LncRNA NNT-AS1靶向miR-199a-5p对口腔鳞癌细胞增殖、迁移和侵袭的影响

doi:10.11958/20251271

摘要/Abstract

摘要：

目的探讨长链非编码RNA（LncRNA）NNT-AS1调节微小RNA（miR）-199a-5p对口腔鳞癌（OSCC）细胞增殖、迁移和侵袭的影响。方法将CAL-27细胞分为对照组、sh-NC组、sh-NNT-AS1组、sh-NNT-AS1+miR-In-NC组、sh-NNT-AS1+miR-199a-5p-In组。分别采用5-乙炔基-2'-脱氧尿苷（EdU）法、克隆形成、划痕愈合、Transwell实验检测CAL-27细胞增殖、集落形成、迁移和侵袭能力。采用qRT-PCR实验检测人角质形成细胞系HaCaT、人OSCC细胞系CAL-27及各组细胞中LncRNA NNT-AS1和miR-199a-5p的表达；Western blot实验检测细胞中上皮间充质转化（EMT）相关蛋白[E-钙黏蛋白（E-cadherin）、N-钙黏蛋白（N-cadherin）、波形蛋白（Vimentin）和表皮生长因子受体（EGFR）]的表达；双萤光素酶报告基因实验验证LncRNA NNT-AS1与miR-199a-5p的靶向关系。结果与HaCaT细胞相比，CAL-27细胞中LncRNA NNT-AS1水平升高，miR-199a-5p水平降低（P<0.05）；与对照组、sh-NC组相比，sh-NNT-AS1组EdU阳性率、集落形成率、划痕愈合率、细胞侵袭数、LncRNA NNT-AS1、N-cadherin、Vimentin和EGFR蛋白表达水平降低，miR-199a-5p和E-cadherin蛋白表达水平升高（P<0.05）；与sh-NNT-AS1组、sh-NNT-AS1+miR-In-NC组相比，sh-NNT-AS1+miR-199a-5p-In组EdU阳性率、集落形成率、划痕愈合率、细胞侵袭数、N-cadherin、Vimentin和EGFR蛋白表达水平升高，miR-199a-5p和E-cadherin蛋白表达水平降低（P<0.05）；与miR-NC组比较，miR-199a-5p mimic组转染NNT-AS1-WT细胞的相对萤光素酶活性降低（P<0.05）。结论 LncRNA NNT-AS1在OSCC中高表达，通过负向调控miR-199a-5p可促进OSCC细胞增殖、迁移、侵袭及EMT进程。

关键词: 头颈鳞癌, 口腔肿瘤, RNA, 长链非编码, 长链非编码RNA NNT-AS1, 微小RNA-199a-5p

Abstract:

Objective To investigate the effect of long non-coding RNA (LncRNA) NNT-AS1 on proliferation, migration and invasion of oral squamous cell carcinoma (OSCC) cells by regulating microRNA (miR)-199a-5p. Methods CAL-27 cells were assigned into the control group, the sh-NC group, the sh-NNT-AS1 group, the sh-NNT-AS1+miR-In-NC group and the sh-NNT-AS1+miR-199a-5p-In group. The proliferation, colony formation, migration and invasion abilities of CAL-27 cells were detected by EdU method, clone formation, scratch healing and Transwell experiments. The expressions of LncRNA NNT-AS1 and miR-199a-5p in human keratinocyte cell line HaCaT, human OSCC cell line CAL-27 and each group of cells were detected by qRT-PCR experiment. Western blot experiments were performed to detect the expressions of epithelial-mesenchymal transition (EMT)-related proteins [E-cadherin, N-cadherin, Vimentin, and epidermal growth factor receptor (EGFR)]. The targeting relationship between LncRNA NNT-AS1 and miR-199a-5p was verified by dual luciferase reporter gene assay. Results Compared with HaCaT cells, the level of LncRNA NNT-AS1 was elevated in CAL-27 cells, while the level of miR-199a-5p was decreased (P<0.05). Compared with the control group and the sh-NC group, the EdU positive rate, colony formation rate, scratch healing rate, cell invasion number, expression levels of LncRNA NNT-AS1, N-cadherin, Vimentin and EGFR proteins were decreased in the sh-NNT-AS1 group, while the expression levels of miR-199a-5p and E-cadherin protein were increased (P<0.05). Compared with the sh-NNT-AS1 group and the sh-NNT-AS1+miR-In-NC group, the EdU positive rate, colony formation rate, scratch healing rate, cell invasion number, N-cadherin, Vimentin and EGFR protein expression levels were increased in the sh-NNT-AS1+miR-199a-5p-In group, while the expression levels of miR-199a-5p and E-cadherin protein were decreased (P<0.05). Compared with the miR-NC group, the relative luciferase activity of NNT-AS1-WT cells transfected with miR-199a-5p mimic was decreased (P<0.05). Conclusion LncRNA NNT-AS1 is highly expressed in OSCC. It can promote the proliferation, migration, invasion and EMT process of OSCC cells by negatively regulating miR-199a-5p.

