肌动蛋白相关蛋白2调控人抗原R稳定性对非小细胞肺癌细胞放疗抵抗的影响

Effect of actin-related protein 2 modulation of human antigenr R stability on radiotherapy resistance in lung cancer cells

目的 探讨肌动蛋白相关蛋白2(ARPC2)通过人抗原R(HuR)对肺癌细胞放疗抵抗的影响.方法 将肺癌细胞根据不同处理,分为7组,ARPC2过表达组(使用ARPC2过表达慢病毒感染肺癌细胞)、ARPC2敲低组(使用shARPC2敲低慢病毒感染肺癌细胞)、HuR过表达组(使用HuR过表达慢病毒感染肺癌细胞)、HuR敲低组(使用shHuR敲低慢病毒感染肺癌细胞)、ARPC2过表达的同时HuR敲低组(同时使用ARPC2过表达和shHuR敲低慢病毒感染肺癌细胞)、ARPC2敲低的同时HuR过表达组(同时使用shHuR敲低和HuR过表达慢病毒感染肺癌细胞)、对照组(使用空载体慢病毒感染肺癌细胞).6 Gy的电离辐射处理后,流式细胞术检测各组肺癌细胞(A549、Calu-1、HCC827、MSTO-211H、NCI-H1299、NCI-H520、SK-MES-1)的凋亡水平.免疫印迹法检测肺癌细胞中HuR的蛋白表达水平.通过凋亡水平指示肺癌细胞的放疗抵抗.通过HuR的表达水平的半衰期以指示HuR的稳定性.比较用非配对双尾t检验,多组间比较则采用方差分析.结果 A549(0.43±0.08、1.88±0.15)、Calu-1(0.38±0.05、1.90±0.14)、HCC827(0.37±0.07、1.89±0.23)、MSTO-211H(0.39±0.06、1.95±0.29)、NCI-H1299(0.40±0.07、1.92±0.32)、NCI-H520(0.42±0.10、1.85±0.30)、SK-MES-1(0.44±0.09、1.90±0.33)中 ARPC2 的 mRNA 和蛋白表达水平高于人正常肺细胞 BEAS-2(0.12±0.03、1.19±0.19)、BNHLF(0.08±0.02、1.16±0.20,t=11.473、14.100、13.825、15.627、18.364、20.594、24.504、9.107、10.335、9.634、11.910、10.882、9.027、10.658,P<0.05).电离辐射处理组中 ARPC2 的蛋白表达水平(0.81±0.15、1.25±0.25、0.63±0.12,0.51±0.10、0.64±0.15、1.86±0.30、0.85±0.14)高于未处理组(0.17±0.04、0.15±0.04、0.16±0.04,0.17±0.05、0.16±0.04、0.17±0.05、0.18±0.06,t=13.037、21.035、13.610、7.331、9.925、28.567、14.814,P<0.05).电离辐射处理后ARPC2过表达组肺癌细胞的凋亡[(16.48±6.11)%、(16.03±5.35)%、(18.28±8.22)%、(24.70±9.71)%、(17.68±9.11%)]低于对照组[(47.92±6.58)%、(55.31±6.94)%、(51.29±7.16)%、(51.96±9.17)%、(54.16±2.24)%,t=11.071、14.183、9.579、6.453、12.311,P<0.05],ARPC2 敲低组肺癌细胞的凋亡[(70.47±8.46)%、(84.16±0.73)%、(76.71±1.87)%、(76.05±0.47)%、(88.07±9.14)%]高于对照组[(55.21±6.29)%、(51.59±7.04)%、(50.07±5.33)%、(47.14±7.53)%、(44.41±9.38)%,t=4.584、14.550、14.912、12.124、10.543,P<0.05].ARPC2 过表达组中 HuR、p-HuR、JAK1、p-STAT3 的蛋白表达水平(0.81±0.11、0.92±0.14、0.55±0.10、0.63±0.12)高于对照组(0.15±0.03、0.14±0.04、0.15±0.03、0.16±0.05,t=18.305、22.614、8.391、11.287,P<0.05),ARPC2 敲低组中 HuR、p-HuR、JAK1、p-STAT3 的蛋白表达水平(0.19±0.05、0.15±0.04、0.14±0.03、0.17±0.05)低于对照组(0.90±0.15、0.85±0.13、0.63±0.12、0.71±0.14,t=14.200、15.628、11.307、13.462,P<0.05).ARPC2 过表达组[(11.32±0.26)、(10.17±0.22)、(10.49±0.21)、(10.17±0.03)、(10.53±0.06)h]相较对照组 HuR 半衰期[(5.61±1.31)、(5.30±2.53)、(5.72±1.95)、(5.34±0.70)、(5.86±1.09)h]延长(t=13.523、6.056、7.691、21.819、13.533,P<0.05),ARPC2 敲低组[(2.02±0.71)、(2.96±1.23)、(2.61±0.68)、(3.04±0.82)、(2.23±0.22)h]相较于对照组 HuR 半衰期[(4.92±1.69)、(5.05±0.45)、(5.38±0.56)、(5.64±0.21)、(4.62±0.37)h]缩短(t=5.012、5.047、9.941、9.714、17.558,P<0.05).电离辐射处理后,HuR 过表达组(23.01±3.83、17.30±6.59、21.10±4.58、16.44±9.04、23.36±4.56)比对照组(47.30±6.59、51.10±4.58、46.44±9.00、53.36±9.56、45.97±2.54)可抑制肺癌细胞的凋亡(t=10.077、13.322、7.935、8.869、13.716,P<0.05),HuR 敲低组(80.90±17.46、72.24±10.84、77.82±18.9、84.16±10.98、83.50±11.54)比对照组能促进肺癌细胞的凋亡(t=5.693、4.918、5.163、6.025、5.925,P<0.05).结论 ARPC2通过提高HuR的蛋白稳定性促进了肺癌细胞的放疗抵抗.

