LncRNANNT-AS1通过调控miR-582-5p/NCKAP1轴激活Hippo-YAP/TAZ信号通路促进膀胱癌细胞增殖、迁移、侵袭和干细胞干性影响

LncRNA NNT-AS1 Activates Hippo-YAP/TAZ Signaling Pathway by Regulating miR-582-5p/NCKAP1 Axis to Promote Bladder Cancer Cell Proliferation,Migration,Invasion and Stem Cell Stemness Effects

目的检测膀胱癌中长链非编码RNA(long non-coding RNA,lncRNA)烟酰胺核苷酸转氢酶反义RNA1(nicotinamide nucleotide transhydrogenase antisense RNA 1,NNT-AS1)表达情况,研究其对膀胱癌细胞增殖、迁移、侵袭及肿瘤干细胞干性的影响及可能分子机制。方法实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)法检测膀胱癌组织标本及细胞中LncRNA NNT-AS1表达情况;将膀胱癌细胞转染分为sh-NC组,sh-NNT-AS1组,sh-NNT-AS1+inh-582-5p组和sh-NNT-AS1+inh-582-5p+si-NCKAP1组。采用CCK-8法检测细胞增殖吸光度值(A值);Transwell实验检测细胞迁移、侵袭穿膜数;细胞成球实验检测干细胞干性。检索starBase和TargetScan数据库,并通过双荧光素酶报告基因实验预测验证LncRNA NNT-AS1和miR-582-5p,miR-582-5p与NCKAP1的靶向结合关系。Western blot检测膀胱癌干细胞标志蛋白(CD44,ALDH1A1,Oct4,Nanog)及Hippo-YAP/TAZ信号通路相关蛋白表达灰度值。结果与癌旁组织相比,膀胱癌组织中LncRNA NNT-AS1表达水平(0.34±0.07 vs 1.15±0.21)明显升高,差异有统计学意义(t=16.364,P<0.001)。与人正常膀胱上皮SV-HUC-1细胞(1.00±0.01)相比,膀胱癌细胞T24,5637,UM-UC-3和TCC-SUP中LncRNA NNT-AS1表达(6.03±0.17,4.66±0.36,5.47±0.26,3.02±0.20)明显升高,差异有统计学意义(t=17.472~51.160,均P<0.001)。与sh-NC组相比,在24,48和72 h时sh-NNT-AS1组细胞增值能力(A值)均显著降低(0.80±0.01 vs 1.07±0.06,1.18±0..07 vs 1.83±0.03,1.89±0.07 vs 2.53±0.06),差异有统计学意义(t=7.688,14.783,12.024,均P<0.05);sh-NNT-AS1组细胞迁移穿膜数(55.00±2.65个vs 354.30±7.84个)、细胞侵袭穿膜数(45.67±2.33个vs 303.00±9.07个)及膀胱癌干细胞成球数(20.85±2.17个vs 41.35±3.67个)显著降低,差异具有统计学意义(t=-62.641,-47.596,8.328,均P<0.001)。与sh-NC组相比,sh-NNT-AS1组细胞中CD44(0.04±0.01 vs 1.12±0.02),ALDH1A1(0.23±0.01 vs 1.16±0.05),Oct4(0.17±0.02 vs 1.10±0.04),Nanog(0.49±0.03 vs 1.24±0.03)的蛋白表达灰度值显著降低,差异具有统计学意义(t=83.656,31.591,36.019,30.619,均P<0.001)。与si-NC组相比,sh-NNT-AS1组CD44+CD133+细胞比例(9.30%±0.79%vs 88.50%±2.77%)明显降低,差异有统计学意义(t=-47.624,P<0.001)。双荧光素酶报告基因检测结果显示miR-582-5p为LncRNANNT-AS1靶基因,NCKAP1为miR-582-5p靶基因;LncRNA NNT-AS1靶向调控miR-582-5p/NCKAP1轴。与sh-NNT-AS1组相比,在24,48,72 h时sh-NNT-AS1+inh-582-5p组细胞增值能力(A值)均明显升高(0.98±0.03 vs 0.73±0.06,1.74±0.04 vs 1.22±0.05,2.33±0.16 vs 1.69±0.14),差异有统计学意义(t=5.977~11.628,均P<0.001)。与sh-NNT-AS1+inh-582-5p组相比,在24,48,72 h时sh-NNT-AS1+inh-582-5p+si-NCKAP1组细胞增值能力(A值)显著降低(0.69±0.04,1.01±0.07,1.39±0.08),差异有统计学意义(t=7.877~16.323,均P<0.001)。与sh-NNT-AS1组相比,sh-NNT-AS1+inh-582-5p组细胞迁移穿膜数(322.31±28.45个vs 81.42±13.22个)、细胞侵袭穿膜数(316.07±30.21个vs 92.13±12.65个)及膀胱癌干细胞成球数(38.55±2.20个vs 18.98±1.16个)显著增加,差异具有统计学意义(t=15.115,13.158,14.592,均P<0.001)。与sh-NNT-AS1组相比,sh-NNT-AS1+inh-582-5p组细胞CD44(1.05±0.08 vs 0.10±0.01),ALDH1A1(1.20±0.16 vs 0.22±0.02),Oct4(1.32±0.14 vs 0.19±0.03),Nanog(0.97±0.12 vs 0.15±0.04),YAP(1.29±0.11 vs 0.42±0.07)和TAZ(1.41±0.16 vs 0.35±0.05)蛋白表达灰度值均显著增加,差异具有统计学意义(t=10.650~21.243,均P<0.001)。与sh-NNT-AS1+inh-582-5p组相比,sh-NNT-AS1+inh-582-5p+si-NCKAP1组细胞迁移穿膜数(65.33±12.60个)、细胞侵袭穿膜数(71.08±15.19个)、膀胱癌干细胞成球数(11.36±1.05个)均显著降低,差异具有统计学意义(t=16.125,14.395,21.365,均P<0.001)。与sh-NNT-AS1+inh-582-5p组相比,sh-NNT-AS1+inh-582-5p+si-NCKAP1组细胞CD44(0.25±0.05),ALDH1A1(0.61±0.11),Oct4(0.22±0.08),Nanog(0.44±0.07),YAP(0.25±0.09)和TAZ(0.30±0.04)蛋白表达灰度值显著降低,差异具有统计学意义(t=6.412~17.889,均P<0.001)。结论膀胱癌中LncRNA NNT-AS1表达上调,其对膀胱癌细胞增殖、侵袭及肿瘤干细胞干性的影响,可能是通过调控miR-582-5p/NCKAP1分子轴,激活Hippo-YAP/TAZ信号通路完成。

