human laryngeal carcinoma cell line HEp-2 were significantly inhibited from proliferation at 10 Gy irradiation,and the proliferation inhibition was more pronounced at 20 Gy and 50 Gy.But in liver cancer cell lines HepG2,Hep3 B and breast cancer cell line MCF-7,the inhibitory effect of irradiation on cell proliferation was not obvious;in the liver cancer cell line Huh7 and human lung cancer cell line A549,the effect of irradiation on cell proliferation In between.2.At irradiation doses of 2 Gy,4 Gy,and 6 Gy,the radiation-tolerant cell line HepG2 was significantly higher compared with the two radiation-sensitive cell lines,CNE-1 and HEp-2,with significant differences（P<0.005）.3.Comet electrophoresis results were shown in three cell lines:CNE-1,HEp-2,and MDA-MB-231.The OTM value of comet assay after irradiation was significantly higher than that of the non-irradiation group;however,The OTM value of HepG2,Hep3 B,and MCF were increased not significantly higher than non-irradiation group.4.After irradiation for 24 h,the apoptotic rate of the irradiation-sensitive and radiation-tolerant strains was not significantly different from that of the non-irradiated group.After irradiation for 72 h,the apoptosis rate of irradiation-sensitive nasopharyngeal carcinoma cell line CNE-1 and human laryngeal carcinoma cell line HEp-2 was significantly higher than that of non-irradiation group,but the apoptosis rate of irradiation-tolerant cell line was a little higher than that of non-irradiation group.5.Western Blot results show that different irradiation-sensitive cells can promote ATM phosphorylation and p65 phosphorylation after irradiation.6.In irradiation-sensitive strains CNE-1 and HEp-2,overexpression of RMP significantly increased its tolerance to irradiation compared with control（P<0.001）;in radiation-tolerant strain HepG2 In MCF-7,overexpression of RMP also increased its tolerance to irradiation,but there was no significant difference.7.The transient transfection of NC,RMPo and RMPi in MCF-7 was treated by irradiation.Compared with the non-irradiated group,the apoptosis rate of NC group and RMPo group had no significant difference before and after irradiation,but the apoptosis rate of RMPi group after irradiation was significant improved.8.The results of subcutaneous xenografts in nude mice showed that the tumor volume in the RMP overexpression group was significantly larger than that in the control group,and the tumor volume in the RMP interference group was significantly smaller than that in the control group.9.The results of HE staining showed that the RMP overexpression group significantly increased the number of tumor cells after irradiation,and the growth status was better than that of the control group.The tumor cell density in the RMP interference group was significantly lower than that in the control group,and the cell growth was poor.Conclusion:1.CNE-1,HEp-2 and MDA-MB-231,were selected as radiation-sensitive cell lines.HepG2,Hep3 B and MCF-7 cells were irradiated tolerant cell lines.2.Irradiation can activate the NFκB pathway in different radiation-sensitive tumor cells.3.RMP can increase the tolerance of tumor cells to radiation,on the contrary interfere with RMP to increase the radiosensitivity of tumor cells.4.Overexpression of RMP can promote the growth of transplanted tumors and increase the tolerance of xenografts to irradiation.