Impacts of CircFN1 on radiosensitivity of esophageal squamous cell carcinoma cells by regulating miR-375/CBX3 axis
Objective To investigate the impacts of CircFN1 on radiosensitivity of esophageal squamous cell carcinoma(ES-CC)cells by regulating miR-3 75/chromobox homologue 3(CBX3)axis.Methods Si-NC,si-CircFN1,si-CircFN1+in-hibitor NC and si-CircFN1+miR-375 inhibitor were transfected into ESCC cells(KYSE-150)respectively,which were classified as si-NC group,si-CircFN1 group,si-CircFN1+inhibitor NC group,and si-CircFN1+miR-375 inhibitor group,respectively.After transfection,the cells were irradiated with 2,4,6 and 8 Gy MV-X rays.The expressions of CircFN1 and miR-375 were detected by real-time fluorescent quantitative-PCR(qRT-PCR).The expression of CBX3 pro-tein was detected by western blot.Clonogenic assays were conducted to evaluate the clonogenic capacity of KYSE-150 cells after irradiation.The activity of KYSE-150 cells was detected by CCK8.Transwell and scratch assays were applied to detect cell invasion and migration.Double luciferase reporter gene experiment was applied to verify the relationship be-tween CircFN1 and miR-375,and between miR-375 and CBX3.Data were expressed as(X)±S of 6 replicates.One-way ANOVA and SNK-q test were used to compare differences among groups.Results After 2,4,6 and 8 Gy irradiation,the colony forming rate of KYSE-150 cells in the si-CircFN1 group was obviously lower than that in the si-NC group,the colony formation rate of si-CircFN1+miR-375 inhibitor group was obviously higher than that of si-CircFN1 group and si-CircFN1+inhibitor NC group(P＜0.05).The sensitization enhancement ratio(SER)of the si-CircFN1 group,si-Cir-cFN1+inhibitor NC group,and si-CircFN1+miR-375 inhibitor group were 1.352,1.374 and 1.011,respectively.Un-der 0 Gy 6 MV-X ray irradiation,compared with the si-CircFN1 group,the level of CircFN1,CBX3,migration rate and number of invasive cells in the si-CircFN1 group were decreased(P＜0.05),the expression of miR-375 was increased(P＜0.05),D(450 nm)value(0.51±0.05 vs 0.97±0.11)was decreased(q=14.196,P＜0.001),apoptosis rate[(16.51±1.45)％vs(5.97±0.62)％]was increased,q=22.412,P＜0.001;Compared with the the si-CircFN1+in-hibitor NC group,the level of CircFN1,CBX3,migration rate and number of invasive cells in the si-CircFN1+miR-375 inhibitor group were higher(P＜0.05),the expression of miR-375 was decreased(P＜0.05),D(450 nm)(0.94±0.09 vs 0.53±0.05),q=12.653,P＜0.001 and apoptosis rate[(6.23±0.67)％vs(15.76±1.55)％]were lower,q=20.207,P＜0.001.Under 4 Gy 6 MV-X ray irradiation,the level of CircFN1 and CBX3,migration rate and number of invasive cells in the si-CircFN1 group were decreased compared with the si-NC group(P＜0.05),the expression of miR-375 was increased(P＜0.05),D(450 nm)value(0.23±0.02 vs 0.77±0.08)decreased(q=25.456,P＜0.001),and the apoptosis rate[(22.23±2.58)％vs(9.77±0.98)％]was increased,q=15.263,P＜0.001;Compared with si-CircFN1+inhibitor NC group,CBX3 level,migration rate and number of invasive cells were increased in si-CircFN1+miR-375 inhibitor group(P＜0.05),miR-375 expression decreased(P＜0.05),D(450 nm)value(0.73±0.06 vs 0.22 ±0.02)increased(q=24.042,P＜0.001),the apoptosis rate[(10.34±1.23)％vs(23.67±2.62)％]decreased,q=16.329,P＜0.001.The levels of CircFN1,CBX3,D(450 nm),migration rate and number of invasive cells under 4 Gy irradiation were lower than those in the non-irradiated group,the expression of miR-375 and apoptosis rate were higher than those in the non-irradiated group,all P＜0.05.Conclusion Silencing CircFN1 may inhibit the expression of CBX3 by up-regulating miR-375,thereby increasing the radiosensitivity of ESCC cells.
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