白细胞介素-37通过调控红细胞膜蛋白带4.1类似物4a的反义RNA1/微小RNA-106b-5p轴降低肾癌细胞的增殖、迁移和侵袭能力

Interleukin-37 reduces the proliferation, migration and invasion of renal cancer cells by regulating the erythrocyte membrane protein band 4.1 like 4a antisense RNA1/microRNA-106b-5p axis

目的:探讨白细胞介素(IL)-37对肾癌细胞786-O增殖、迁移和侵袭的影响及其机制。方法:将肾癌细胞786-O分为对照组、不同剂量(12.5、50.0、200.0 ng/ml)重组IL-37蛋白组、pcDNA组、pcDNA-红细胞膜蛋白带4.1类似物4a的反义RNA1(EPB41L4A-AS1)组、200 ng/ml IL-37+si-NC组和200 ng/ml IL-37+si-EPB41L4A-AS1组,细胞计数试剂盒(CCK-8)法和克隆形成实验检测细胞增殖能力,划痕实验检测细胞迁移能力,Transwell检测细胞侵袭,实时定量反转录聚合酶链反应(RT-qPCR)法检测EPB41L4A-AS1和miR-106b-5p表达。双荧光素酶报告基因实验验证EPB41L4A-AS1和miR-106b-5p调控关系。两组间比较行t检验;多组间比较采用单因素方差分析,组间进一步两两比较行LSD-t检验。结果:12.5、50、200 ng/ml IL-37组786-O细胞吸光度(A)值(0.58±0.02、0.43±0.03、0.29±0.02比0.69±0.06,F=207.113,P<0.05)、克隆形成数[(114.33±6.61)、(87.33±5.22)、(51.33±4.92)个比(139.44±9.49)个,F=277.041,P<0.05]、划痕愈合率[(69.03±0.35)%、(54.9±1.84)%、(38.81±2.73)%比(85.08±0.91)%,F=1191.221,P<0.05]和侵袭数[(91.44±5.68)、(70.89±2.85)、(48.78±6.00)个比(112.44±6.62)个,F=223.386,P<0.05]均低于对照组,差异均有统计学意义,细胞中EPB41L4A-AS1表达高于对照组(1.39±0.15、1.71±0.18、2.18±0.20比1.01±0.10,F=84.386,P<0.05),差异均有统计学意义,miR-106b-5p表达低于对照组(0.82±0.06、0.60±0.09、0.40±0.06比1.00±0.12,F=82.545,P<0.05),差异均有统计学意义,且呈剂量依赖性。pcDNA-EPB41L4A-AS1组786-O细胞(0.23±0.02比0.69±0.05,t=25.626,P<0.05)、克隆形成数[(45.33±3.32)个比(137.78±7.01)个,t=35.757,P<0.05]、划痕愈合率[(35.01±3.27)%比(85.11±1.29)%,t=42.757,P<0.05]和侵袭数[(44.56±4.48)个比(109.44±8.59)个,t=20.091,P<0.05]均降低,差异均有统计学意义。EPB41L4A-AS1可靶向负调控miR-106b-5p。200 ng/ml IL-37+si-EPB41L4A-AS1组786-O细胞中EPB41L4A-AS1表达低于200 ng/ml IL-37+si-NC组(1.14±0.13比2.49±0.19,t=17.592,P<0.05),差异均有统计学意义,miR-106b-5p表达高于200 ng/ml IL-37+si-NC组(0.91±0.09比0.43±0.08,t=11.959,P<0.05),差异均有统计学意义,细胞A值(0.64±0.06比0.31±0.04,t=13.729,P<0.05)、克隆形成数[(123.33±6.18)个比(49.22±3.23)个,t=31.884,P<0.05]、划痕愈合率[(74.90±0.95)%比(38.15±2.17)%,t=46.542,P<0.05]和侵袭数[(92.89±5.25)个比(47.44±4.13)个,t=20.412,P<0.05]均高于200 ng/ml IL-37+si-NC组,差异均有统计学意义。结论:IL-37可能通过调控EPB41L4A-AS1/miR-106b-5p轴抑制肾癌细胞786-O增殖、迁移和侵袭。

