miR-106a-5p调控<i>PTEN</i>对鼻咽癌细胞SUNE2增殖和迁移的抑制作用研究

doi:10.3877/cma.j.issn.2095-1221.2020.04.004

目的

研究miR-106a-5p对鼻咽癌细胞SUNE2增殖和迁移的影响。

方法

将体外培养的鼻咽癌细胞SUNE2分成对照组（细胞未做任何处理）、Anti-NC组（转染阴性对照抑制剂）、Anti-miR-106a-5p组（转染miR-106a-5p抑制剂）、后期实验另设Anti-miR-106a-5p-inhibitor+si-NC组（转染miR-106a-5p抑制剂、siRNA阴性对照）、Anti-miR-106a-5p-inhibitor+si-PTEN组（转染miR-106a-5p抑制剂、PTEN siRNA），采用Realtime PCR测定miR-106a-5p表达，MTT法检测增殖，Transwell小室检测细胞侵袭和迁移能力变化，用Western blot方法测定vimentin、E-cadherin蛋白表达。在线靶基因预测软件预测miR-106a-5p的靶基因可能为PTEN，双荧光素酶报告系统鉴定miR-106a-5p和PTEN的靶向关系。两组间比较用独立样本t检验，多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。

结果

与正常鼻咽上皮细胞NP69比较，鼻咽癌细胞CNE-2、HK1、SUNE2中miR-106a-5p水平（1.00±0.11比1.84±0.13、2.19±0.14、2.87±0.25）升高，差异具有统计学意义（P < 0.05）。与对照组、Anti-NC组比较，Anti-miR-106a-5p组鼻咽癌细胞miR-106a-5p水平（1.00±0.10、0.99±0.12比0.76±0.08）降低，OD值（0.56±0.05、0.57±0.04比0.32±0.02），细胞侵袭数[（128.47±11.65）个、（129.84±10.93）个比（85.12±6.75）个]，迁移数[（182.51±14.81）个、（180.68±17.64）个比（122.01±11.62）个]，vimentin蛋白表达水平（0.84±0.09、0.82±0.07比0.30±0.05）降低，E-cadherin蛋白表达水平（0.29±0.04、0.28±0.05比0.76±0.08）升高，差异具有统计学意义（P均< 0.05）。与Anti-miR-106a-inhibitor+si-NC组比较，Anti-miR-106a-inhibitor+si-PTEN组细胞OD值（0.33±0.03比0.52±0.05）、侵袭数[（84.16±5.91）个比（105.79±8.63）个]、迁移数[（118.42±10.25）个比（164.28±12.05）个]、vimentin蛋白表达水平（0.34±0.06比0.68±0.05）均升高，E-cadherin蛋白表达水平（0.72±0.06比0.29±0.05）降低，差异具有统计学意义（P均< 0.05）。

结论

miR-106a-5p可通过靶向调控PTEN抑制鼻咽癌细胞SUNE2增殖和迁移潜能。

关键词: 鼻咽癌, miR-106a-5p, PTEN, 侵袭, 增殖

Objective

To investigate the effect of miR-106a-5p on the proliferation and migration of nasopharyngeal carcinoma cells SUNE2.

Methods

The nasopharyngeal carcinoma cells SUNE2 cultured in vitro were divided into Control group (without treatment), Anti-NC group (transfected with inhibitor control) and Anti-miR-106a-5p group (transfected with miR-106a-5p inhibitor), as well as Anti-miR-106a-5p+si-NC group (transfected with miR-106a-5p inhibitor and siRNA control) and Anti-miR-106a-5p+si-PTEN group (transfected with miR-106a-5p inhibitor and PTEN siRNA). Realtime PCR was used to determine the expression of miR-106a-5p, MTT and Transwell were used to detect proliferation rate, invasion and migration ability of cells, respectively. The expression levels of vimentin and E-cadherin protein were detected by Western blot. The online target gene prediction software suggests that the target gene of miR-106a-5p may be PTEN, and the dual luciferase reporter system examines the targeting relationship between miR-106a-5p and PTEN. The independent sample t-test was used for the comparison between two groups, the ANOVA analysis was used for the multiple group comparison, and the LSD-t test was used for the pairwise comparison of the components.

