CircFN1调节miR-375/CBX3轴对食管鳞状细胞癌细胞放射敏感性的影响

Impacts of CircFN1 on radiosensitivity of esophageal squamous cell carcinoma cells by regulating miR-375/CBX3 axis

目的 探讨CircFN1调节miR-375/染色框同源物3(CBX3)轴对食管鳞状细胞癌(ESCC)细胞放射敏感性的影响。方法 将si-NC、si-CircFN1、si-CircFN1+inhibitor NC和si-CircFN1+miR-375 inhibitor分别转染至ESCC细胞(KYSE-150),分别作为si-NC组、si-CircFN1组、si-CircFN1+inhibitor NC组和si-CircFN1+miR-375 inhibitor组。细胞转染后用2、4、6和8 Gy 6 MV-X射线照射。采用实时荧光定量PCR(RT-qPCR)检测细胞CircFN1和miR-375表达;蛋白质印迹法检测细胞CBX3蛋白表达;克隆形成实验检测KYSE-150细胞受照后的克隆形成能力;CCK8检测KYSE-150细胞活力;Transwell和划痕实验检测细胞侵袭和迁移能力;双荧光素酶报告基因实验验证CircFN1与miR-375以及miR-375与CBX3的关系。数据以6次重复实验的X±S表示,单因素方差分析和SNK-q检验比较多组间差异。结果 2、4、6和8 Gy照射后,si-CircFN1组较si-NC组KYSE-150细胞克隆形成率降低,si-CircFN1+miR-375 inhibitor组较si-CircFN1组和si-CircFN1+inhibitor NC组克隆形成率升高(P<0.05),si-CircFN1组、si-CircFN1+inhibitor NC组和si-CircFN1+miR-375 inhibitor组放射增敏比(SER)分别为为1.352、1.374和1.011。在0 Gy 6 MV-X射线照射下,si-CircFN1组较si-NC组相比,CircFN1与CBX3水平、迁移率和侵袭细胞数量均下降(P<0.05),miR-375表达量增高(P<0.05);D(450 nm)值(0.51±0.05 vs 0.97±0.11)降低,q=14.196,P<0.001;凋亡率[(16.51±1.45)%vs(5.97±0.62)%]升高,q=22.412,P<0.001。与si-CircFN1+inhibitor NC组相比,si-CircFN1+miR-375 inhibitor组CBX3水平、迁移率、侵袭细胞数量上升(P<0.05),miR-375表达量下降(P<0.05),D(450 nm)值(0.94±0.09 vs 0.53±0.05)升高(q=12.653,P<0.001),凋亡率[(6.23±0.67)%vs(15.76±1.55)%]下降,q=20.207,P<0.001。在4 Gy 6 MV-X射线照射下,si-CircFN1组与si-NC组相比,CircFN1与CBX3水平、迁移率和侵袭细胞数量均下降(P<0.05),miR-375表达量升高(P<0.05),D(450 nm)值(0.23±0.02 vs 0.77±0.08)下降(q=25.456,P<0.001),凋亡率[(22.23±2.58)%vs(9.77±0.98)%]升高,q=15.263,P<0.001;与si-CircFN1+inhibitor NC组相比,si-CircFN1+miR-375 inhibitor组CBX3水平、迁移率、侵袭细胞数量上升(P<0.05),miR-375表达量均下降(P<0.05),D(450 nm)值(0.73±0.06 vs 0.22±0.02)上升(q=24.042,P<0.001),凋亡率[(10.34±1.23)%vs(23.67±2.62)%]下降,q=16.329,P<0.001。且4 Gy照射后各组KYSE-150细胞CBX3水平、D(450 nm)、迁移率和侵袭细胞数量低于对应的0 Gy各组,miR-375表达量和凋亡率高于对应的0 Gy各组,均P<0.05。结论 沉默CircFN1可能通过上调miR-375来抑制CBX3表达,进而增加ESCC细胞放射敏感性。

Objective To investigate the impacts of CircFNl on radiosensitivity of esophageal squamous cell carcinoma(ESCC)cells by regulating miR-375/chromobox homologue 3(CBX3)axis.Methods Si-NC,si-CircFN1,si-CircFN1+inhibitor 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 protein 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 between 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-CircFNl 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-CircFNl group and si-CircFN1+inhibitor NC group(P<0.05).The sensitization enhancement ratio(SER)of the si-CircFNl group,si-CircFN1+inhibitor NC group,and si-CircFN1+miR-375 inhibitor group were 1.352,1.374 and 1.01l,respectively.Under O Gy 6 MV-X ray irradiation,compared with the si-CircFNl group,the level of CircFN1,CBX3,migration rate and number of invasive cells in the si-CircFNl group were decreased(P<0.05),the expression of miR-375 was increased(P<0.05),D(450 nm)value(0.51±0.05 us 0.97±0.11)was decreased(q=14.196,P<0.001),apoptosis rate[(16.51±1.45)%us(5.97±0.62)%]was increased,q=22.412,P<0.001;Compared with the the si-CircFN1+inhibitor 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 us 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-CircFNl 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)%us(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 s 0.22±0.02)increased(q=24.042,P<0.001),the apoptosis rate[(10.34±1.23)%us(23.67±2.62)%]decreased,q=16.329,P<0.001.The levels of CircFNl,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.