Objective To detect the expression of long non coding RNA(LncRNA)nicotinamide nucleotide transhydrogenase antisense RNA1(NT-AS1)in bladder cancer,and investigate its effect on the proliferation,migration and invasion of bladder cancer cells and the stemness of tumor stem cells and its possible molecular mechanism.Methods LncRNA NNT-AS1 expression in bladder cancer tissues and cells was detected by quantitative real-time PCR(qRT-PCR).Bladder cancer cells were transfected into sh-NC group,sh-NNT-AS1 group,sh-NNT-AS1+inh-582-5p group and sh-NNT-AS1+inh-582-5p+si-NCKAP1 group.Cell proliferation absorbance(A value)was detected by CCK-8 method.Transwell assay was used to detect cell migration and invasion through membrane.The number of cell pellet formation was measured by tumor stem cell pellet formation assay.The starBase and TargetScan databases were searched,and the targeted binding relationship between LncRNA NNT-AS1 and miR-582-5p,miR-582-5p and NCKAP1 was predicted and verified by double luciferase reporter gene experiment.Western blot analysis was performed to detect the expression gray values of marker proteins(CD44,ALDH1A1,Oct4,Nanog)and Hippo-YAP/TAZ signaling pathways.Results LncRNA NNT-AS1 expression in bladder cancer tissues was significantly higher than that in adjacent tissues(0.34±0.07 vs 1.15±0.21),and the difference was statistically significant(t=16.364,P<0.001).LncRNA NNT-AS1 expression in bladder cancer cells T24,5637,UM-UC-3 and TCC-SUP(6.03±0.17,4.66±0.36,5.47±0.26,3.02±0.20)were significantly higher than that in normal human bladder epithelial SV-HUC-1 cells(1.00±0.01),and the differences were statistically significant(t=17.472~51.160,all P<0.001).Compared with sh-NC group,cell A value in sh-NNT-AS1 group(0.80±0.01 vs 1.07±0.06,1.18±0.07 vs 1.83±0.03,1.89±0.07 vs 2.53±0.06)was significantly decreased at 24,48 and 72 h,and the differences were statistically significant(t=7.688,14.783,12.024,all P<0.05).Compared with sh-NC group,the number of cell migration through the membrane(55.00±2.65 vs 354.30±7.84),the number of cell invasions through the membrane(45.67±2.33 vs 303.00±9.07),and the number of bladder cancer stem cells bulging(20.85±2.17 vs 41.35±3.67)were significantly reduced in sh-NNT-AS1 group(t=-62.641,-47.596,8.328,all P<0.001).Compared with sh-NC group,the expression gray values of CD44(0.04±0.01 vs 1.12±0.02),ALDH1A1(0.23±0.01 vs 1.16±0.05),Oct4(0.17±0.02 vs 1.10±0.04)and Nanog(0.49±0.03 vs 1.24±0.03)protein in sh-NNT-AS1 group were