LncRNA SNHG17通过miR-384靶向AEG1对非小细胞肺癌细胞生物学行为的调控作用

doi:10.13481/j.1671-587X.20230205

摘要/Abstract

摘要：

目的研究长链非编码RNA小核仁RNA宿主基因17（lncRNA SNHG17）对非小细胞肺癌（NSCLC）细胞生物学行为的影响，并阐明其作用机制。方法培养人NSCLC A549细胞和H1299细胞，细胞按照分组要求分别转染pcDNA3.1-NC、SNHG17过表达质粒（pcDNA3.1-SNHG17）、si-NC、SNHG17小干扰RNA（si-SNHG17）、mimics NC、微小RNA-384模拟物（miR-384 mimics）、inhibitor NC、miR-384抑制剂（miR-384 inhibitor）、pcDNA3.1-星形细胞上调基因1（AEG1）和si-AEG1。实时荧光定量PCR（RT-qPCR）法检测A549细胞和H1299细胞及各组转染后细胞中lncRNA SNHG17、miR-384和AEG1 mRNA表达水平；ENCORI和TargetScan数据库预测lncRNA SNHG17与miR-384、miR-384与AEG1 mRNA之间的靶向结合作用。A549细胞分为si-NC组、si-SNHG17组、inhibitor NC组、miR-384 inhibitor组、si-NC+inhibitor NC组、si-SNHG17+inhibitor NC组、si-SNHG17+miR-384 inhibitor组、si-AEG1组、inhibitor NC+si-NC组、miR-384 inhibitor+si-NC组和miR-384 inhibitor+si-AEG1组。H1299细胞分为pcDNA3.1-NC组、pcDNA3.1-SNHG17组、mimics NC组、miR-384 mimics组、pcDNA3.1-NC+mimics NC组、pcDNA3.1-SNHG17+mimics NC组、pcDNA3.1-SNHG17+miR-384 mimics组、pcDNA3.1-AEG1组、mimics NC+pcDNA3.1-NC组、miR-384 mimics+pcDNA3.1-NC组和miR-384 mimics+pcDNA3.1-AEG1组。采用CCK-8法检测各组细胞活力，Transwell法检测各组细胞中迁移细胞数和侵袭细胞数，Western blotting法检测各组细胞中AEG1蛋白表达水平。结果 H1299细胞中lncRNA SNHG17表达水平明显低于A549细胞（P<0.01）。在A549细胞中，与si-NC组比较，si-SNHG17组细胞中SNHG17表达水平明显降低（P<0.01），细胞活力、迁移细胞数和侵袭细胞数明显降低（P<0.01）；在H1299细胞中，与pcDNA3.1-NC组比较，pcDNA3.1-SNHG17组细胞中SNHG17表达水平明显升高（P<0.01），细胞活力、迁移细胞数和侵袭细胞数明显升高（P<0.01）。