Key words: squamous cell carcinoma of head and neck, mouth neoplasms, RNA, long noncoding, long non-coding RNA NNT-AS1, microRNA-199a-5p

中图分类号:

R739.85

图/表 11

表1 引物序列

Tab.1 Primer sequences

基因名称	引物序列（5'→3'）	产物大小/bp
LncRNA NNT-AS1	上游：ACGTGCAGACAACATCTACCT	168
LncRNA NNT-AS1	下游：TACAACACCTTCCCGCAT	168
GAPDH	上游：GGAGCGAGATCCCTCCAAAAT	189
GAPDH	下游：CTGGCCCGGAAGACATCTG	189
miR-199a-5p	上游：CGCGCCCAGTGTTCAGACTAC	124
miR-199a-5p	下游：AGTGCAGGGTCCGAGGTATT	124
U6	上游：CTCGCTTCGGCAGCACA	116
U6	下游：AACGCTTCACGAATTTGCGT	116

图1 EdU法测定各组CAL-27细胞增殖情况（EdU染色，×200）

Fig.1 Proliferation of CAL-27 cells in each group determined by EdU method (EdU staining, ×200)

表2 各组CAL-27细胞EdU阳性率、集落形成率、划痕愈合率及细胞侵袭数量比较（n=6，$\bar{x}\pm s$）

Tab.2 Comparison of EdU positive rate，colony formation rate，scratch healing rate and cell invasion quantity between five groups of CAL-27 cells

组别	EdU阳性率/%	集落形成率/%	划痕愈合率/%	细胞侵袭数量/个
对照组	41.95±5.81	36.67±4.33	82.15±10.36	123.67±14.82
sh-NC组	40.26±5.37	34.83±4.17	79.72±9.19	119.83±12.38
sh-NNT-AS1组	17.42±2.23^ab	16.50±2.50^ab	38.41±4.54^ab	57.67±6.23^ab
sh-NNT-AS1+miR-In-NC组	18.35±2.19	15.17±1.83	36.67±4.83	55.33±5.96
sh-NNT-AS1+miR-199a-5p-In组	39.73±5.54^cd	31.33±4.67^cd	80.53±9.61^cd	106.50±12.14^cd
F	45.474^＊＊	47.048^＊＊	51.442^＊＊	56.876^＊＊

图2 克隆形成实验评估CAL-27细胞集落形成能力

Fig.2 Colony formation ability of CAL-27 cells evaluated by clone formation assay

图3 划痕愈合实验评估CAL-27细胞迁移能力

Fig.3 The migration ability of CAL-27 cells evaluated by scratch healing assay

图4 Transwell实验测定CAL-27细胞侵袭情况（结晶紫染色，×200）

Fig.4 The invasion of CAL-27 cells detected by Transwell assay (crystal violet staining, ×200)

表3 各组CAL-27细胞中LncRNA NNT-AS1和miR-199a-5p水平比较（n=6，$\bar{x}\pm s$）

Tab.3 Comparison of LncRNA NT-AS1 and miR-199a-5p levels between five groups of CAL-27 cells

组别	LncRNA NNT-AS1	miR-199a-5p
对照组	1.00±0.00	1.00±0.00
sh-NC组	1.02±0.13	0.98±0.11
sh-NNT-AS1组	0.42±0.07^ab	3.05±0.43^ab
sh-NNT-AS1+miR-In-NC组	0.45±0.08	2.89±0.38
sh-NNT-AS1+miR-199a-5p-In组	0.44±0.08	1.12±0.14^cd
F	85.647^＊＊	94.011^＊＊

图5 CAL-27细胞中E-cadherin、N-cadherin、Vimentin、EGFR蛋白表达 A：对照组；B：sh-NC组；C：sh-NNT-AS1组；D：sh-NNT-AS1+miR-In-NC组；E：sh-NNT-AS1+miR-199a-5p-In组。

Fig.5 Expressions of E-cadherin，N-cadherin，Vimentin，EGFR and GAPDH proteins in CAL-27 cells

表4 各组CAL-27细胞中E-cadherin、N-cadherin、 Vimentin和EGFR蛋白表达水平比较（n=6，$\bar{x}\pm s$）

Tab.4 Comparison of expression levels of E-cadherin， N-cadherin，Vimentin and EGFR proteins between five groups of CAL-27 cells

组别	E-cadherin/ GAPDH	N-cadherin/ GAPDH
对照组	0.24±0.03	0.78±0.10
sh-NC组	0.26±0.05	0.75±0.11
sh-NNT-AS1组	0.72±0.11^ab	0.41±0.06^ab
sh-NNT-AS1+miR-In-NC组	0.69±0.09	0.43±0.08
sh-NNT-AS1+miR-199a-5p-In组	0.38±0.05^cd	0.70±0.10^cd
F	61.862^＊＊	22.967^＊＊
组别	Vimentin/ GAPDH	EGFR/ GAPDH
对照组	0.81±0.14	1.18±0.16
sh-NC组	0.78±0.13	1.15±0.17
sh-NNT-AS1组	0.23±0.04^ab	0.76±0.12^ab
sh-NNT-AS1+miR-In-NC组	0.26±0.05	0.72±0.13
sh-NNT-AS1+miR-199a-5p-In组	0.75±0.12^cd	1.07±0.15^cd
F	47.144^＊＊	13.360^＊＊

图6 预测miR-199a-5p与LncRNA NNT-AS1 3′-UTR之间的靶向结合位点

Fig.6 Predicting the targeted binding sites between miR-199a-5p and LncRNA NNT-AS1 3'-UTR

表5 2组CAL-27细胞中相对萤光素酶活性检测结果比较（n=6，$\bar{x}\pm s$）

Tab.5 Comparison of relative luciferase activity detection results between two groups of CAL-27 cells

组别	NNT-AS1-MUT	NNT-AS1-WT
miR-NC组	1.02±0.14	0.97±0.12
miR-199a-5p mimic组	0.99±0.15	0.56±0.10
t	0.358	6.429^＊

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