Objective To investigate the effect of interleukin 37(IL-37)on the proliferation,migration and invasion of renal cancer cells 786-O and its mechanism.Methods 786-O cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and divided into the control group,different doses(12.5,50,200 ng/ml)of recombinant IL-37 protein groups,pcDNA group,pcDNA-erythrocyte membrane protein band 4.1 like 4a antisense RNA1(EPB41L4A-AS1)group,200 ng/ml IL-37+si-NC group and 200 ng/ml IL-37+si-EPB41L4A-AS1 group.Cell counting kit-8(CCK-8)method and clone formation test were used to detect cell proliferation,scratch test was used to detect cell migration,Transwell was used to detect cell invasion,and real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was used to detect the expression of EPB41L4A-AS1 and miR-106b-5p.The dual luciferase reporter gene experiment verified the regulatory relationship between EPB41L4A-AS1 and miR-106b-5p.The comparison between the two groups was performed by t test;the comparison between multiple groups was performed by one-way analysis of variance,and the pairwise comparison between groups was performed by LSD-t test.Results The absorbance(A)values of 786-O cells in the 12.5,50,200 ng/ml IL-37 groups(0.58±0.02,0.43±0.03,0.29±0.02 vs.0.69±0.06,F=207.113,P<0.05),the number of clones formed(114.33±6.61,87.33±5.22,51.33±4.92 vs.139.44±9.49,F=277.041,P<0.05),scratch healing rate[(69.03±0.35)%,(54.9±1.84)%,(38.81±2.73)%vs.(85.08±0.91)%,F=1191.221,P<0.05]and the number of invasive cells(91.44±5.68,70.89±2.85,48.78±6.00 vs.112.44±6.62,F=223.386,P<0.05)were reduced as compared with those in the control group,and the expression of EPB41L4A-AS1 in 786-O cells was higher than that in the control group(1.39±0.15,1.71±0.18,2.18±0.20 vs.1.01±0.10,F=84.386,P<0.05),but the expression of miR-106b-5p was lower than that in the control group(0.82±0.06,0.60±0.09,0.40±0.06 vs.1.00±0.12,F=82.545,P<0.05),and they were dose-dependent.The cell A value(0.23±0.02 vs.0.69±0.05,t=25.626,P<0.05),number of clones formed(45.33±3.32 vs.137.78±7.01,t=35.757,P<0.05),scratch healing rate[(35.01±3.27)%vs.(85.11±1.29)%,t=42.757,P<0.05]and the number of invasive cells(44.56±4.48 vs.109.44±8.59,t=20.091,P<0.05)in the pcDNA-EPB41L4A-AS1 group were reduced as compared with those in the pcDNA group.EPB41L4A-AS1 could target and negatively regulate miR-106b-5p.The expression of EPB41L4A-AS1 in the 786-O cells of the 200 ng/ml IL-37+si-EPB41L4A-AS1 group was lower than that in the 200 ng/ml IL-37+si-NC group(1.14±0.13 vs.2.49±0.19,t=17.592,P<0.05),but the expression of miR-106b-5p was higher than that in the 200 ng/ml IL-37+si-NC group(0.91±0.09 vs.0.43±0.08,t=11.959,P<0.05).The A value(0.64±0.06 vs.0.31±0.04,t=13.729,P<0.05),the number of clones formed(123.33±6.18 vs.49.22±3.23,t=31.884,P<0.05),scratch healing rate[(74.90±0.95)%vs.(38.15±2.17)%,t=46.542,P<0.05]and the number of invasive cells(92.89±5.25 vs.47.44±4.13,t=20.412,P<0.05)in the 200 ng/ml IL-37+si-EPB41L4A-AS1 group were increased as compared with those in the 200 ng/ml IL-37+si-NC group.Conclusion IL-37 may inhibit the proliferation,migration and invasion of 786-O cells by regulating the EPB41L4A-AS1/miR-106b-5p axis.