Results

Compared with NP69 in normal nasopharyngeal epithelial cells, the expression level of miR-106a-5p in nasopharyngeal carcinoma cells CNE-2, HK1, and SUNE2 were increased (1.00±0.11 vs 1.84±0.13, 2.19±0.14, 2.87±0.25), the difference was statistically significant (P < 0.05) .Compared with the Control and Anti-NC group, the expression level of miR-106a-5p (1.00±0.10, 0.99±0.12 vs 0.76±0.08), the OD values (0.56±0.05, 0.57±0.04 vs 0.32±0.02), cell invasion (128.47±11.65, 129.84±10.93 vs 85.12±6.75), the number of migrations (182.51±14.81, 180.68±17.64 vs 122.01±11.62) as well as the expression level of vimentin protein (0.84±0.09, 0.82±0.07 vs 0.30±0.05) of nasopharyngeal cancer cells in the Anti-miR-106a-5p group were significantly reduced. While the expression level of E-cadherin protein was increased (0.29±0.04, 0.28±0.05 vs 0.76±0.08) (P < 0.05). Compared with the Anti-miR-106a-inhibitor+si-NC group, in the Anti-miR-106a-inhibitor+si-PTEN group, the OD value (0.33±0.03 vs 0.52±0.05), the number of invasions (84.16±5.91 vs 105.79±8.63) and migrations (118.42±10.25 vs 164.28±12.05) as well as the expression of vimentin protein (0.34±0.06 vs 0.68±0.05) were all significantly increased. While the expression of E-cadherin protein was decreased (0.72±0.06 vs 0.29±0.05) (P < 0.05) .

Conclusion

miR-106a-5p could inhibit the proliferation and migration of nasopharyngeal carcinoma cells SUNE2 by targeted regulation of PTEN.

Key words: Nasopharyngeal carcinoma, MiR-106a-5p, PTEN, Invasion, Proliferation

表1 引物序列信息

基因	序列（5'-3'）	预期扩增产物长度（bp）
miR-106a-5p	上游ATCCAGTGCTGTCGTG	92
?	下游TGCTAAAAGTGCTTACAGTG	?
U6（内参）	上游TGCGGGTGCTCGCTTCGGCAGC	96
?	下游GTGCAGGGTCCGAGGT	?

表1 引物序列信息

基因	序列（5'-3'）	预期扩增产物长度（bp）
miR-106a-5p	上游ATCCAGTGCTGTCGTG	92
?	下游TGCTAAAAGTGCTTACAGTG	?
U6（内参）	上游TGCGGGTGCTCGCTTCGGCAGC	96
?	下游GTGCAGGGTCCGAGGT	?

表2 鼻咽癌细胞和正常鼻咽上皮细胞中miR-106a-5p表达水平（±s，n = 3）

细胞	miR-106a-5p
NP69	1.00±0.11
CNE-2	1.84±0.13^a
HK1	2.19±0.14^a
SUNE2	2.87±0.25^abc
F值	196.158
P值	<0.001

表2 鼻咽癌细胞和正常鼻咽上皮细胞中miR-106a-5p表达水平（±s，n = 3）

细胞	miR-106a-5p
NP69	1.00±0.11
CNE-2	1.84±0.13^a
HK1	2.19±0.14^a
SUNE2	2.87±0.25^abc
F值	196.158
P值	<0.001

表3 miR-106a-5p抑制剂转染后鼻咽癌细胞中miR-106a-5p水平（±s，n = 3）

分组	miR-106a-5p
对照组	1.00±0.10
Anti-NC组	0.99±0.12
Anti-miR-106a-5p组	0.31±0.05^ab
F值	157.015
P值	< 0.001

表3 miR-106a-5p抑制剂转染后鼻咽癌细胞中miR-106a-5p水平（±s，n = 3）

分组	miR-106a-5p
对照组	1.00±0.10
Anti-NC组	0.99±0.12
Anti-miR-106a-5p组	0.31±0.05^ab
F值	157.015
P值	< 0.001

图1 倒置显微镜下观察Transwell小室检测miR-106a抑制剂转染后鼻咽癌细胞侵袭和迁移结果（结晶紫染色，×200）

图2 Western blot检测miR-106a抑制剂转染后鼻咽癌细胞中vimentin、E-cadherin蛋白水平

表4 miR-106a抑制剂转染后鼻咽癌细胞OD值、侵袭数目、迁移数目和vimentin、E-cadherin蛋白水平（±s，n = 3）

分组	OD值	侵袭数目（个）	迁移数目（个）	vimentin	E-cadherin
对照组	0.56±0.05	128.47±11.65	182.51±14.81	0.84±0.09	0.29±0.04
Anti-NC组	0.57±0.04	129.84±10.93	180.68±17.64	0.82±0.07	0.28±0.05
Anti-miR-106a-5p组	0.32±0.02^ab	85.12±6.75^ab	122.01±11.62^ab	0.30±0.05^ab	0.76±0.08^ab
F值	120.200	58.070	48.046	163.277	193.457
P值	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