在A549细胞中，与si-NC组比较，si-SNHG17组细胞中miR-384表达水平明显升高（P<0.01），AEG1 mRNA表达水平明显降低（P<0.01）；在H1299细胞中，与pcDNA3.1-NC组比较，pcDNA3.1-SNHG17组细胞中miR-384表达水平明显降低（P<0.01），AEG1 mRNA表达水平明显升高（P<0.01）。ENCORI数据库预测SNHG17与miR-384有2个结合位点。在A549细胞中，与si-NC+inhibitor NC组比较，si-SNHG17+inhibitor NC组细胞中AEG1蛋白表达水平和细胞活力明显降低（P<0.01），迁移细胞数和侵袭细胞数明显减少（P<0.01）；与si-SNHG17+inhibitor NC组比较，si-SNHG17+miR-384 inhibitor组细胞中AEG1蛋白表达水平和细胞活力明显升高（P<0.01），迁移细胞数和侵袭细胞数明显增加（P<0.01）。在H1299细胞中，与pcDNA3.1-NC+mimics NC组比较，pcDNA3.1-SNHG17+mimics NC组细胞中AEG1蛋白表达水平和细胞活力明显升高（P<0.01），迁移细胞数和侵袭细胞数明显增加（P<0.01）；与pcDNA3.1-SNHG17+mimics NC组比较，pcDNA3.1-SNHG17+miR-384 mimics组细胞中AEG1蛋白表达水平和细胞活力明显降低（P<0.01），迁移细胞数和侵袭细胞数明显减少（P<0.01）。在A549细胞中，与si-NC组比较，si-AEG1组细胞中AEG1蛋白表达水平和细胞活力明显降低（P<0.01），迁移细胞数和侵袭细胞数明显减少（P<0.01）；在H1299细胞中，与pcDNA3.1-NC组比较，pcDNA3.1-AEG1组细胞中AEG1蛋白表达水平和细胞活力明显升高（P<0.01），迁移细胞数和侵袭细胞数明显增加（P<0.01）。ENCORI数据库预测miR-384与AEG1有1个结合位点。在A549细胞中，与inhibitor NC+si-NC组比较，miR-384 inhibitor+si-NC组细胞中细胞活力明显升高（P<0.01），迁移细胞数和侵袭细胞数明显增加（P<0.01）；与miR-384 inhibitor+si-NC组比较，miR-384 inhibitor+si-AEG1组细胞中细胞活力明显降低（P<0.01），迁移细胞数和侵袭细胞数明显减少（P<0.01）。在H1299细胞中，与mimics NC+pcDNA3.1-NC组比较，miR-384 mimics+pcDNA3.1-NC组细胞中细胞活力明显降低（P<0.01）、迁移细胞数和侵袭细胞数明显减少（P<0.01）；与miR-384 mimics+pcDNA3.1-NC组比较，miR-384 mimics+pcDNA3.1-AEG1组细胞中细胞活力明显升高（P<0.01），迁移细胞数和侵袭细胞数明显增加（P<0.01）。结论 lncRNA SNHG17通过miR-384靶向AEG1调控NSCLC细胞的增殖、迁移和侵袭。

