URG11基因介导Wnt/β-catenin信号通路调控EMT参与非小细胞肺癌转移的分子机制研究

The Molecular Mechanisms of URG11 Regulation of EMT through Wnt/β-catenin Signaling in Non-small Cell Lung Cancer Metastasis

目的探讨URG11基因介导的Wnt/β-Catenin信号通路调控EMT参与非小细胞肺癌转移的分子机制。方法非小细胞肺癌细胞系A549经shRNA质粒转染48h,敲除URG11后收集细胞行后续实验。实验共分两组,分别为敲除URG11的A549细胞组(实验组)和未敲除URG11的A549细胞组(空白对照组)。采用Western blott测定β-Catenin及其下游基因cyclinD1、c-myc的蛋白表达,同时用RT-qPCR检测β-Catenin、cyclinD1和c-myc的mRNA表达。并用裸鼠成瘤模型评估URG11基因对NSCLC侵袭性生长的影响。结果①敲除URG11显著抑制Wnt/β-Catenin信号通路在NSCLC细胞中激活。Western blot蛋白质印迹分析显示β-Catenin、cyclin D1和c-myc的蛋白水平在敲除实验组中的表达显著低于对照组(DPI值分别为0.22±0.06 VS 0.77±0.21,0.61±0.09 VS 1.52±0.23,0.42±0.07 VS 0.84±0.14,P<0.05)。RT-qPCR结果显示β-Catenin、cyclinD1和c-myc的mRNA在对照组中的表达显著高于实验组(分别为3.52倍,2.58倍和2.19倍,P<0.05)。②敲除URG11显著减少异种移植肿瘤体在裸鼠体内侵袭性生长。通过裸鼠异种移植肿瘤模型动态观察了URG11对肿瘤的体内生长作用。与对照组相比,敲除URG11能显著抑制Balb/C裸鼠体内新生肿瘤的重量,第35天实验组VS对照组(0.21±0.04)g VS(0.58±0.08)g,P<0.05,平均抑瘤率为63.79%;同时敲除URG11能显著减少异种移植肿瘤的体积:第21、28、35天的新生瘤体体积,实验组VS对照组(258.33±0.24)mm^(3)VS(512.86±0.18)mm^(3),(414.59±0.17)mm^(3)VS(685.78±0.23)mm^(3),(423.21±0.36)mm^(3)VS(986.73±0.14)mm^(3),P<0.05。结论URG11导致的EMT与NSCLC的发生和进展密切相关。

Objective To investigate the molecular mechanisms of URG11 regulation of EMT through Wnt/β-catenin signaling in non-small cell lung cancer(NSCLC)metastasis.Method NSCLC cell line A549 was transfected with shRNA plasmid for 48 hours.After knockout of URG11,the cells were collected for follow-up experiments.They were divided into A549 cell group with knockout of URG11(the experimental group)and A549 cell group without knockout of urg11(the blank control group).Western blotting assay and RT qPCR was performed to detect the expression ofβ-catenin and its downstream genes Cyclin D1 and c-myc.The effect of URG11 gene on the invasive growth of NSCLC was evaluated by nude mouse tumorigenesis model.Results①Knockout of URG11 significantly inhibited Wnt/β-Catenin signaling pathway in NSCLC cells.Western blot analysis showed that the protein level ofβ-catenin,CyclinD1 and c-myc expression in the experimental group was significantly lower than that in thecontrol group,with DPI values of 0.22±0.06 VS 0.77±0.21,0.61±0.09 VS 1.52±0.23,0.42±0.07 VS 0.84±0.14 respectively(P<0.05).RT qPCR results showed that mRNA level ofβ-catenin,Cyclin D1 and c-myc in the control groupwas significantly higher than that in the experimental group(3.52 times,2.58 times and 2.19 times respectively,P<0.05).②Knockout of URG11 significantly reduced xenotransplantation tumor growth in nude mice.It was dynamically observed the effect of URG11 on tumor growth in vivo by xenotransplantation tumor model in nude mice.Compared with the control group,knockout of urg11 could significantly inhibit the weight of new tumors in BALB/C nude mice on the 35th day,the weight of new tumors in the experimental group VS the control group was(0.21±0.04)g VS(0.58±0.08)g(P<0.05),and the average tumor inhibition rate was 63.79%.At the same time,knockout of urg11 could significantly reduce the volume of xenograft tumors,which was(258.33±0.24)mm^(3)VS(512.86±0.18)mm^(3),(414.59±0.17)mm^(3)VS(685.78±0.23)mm^(3)and(423.21±0.36)mm^(3)VS(986.73±0.14)mm^(3)on the 21st,28th and 35th day,P<0.05.Conclusion It is proved that URG11-mediated EMT is closely related to the occurrence and progression of NSCLC.