表4 miR-106a抑制剂转染后鼻咽癌细胞OD值、侵袭数目、迁移数目和vimentin、E-cadherin蛋白水平（±s，n = 3）

分组	OD值	侵袭数目（个）	迁移数目（个）	vimentin	E-cadherin
对照组	0.56±0.05	128.47±11.65	182.51±14.81	0.84±0.09	0.29±0.04
Anti-NC组	0.57±0.04	129.84±10.93	180.68±17.64	0.82±0.07	0.28±0.05
Anti-miR-106a-5p组	0.32±0.02^ab	85.12±6.75^ab	122.01±11.62^ab	0.30±0.05^ab	0.76±0.08^ab
F值	120.200	58.070	48.046	163.277	193.457
P值	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

图3 miR-106a与PTEN的3'UTR端结合位点

图4 Western blot检测miR-106a抑制剂转染后鼻咽癌细胞中PTEN蛋白水平

表5 荧光素酶活性（±s，n = 3）

分组	MUT	WT
mimics control	1.00±0.10	1.00±0.12
miR-106a mimics	1.01±0.12	0.46±0.05
t值	0.192	12.462
P值	0.850	< 0.001

表5 荧光素酶活性（±s，n = 3）

分组	MUT	WT
mimics control	1.00±0.10	1.00±0.12
miR-106a mimics	1.01±0.12	0.46±0.05
t值	0.192	12.462
P值	0.850	< 0.001

表6 miR-106a抑制剂转染后鼻咽癌细胞中PTEN蛋白水平（±s，n = 3）

分组	PTEN
对照组	0.36±0.05
Anti-NC组	0.37±0.04
Anti-miR-106a-5p组	0.69±0.06^ab
F值	123.546
P值	< 0.001

表6 miR-106a抑制剂转染后鼻咽癌细胞中PTEN蛋白水平（±s，n = 3）

分组	PTEN
对照组	0.36±0.05
Anti-NC组	0.37±0.04
Anti-miR-106a-5p组	0.69±0.06^ab
F值	123.546
P值	< 0.001

图5 Western blot检测miR-106a-5p抑制剂和PTEN siRNA转染后鼻咽癌细胞中vimentin、E-cadherin、PTEN蛋白水平

表7 miR-106a-5p抑制剂和PTEN siRNA转染后鼻咽癌细胞OD值、侵袭数目、迁移数目和vimentin、E-cadherin、PTEN蛋白水平（±s，n = 3）

分组	OD值	侵袭数目（个）	迁移数目（个）	vimentin	E-cadherin	PTEN
Anti-miR-106a-5p-inhibitor+si-NC组	0.33±0.03	84.16±5.91	118.42±10.25	0.34±0.06	0.72±0.06	0.67±0.08
Anti-miR-106a-5p-inhibitor+si-PTEN组	0.52±0.05	105.79±8.63	164.28±12.05	0.68±0.05	0.29±0.05	0.35±0.06
t值	9.775	6.204	8.697	13.060	16.517	9.600
P值	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

表7 miR-106a-5p抑制剂和PTEN siRNA转染后鼻咽癌细胞OD值、侵袭数目、迁移数目和vimentin、E-cadherin、PTEN蛋白水平（±s，n = 3）

分组	OD值	侵袭数目（个）	迁移数目（个）	vimentin	E-cadherin	PTEN
Anti-miR-106a-5p-inhibitor+si-NC组	0.33±0.03	84.16±5.91	118.42±10.25	0.34±0.06	0.72±0.06	0.67±0.08
Anti-miR-106a-5p-inhibitor+si-PTEN组	0.52±0.05	105.79±8.63	164.28±12.05	0.68±0.05	0.29±0.05	0.35±0.06
t值	9.775	6.204	8.697	13.060	16.517	9.600
P值	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

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Inhibitory of miR-106a-5p regulating PTEN on the proliferation and migration of nasopharyngeal carcinoma cells SUNE2

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Inhibitory of miR-106a-5p regulating PTEN on the proliferation and migration of nasopharyngeal carcinoma cells SUNE2