微小RNA-1910-3p调控三方基序包含蛋白37影响非小细胞肺癌细胞生物学行为

Effects of MicroRNA-1910-3p on the biological behavior of non-small cell lung cancer cells by regulating tripartite motif-containing 37

目的探讨微小RNA-1910-3p(miR-1910-3p)调控三方基序包含蛋白37(TRIM37)对非小细胞肺癌(NSCLC)细胞生物学行为的影响。方法通过实时荧光定量聚合酶链反应(RT-qPCR)法评估人非小细胞肺癌细胞(A549细胞系)和人正常肺上皮细胞(BEAS-2B细胞系)中miR-1910-3p的表达水平。采用生物信息学分析、RT-qPCR、蛋白质印迹法(Western blot)分析miR-1910-3p对TRIM37的靶向调控关系。A549细胞分别转染阴性对照模拟物(NC组)、miR-1910-3p模拟物(miRNA-mimic组)、miR-1910-3p抑制剂(miRNA-inhibitor组), 细胞计数试剂盒(CCK-8)和克隆形成实验检测各组细胞增殖能力、膜联蛋白V(Annexin V)-异硫氰酸荧光素(FITC)实验检测各组细胞凋亡率、Transwell小室和细胞划痕实验分别检测各组细胞侵袭率和迁移率。两组间比较采用独立样本t检验。结果 RT-qPCR结果显示, A549细胞组中miR-1910-3p表达水平高于BEAS-2B细胞组(1.566±0.019比1.114±0.078, t=10.920, P<0.01)。生物信息分析结果显示, TRIM37满足与miR-1910-3p的5′端2~8位种子区完全互补配对。RT-qPCR及Western blot结果显示, miR-1910-3p mimic组TRIM37表达水平低于mimic NC组[RT-qPCR(0.787±0.050)比(1.025±0.024), t=10.446, P<0.01;Western blot(0.124±0.003)比(0.223±0.003), t=47.047, P<0.01];miR-1910-3p inhibitor组TRIM37表达水平高于inhibitor NC组[RT-qPCR(1.518±0.067)比(1.074±0.077), t=10.620, P<0.01;Western blot(0.730±0.005)比(0.243±0.004), t=123.124, P<0.01]。在NSCLC细胞中, miR-1910-3p mimic组细胞增殖能力、侵袭细胞数和细胞迁移率高于mimic NC组[相对细胞活力(122.50±4.08)%比(100.00±3.36)%, t=14.632, P<0.01;克隆形成率(10.625±0.917)%比(6.000±0.500)%, t=13.100, P<0.01;侵袭细胞数(195.800±25.762)个比(139.400±15.421)个, t=4.200, P<0.01;细胞迁移率(46.330±4.163)%比(37.330±1.528)%, t=3.515, P<0.05], 细胞凋亡率低于mimic NC组[(3.393±0.486)%比(8.227±0.465)%, t=12.451, P<0.05];miR-1910-3p inhibitor组细胞增殖能力、侵袭细胞数和细胞迁移率低于inhibitor NC组[相对细胞活力(79.79±4.76)%比(100.00±2.31)%, t=12.914, P<0.01;克隆形成率(5.169±0.391)%比(6.981±0.163)%, t=7.407, P<0.05;侵袭细胞数(62.800±6.760)个比(138.400±14.673)个, t=10.464, P<0.01;迁移率(32.670±3.215)%比(39.330±1.528)%, t=3.244, P<0.01], 细胞凋亡率高于inhibitor NC组[(11.910±0.898)%比(6.320±0.584)%, t=9.041, P<0.05]。结论 miR-1910-3p通过负调控TRIM37影响非小细胞肺癌细胞增殖、侵袭、迁移和凋亡。

Objective To investigate the effect of MicroRNA-1910-3p(miR-1910-3p)on the biological behavior of non-small cell lung cancer(NSCLC)cells by regulating tripartite motif-containing 37(TRIM37).Methods The expression levels of miR-1910-3p were detected in human NSCLC cells(A549 cells)and normal lung epithelial cells(BEAS-2B cells)using real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).Bioinformatics analysis,RT-qPCR,and Western blotting were used to analyze the targeted regulatory relationship of miR-1910-3p on TRIM37.A549 cells were transfected with negative control mimics(NC group),miR-1910-3p mimics(miRNA mimic group),miR-1910-3p inhibitors(miRNA inhibitor group).The cell counting kit-8(CCK-8)and clone formation assays were used to test the cell proliferation ability of each group.Annexin V-fluoresceine isothiocyanate(FITC)assay was used to detect the cell apoptosis rate.Transwell chamber,and cell scratch assays were used to detect the cell invasion rate and migration rate of each group,respectively.The data analysis was conducted using SPSS 20.0 statistical software,and independent sample t-tests were used for comparison between the two groups.P<0.05 indicates a statistically significant difference.Results RT-qPCR results showed that the expression level of miR-1910-3p in the A549 cell group was higher than that in the BEAS-2B cell group(1.566±0.019 vs.1.114±0.078,t=10.920,P<0.01).Bioinformatic analysis showed that TRIM37 met the requirement of complete complementary pairing with the 2-8 seed region at the 5′end of miR-1910-3p.RT-qPCR and Western blotting results showed that the expression level of TRIM37 in miR-1910-3p mimic group was lower than that in mimic NC group[RT-qPCR:(0.787±0.050)vs.(1.025±0.024),t=10.446,P<0.01;Western blotting:(0.124±0.003)vs.(0.223±0.003),t=47.047,P<0.01].The expression level of TRIM37 in miR-1910-3p inhibitor group was higher than that in inhibitor NC group[RT-qPCR:(1.518±0.067)vs.(1.074±0.077),t=10.620,P<0.01;Western blotting:(0.730±0.005)vs.(0.243±0.004),t=123.124,P<0.01].In NSCLC cells,miR-1910-3p mimics group had higher cell proliferation ability,greater invasive cell number and higher cell migration rate than mimics NC group[the relative cell viability:(122.50±4.08)%vs.(100.00±3.36)%,t=14.632,P<0.01;the clonogenic rate:(10.625±0.917)%vs.(6.000±0.500)%,t=13.100,P<0.01;the number of invasive cells:(195.800±25.762)%vs.(139.400±15.421),t=4.200,P<0.01;the cell migration rate:(46.330±4.163)%vs.(37.330±1.528)%,t=3.515,P<0.05].The apoptosis rate in miR-1910-3p mimics group was lower than that in mimic NC group[(3.393±0.486)%vs.(8.227±0.465)%,t=12.451,P<0.05].The cell proliferation ability,invasive cell number and cell migration rate in miR-1910-3p inhibitor group were lower than those in inhibitor NC group[relative cell viability:(79.79±4.76)%vs.(100.00±2.31)%,t=12.914,P<0.01;clonogenic rate:(5.169±0.391)%vs.(6.981±0.163)%,t=7.407,P<0.05;number of invasive cells:(62.800±6.760)%vs.(138.400±14.673),t=10.464,P<0.01;migration rate:(32.670±3.215)%vs.(39.330±1.528)%,t=3.244,P<0.01].The apoptosis rate in miR-1910-3p inhibitor group was higher than that in inhibitor NC group[(11.910±0.898)%vs.(6.320±0.584)%,t=9.041,P<0.05].Conclusion TRIM37 can negatively regulate miR-1910-3p,affecting proliferation,invasion,migration and apoptosis of NSCLC cells.