Role Of Crif1Played In The Radiation Resistance Of U2OS Osteosarcoma Cells And Its Possible Mechanism

Objectives:To identify the role of Crif1played in radiation resistance of osteosarcoma cells andexplore its possible molecular mechanism.Methods:1. Crif1expression was detected by immunohistochemistry in osteosarcoma and lungcancer tissues, and both mRNA and protein levels were detected in U2OS, HepG,PLC,A549,Jurkat,BMSCs and HK2cells by real time PCR and western blot.2. In vitro radiation damage was simulated and Crif1was interferenced by RNAilentivirus effection. Cell proliferation and apoptosis of U2OS cells were detected by CCK8,flow cytometry and TUNEL assay before and after radiation.3. Cell cycle of U2OS cells was examined by flow cytometry before and after radiation.Prokaryotic expression vector of CDK2and Crif1were constructed, GST pull downexperiments were carried out to identify the direct binding of both proteins. Localization ofCDK2was observed by immunofluorescence.4. Accumulation of ROS were detected by flow cytometry and immunofluorescence,while expression of Crif1and NRF2was detected by real time PCR and western blot.Expression of phase II detoxification enzymes including HO-1,GSTA and GCLC weremeasured by real time PCR.Results:1. Crif1expression was significantly higher in osteosarcoma tissue than lung cancer.In different cell lines, Crif1also showed much higher expression in U2OS cell line. To theexpression of Crif1in U2OS, the relative quantity of other cells were BMSCs0.48±0.05,PLC0.19±0.02, HepG20.14±0.02, HK20.08±0.01, A5490.05±0.007, Jurkat0.02±0.005, Crif1expression of U2OS cells was significantly higher than other cell lines (p<0.01), Western Blot showed consistent results with PCR.2. After4Gy radiation，cell proliferation was significantly slowed down, especially in the RNAi group when compared with the control and empty virus group (p <0.01).Apoptosis detection showed that prior exposure, no significant difference among the threegroups (p>0.05). Apoptosis can be induced after9Gy radiation，and the apoptosis rate ofinterference group increased over time significantly(0h5.43±1.52%，24h31.67±4.28%，48h44.30±7.54%，p＜0.01).3. G0/G1arrest was observed after9Gy radiation(control group0h48.8±4.72%，24h55.32±6.17%，48h56.58±6.22%，p＜0.05； empty virus group0h48.2±3.98%，24h52.63±5.77%，48h57.90±7.18%，p＜0.05), and interfering Crif1expression can partly reduce this arrest.GST pull down showed that Crif1can be combined directly with CDK2. After radiation,CDK2nuclear transfer can be clearly observed.4. ROS increasing appeared in U2OS cells after radiation, which is particularlysignificant in RNAi group(p <0.05). Accumulation of ROS negatively correlated with theexpression of NRF2. Expression of HO-1,GSTA and GCLC showed a similar trend withCrif1and NRF2, both increased significantly in the control and empty virus group afterradiation while no increaseing or even lower expression was observed in RNAi group.Conclusions:1. High expression of Crif1in osteosarcoma cells is related to their radiationresistance.2. Knock down Crif1expression can enhance the radiation sensitivity of U2OS cells,inhibition cell proliferation and promote apoptosis.3. Crif1can protect U2OS from radiation damage by negatively regulate cell cycle andinduce G0/G1arrest, which is carried out by directly interacting with CDk2and controllingits nuclear translocation.4. Crif1plays a protective role in oxidative damage caused by radiation in U2OS cellsby regulating NRF2and the downstream phase II detoxifying enzymes.