Objective To investigate the effect of actin-related protein 2 on radiotherapy resistance of lung cancer cells through HuR.Methods After treatment with ionizing radiation of 6 Gy,the apoptosis level of lung cancer cells in each group was detected by flow cytometry.The protein expression level of HuR in lung cancer cells was detected by immunoblotting.Radiotherapy resistance of lung cancer cells was indicated by apoptosis level.The half-life of HuR expression level was passed to indicate the stability of HuR.All data were statistically analyzed using prism7,in which the measurement data in line with normal distribution were expressed as mean±standard deviation((x)±s),and the comparison was performed using unpaired two tailed t-test,while the comparison between multiple groups was performed using analysis of variance.Results The expression levels of ARPC2 mRNA and protein in A549(0.43±0.08,1.88±0.15),Calu-1(0.38±0.05,1.90±0.14),HCC827(0.37±0.07,1.89±0.23),MSTO-211H(0.39±0.06,1.95±0.29),NCI-H1299(0.40±0.07,1.92±0.32),NCI-H520(0.42±0.10,1.85±0.30),SK-MES-1(0.44±0.09,1.90±0.30)were higher than in BEAS-2(0.12±0.03,1.19±0.19)and BNHLF(0.08±0.02,1.16±0.20)(t=11.473,14.100,13.825,15.627,18.364,20.594,24.504,9.107,10.335,9.634,11.910,10.882,9.027,10.658,P<0.05).The protein expression level of ARPC2 in the ionizing radiation group(0.81±0.15,1.25±0.25,0.63±0.12,0.51±0.10,0.64±0.15,1.86±0.30,0.85±0.14)was higher than that in the untreated group(0.17±0.04,0.15±0.04,0.16±0.04,0.17±0.05,0.16±0.04,0.17±0.05,0.18±0.06,t=13.037,21.035,13.610,7.331,9.925,28.567,14.814,P<0.05).The apoptosis of lung cancer cells in ARPC2 overexpression group[(16.48±6.11)%,(16.03±5.35)%,(18.28±8.22)%,(24.70±9.71)%,(17.68±9.11)%after ionizing radiation treatment]was lower than that in control group[(47.92±6.58)%,(55.31±6.94)%,(51.29±7.16)%,(51.96±9.17)%,(54.16±2.24)%,t=11.071,14.183,9.579,6.453,12.311,P<0.05].The apoptosis of lung cancer cells in ARPC2 knockdown group[(70.47±8.46)%,(84.16±0.73)%,(76.71±1.87)%,(76.05±0.47)%,(88.07±9.14)%]was higher than that in control group[(55.21±6.29)%,(51.59±7.04)%,(50.07±5.33)%,(47.14±7.53)%,(44.41±9.38)%,t=4.584,14.550,14.912,12.124,10.543,P<0.05].The protein expression levels of HuR,p-HuR,JAK1 and p-STAT3 in ARPC2 overexpression group(0.81±0.11,0.92±0.14,0.55±0.10,0.63±0.12)were higher than those in control group(0.15±0.03,0.14±0.04,0.15±0.03,0.16±0.05,t=18.305,22.614,8.391,11.287,P<0.05).The protein expression levels of p-HuR,JAK1,p-STAT3 in ARPC2 overex-pression group(0.19±0.05,0.15±0.04,0.14±0.03,0.17±0.05)were lower than those in the con-trol group(0.90±0.15,0.85±0.13,0.63±0.12,0.71±0.14,t=14.200,15.628,11.307,13.462,P<0.05).The half-life of HuR in ARPC2 overexpression group[(11.32±0.26),(10.17±0.22),(10.49±0.21),(10.17±0.03),(10.53±0.06)h]was significantly longer than that in the control group[(5.61±1.31),(5.30±2.53),(5.72±1.95),(5.34±0.70),(5.86±1.09)h,t=13.523,6.056,7.691,21.819,13.533,P<0.05].The half-life of HuR in the ARPC2 knockdown group[(2.02±0.71),(2.96±1.23),(2.61±0.68),(3.04±0.82),(2.23±0.22)h]was signifi-cantly shorter than that in the control group[(4.92±1.69),(5.05±0.45),(5.38±0.56),(5.64±0.21),(4.62±0.37)h,t=5.012,5.047,9.941,9.714,17.558,P<0.05].As compared with the control group(47.30±6.59,51.10±4.58,46.44±9.00,53.36±9.56,45.97±2.54),the apoptosis of lung cancer cells was significantly inhibited in the HuR overexpression group(23.01±3.83,17.30±6.59,21.10±4.58,16.44±9.04,23.36±4.56)(t=10.077,13.322,7.935,8.869,13.716,P<0.05).The apoptosis of lung cancer cells in the HuR knockdown group(80.90±17.46、72.24±10.84、77.82±18.9、84.16±10.98、83.50±11.54)was significantly increased as compared with the control group(t=5.693,4.918,5.163,6.025,5.925,P<0.05).Conclusion ARPC2 promotes the radio-therapy resistance of lung cancer cells by improving the protein stability of HuR.