关键词: 长链非编码RNA, 小核仁RNA宿主基因17, 微小RNA-384, 星形细胞上调基因1, 癌，非小细胞肺, 生物学行为

Abstract:

Objective To investigate the effect of long non-coding RNA small nucleolar RNA host gene 17 （lncRNA SNHG17） on the biological behavior of the non-small cell lung cancer （NSCLC） cells， and to clarify its mechanism. Methods The human NSCLC A549 cells and H1299 cells were cultured，and the cells were transfected with pcDNA3.1-NC， SNHG17 over-expression plasmid（pcDNA3.1-SNHG17）， si-NC， SNHG17 small interference RNA（si-SNHG17），mimics NC，microRNA-384 mimics（miR-384 mimics）， inhibitor NC， miR-384 inhibitor（miR-384 inhibitor）， pcDNA3.1-astrocyte elevated gene 1 （AEG1）， and si-AEG1， respectively. Real-time quantitative PCR （RT-qPCR） method was used to detect the expression levels of lncRNA SNHG17，miR-384， and AEG1 mRNA in the A549 cells， H1299 cells and cells in various groups after transfection； ENCORI and TargetScan Databases were used to predicte the targeted bindings between lncRNA SNHG17 and miR-384，miR-384 and AEG1 mRNA. The A549 cells were divided into si-NC group， si-SNHG17 group， inhibitor NC group， miR-384 inhibitor group， si-NC+inhibitor NC group， si-SNHG17+inhibitor NC group， and si-SNHG17+miR-384 inhibitor group， si-AEG1 group， inhibitor NC+si-NC group， miR-384 inhibitor+si-NC group and miR-384 inhibitor+si-AEG1 group.The H1299 cells were divided into pcDNA3.1-NC group， pcDNA3.1-SNHG17 group， mimics NC group， miR-384 mimics group， pcDNA3.1-NC+mimics NC group， pcDNA3.1-SNHG17+mimics NC group，pcDNA3.1-SNHG17+miR-384 mimics group， pcDNA3.1-AEG1 group， mimics NC+pcDNA3.1-NC group， miR-384 mimics+pcDNA3.1-NC group， and miR-384 mimics+pcDNA3.1-AEG1 group. CCK-8 method was used to detect the cell viabilities in various groups； Transwell method was used to detect the numbers of migration and invasion cells in various groups； Western blotting method was used to detect the expression levels of AEG1 protein in the cells in various groups. Results The expression level of lncRNA SNHG17 in the H1299 cells was significantly lower than that in the A549 cells （P<0.01）. In the A549 cells， compared with si-NC group， the expression level of SNHG17 in the cells in si-SNHG17 group was significantly decreased （P<0.01），the cell viability， number of migration cells， and number of invasion cells were significantly decreased （P<0.01）； in the H1299 cells， compared with pcDNA3.1-NC group， the expression level of SNHG17 in the cells in pcDNA3.1-SNHG17 group was significantly increased （P<0.01）， the cell viability，number of migration cells， and number of invasion cells were increased significantly （P<0.01）. In the A549 cells， compared with si-NC group， the expression level of miR-384 in the cells in si-SNHG17 group was significantly increased（P<0.01）， and the expression level of AEG1 mRNA was significantly decreased（P<0.01）； in the H1299 cells， compared with pcDNA3.1-NC group， the expression level of miR-384 in the cells in pcDNA3.1-SNHG17 group was significantly decreased（P<0.01）， and the expression level of AEG1 mRNA was significantly increased （P<0.01）. The ENCORI Database results predicted that there were two binding sites between SNHG17 and miR-384. The rescue assay results showed that in the A549 cells， compared with si-NC+inhibitor NC group， the expression level of AEG1 protein， cell viability， number of migration cells， and number of invasion cells in si-SNHG17+inhibitor NC group were significantly decreased（P<0.01）； compared with si-SNHG17+inhibitor NC group， the expression level of AEG1 protein， cell viability， number of migration cells， and number of invasion cells in si-SNHG17+miR-384 inhibitor group were significantly increased（P<0.01）. In the H1299 cells， compared with pcDNA3.1-NC+mimics NC group， the expression level of AEG1 protein， cell viability， number of migration cells， and number of invasion cells in pcDNA3.1-SNHG17+mimics NC group were significantly increased （P<0.01）； compared with pcDNA3.1-SNHG17+mimics NC group， the expression level of AEG1 protein， cell viability， number of migration cells， and number of invasion cells in pcDNA3.1-SNHG17+miR-384 mimics group were significantly decreased （P<0.01）. In the A549 cells， compared with si-NC group， the expression level of AEG1 protein in the cells， cell viability， number of migration cells， and number of invasion cells in si-AEG1 group were significantly decreased （P<0.01）； in the H1299 cells， compared with pcDNA3.1-NC group， the expression level of AEG1 protein cell viability， number of migration cells， and number of invasion cells were significantly increased （P<0.01）. The ENCORI Database results predicted that there was one binding site between miR-384 and AEG1.In the A549 cells， compared with inhibitor NC+si-NC group， the cell viability， number of migration cells and number of invasion cells in miR-384 inhibitor+si-NC group were significantly increased （P<0.01）； compared with miR-384 inhibitor+si-NC group， the cell viability， number of migration cells and number of invasion cells in miR-384 inhibitor+si-AEG1 group were significantly decreased （P<0.01）. In the H1299 cells， compared with the mimics NC+pcDNA3.1-NC group， the cell viability， number of migration cells， and number of invasion cells in miR-384 mimics+pcDNA3.1-NC group were significantly decreased （P<0.01）； compared with miR-384 mimics+pcDNA3.1-NC group， the cell viability， number of migration cells， and number of invasion cells in miR-384 mimics+pcDNA3.1-AEG1 group were significantly increased （P<0.01）. Conclusion LncRNA SNHG17 regulates the proliferation， migration， and invasion of the NSCLC cells by targeting AEG1 through miR-384.