significantly decreased,and the differences were statistically significant(t=83.656,31.591,36.019,30.619,all P<0.001).Compared with si-NC group,the proportion of CD44+CD133+cells in sh-NNT-AS1 group was significantly decreased(9.30%±0.79%vs 88.50%±2.77%),and the difference was statistically significant(t=-47.624,P<0.001).The results of double luciferase reporter gene detection showed that miR-582-5p was the target gene of LncRNA NNT-AS1,and NCKAP1 was the target gene of miR-582-5p.LncRNA NNT-AS1 targets miR-582-5p/NCKAP1 axis.Compared with sh-NNT-AS1 group,cell A value in sh-NNT-AS1+inh-582-5p group(0.98±0.03 vs 0.73±0.06,1.74±0.04 vs 1.22±0.05,2.33±0.16 vs 1.69±0.14)was significantly increased at 24,48 and 72 h,and the differences were statistically significant(t=5.977~11.628,all P<0.001).Compared with sh-NNT-AS1+inh-582-5p group,cell A value in sh-NNT-AS1+inh-582-5p+si-NCKAP1 group(0.69±0.04,1.01±0.07,1.39±0.08)was significantly decreased at 24,48 and 72h,and the differences were statistically significant(t=7.877~16.323,all P<0.001).Compared with the sh-NNT-AS1 group,the number of cell migration through the membrane(322.31±28.45 vs 81.42±13.22),the number of cell invasion through the membrane(316.07±30.21 vs 92.13±12.65),and the number of bladder cancer stem cells forming balls(38.55±2.20 vs 18.98±1.16)in the sh-NNT-AS1+inh-582-5p group were significantly increased(t=15.115,13.158,14.592,P<0.001).Compared with sh-NNT-AS1 group,the expression gray values of CD44(1.05±0.08 vs 0.10±0.01),ALDH1A1(1.20±0.16 vs 0.22±0.02),Oct4(1.32±0.14 vs 0.19±0.03),Nanog(0.97±0.12 vs 0.15±0.04),YAP(1.29±0.11 vs 0.42±0.07)and TAZ(1.41±0.16 vs 0.35±0.05)protein in sh-NN-T-AS1+inh-582-5p group were significantly increased,and the differences were statistically significant(t=10.650~21.243,all P<0.001).Compared with the sh-NNT-AS1+inh-582-5p group,the number of cell migration through the membrane(65.33±12.60),the number of cell invasion through the membrane(71.08±15.19),and bladder cancer stem cell spherulation number(11.36±1.05)of the sh-NNT-AS1+inh-582-5p+si-NCKAP1 group were significantly reduced(t=16.125,14.395,21.365,all P<0.001).Compared with sh-NNT-AS1+inh-582-5p group,the expression gray values of CD44(0.25±0.05),ALDH1A1(0.61±0.11),Oct4(0.22±0.08),Nanog(0.44±0.07),YAP(0.25±0.09)and TAZ(0.30±0.04)protein in sh-NNT-AS1+inh-582-5p+si-NCKAP1 group were significantly increased,and the differences were statistically significant(t=6.412~17.889,all P<0.001).Conclusion The expression of LncRNA NNT-AS1 was up-regulated in bladder cancer,and its influence on the proliferation and invasion of bladder cancer cells and the stemness of tumor stem cells may be achieved through the activation of Hippo-YAP/TAZ signaling pathway by regulating the molecular axis of miR-582-5p/NCKAP1.