Key words: Long non-coding RNA, Small nucleolar RNA host gene 17, MicroRNA-384, Astrocyte elevated gene 1, Cancer，Non-small cell lung, Biological behavior

中图分类号:

R734.2

刘韵鹏,刘子豪,康伯铭,杨志广. LncRNA SNHG17通过miR-384靶向AEG1对非小细胞肺癌细胞生物学行为的调控作用[J]. 吉林大学学报(医学版), 2023, 49(2): 298-307.

Yunpeng LIU,Zihao LIU,Boming KANG,Zhiguang YANG. Regulatory effect of lncRNA SNHG17 on biological behavior of non-small cell lung cancer cells by targeting AEG1 through miR-384[J]. Journal of Jilin University(Medicine Edition), 2023, 49(2): 298-307.

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Group	lncRNA SNHG17	Cell viability (η/%)	Number of migration cells	Number of invasion cells
si-NC (A549 cells)	1.03±0.24	95.32±5.45	384.00±12.00	191.00±5.03
si-SNHG17 (A549 cells)	0.21±0.02^*	45.68±10.22^*	289.00±6.08^*	111.00±3.06^*
pcDNA3.1-NC (H1299 cells)	1.02±0.19	96.56±3.89	499.00±13.53	147.00±6.11
pcDNA3.1-SNHG17 (H1299 cells)	6.35±1.42^△	136.23±10.93^△	716.00±11.27^△	271.00±7.21^△

Group	miR-384	AEG1 mRNA
si-NC(A549 cells)	1.01±0.10	1.03±0.24
si-SNHG17(A549 cells)	1.46±0.20^*	0.06±0.02^*
pcDNA3.1-NC(H1299 cells)	1.01±0.13	1.02±0.19
pcDNA3.1-SNHG17(H1299 cells)	0.64±0.14^△	2.34±0.27^△

Group	miR-384	Cell viability (η/%)	Number of migration cells	Number of invasion cells
Inhibitor NC(A549 cells)	1.02±0.21	97.16±4.22	417.00±11.14	160.00±7.00
miR-384 inhibitor(A549 cells)	0.02±0.01^*	145.69±22.24^*	489.00±16.64^*	243.00±3.79^*
Mimics NC(H1299 cells)	1.02±0.21	96.37±3.22	518.00±11.53	207.00±6.03
miR-384 mimics(H1299 cells)	4.68±1.00^△	65.67±6.40^△	406.00±17.67^△	116.00±8.00^△

Group	Cell viability (η/%)	Number of migration cells	Number ofinvasion cells	Expression level of AEG1 protein
si-NC+inhibitor NC(A549 cells)	98.07±4.84	364.00±18.15	195.00±9.85	1.00±0.03
si-SNHG17+inhibitor NC(A549 cells)	63.26±6.04^*	314.00±8.72^*	108.00±6.11^*	0.72±0.01^*
si-SNHG17+miR-384 inhibitor(A549 cells)	78.69±6.65^△	350.00±8.74^△	199.00±11.15^△	0.81±0.01^△
pcDNA3.1-NC+mimics NC(H1299 cells)	101.26±5.91	496.00±18.23	148.00±4.58	1.00±0.02
pcDNA3.1-SNHG17+mimics NC(H1299 cells)	138.61±9.05^#	674.00±6.93^#	271.00±9.07^#	2.26±0.04^#
pcDNA3.1-SNHG17+miR-384 mimics(H1299 cells)	108.64±7.87^○	582.00±14.53^○	221.00±3.79^○	1.56±0.02^○

Group	AEG1 mRNA	Cell viability (η/%)	Number of migration cells	Number of invasion cells
si-NC(A549 cells)	1.02±0.22	98.67±4.76	381.00±13.08	167.00±6.03
si-AEG1(A549 cells)	0.56±0.08^*	71.32±8.98^*	308.00±10.26^*	115.00±10.02^*
pcDNA3.1-NC(H1299 cells)	1.01±0.11	99.21±7.66	499.00±11.53	137.00±7.09
pcDNA3.1-AEG1(H1299 cells)	3.15±0.90^△	132.32±9.33^△	609.00±11.14^△	282.00±3.79^△