金丝桃苷抑制鼻咽癌细胞生物学行为的分子机制研究

doi:10.11958/20220805

摘要/Abstract

摘要：

目的探讨金丝桃苷对鼻咽癌细胞增殖、迁移和侵袭等生物学行为的影响和机制。方法采用10、20及40 mg/L金丝桃苷的培养液孵育SUNE1细胞，依次记为低、中、高剂量组；将长链非编码RNA（lncRNA）配对盒基因8反义RNA 1（PAX8-AS1）过表达或敲低质粒、miR-494-3p模拟物及其阴性对照分别转染至40 mg/L金丝桃苷处理的SUNE1细胞，采用CCK-8实验、平板克隆实验、Transwell实验检测SUNE1细胞活力、克隆形成以及迁移和侵袭。蛋白质印记法分析基质金属蛋白酶（MMP）-2和MMP-9蛋白表达。实时荧光定量聚合酶链反应（RT-qPCR）检测lncRNA PAX8-AS1和miR-494-3p表达。双荧光素酶报告实验分析lncRNA PAX8-AS1和miR-494-3p靶向关系。结果低、中、高剂量金丝桃苷降低SUNE1细胞活力、迁移数和侵袭数，下调MMP-2蛋白、MMP-9蛋白以及miR-494-3p表达水平，上调lncRNA PAX8-AS1表达水平（P<0.05）。lncRNA PAX8-AS1靶向负调控miR-494-3p表达。过表达lncRNA PAX8-AS1增强40 mg/L金丝桃苷对SUNE1细胞活力、迁移、侵袭以及MMP-2、MMP-9蛋白表达的抑制作用（P<0.05）。敲低lncRNA PAX8-AS1和过表达miR-494-3p均减弱40 mg/L金丝桃苷对SUNE1细胞活力、迁移、侵袭以及MMP-2、MMP-9蛋白表达的抑制作用（P<0.05）。结论金丝桃苷可能通过上调lncRNA PAX8-AS1/miR-494-3p轴来抑制鼻咽癌细胞增殖、迁移和侵袭。

关键词: 鼻咽肿瘤, 药理作用分子作用机制, 基因，肿瘤抑制, 细胞增殖, 肿瘤侵润, 金丝桃苷, lncRNA PAX8-AS1, miR-494-3p

Abstract:

Objective To investigate the effect and mechanism of hyperoside on the biological behavior such as cell proliferation, migration and invasion of nasopharyngeal carcinoma cells. Methods SUNE1 cells were incubated with hyperin medium of 10 mg/L, 20 mg/L and 40 mg/L, and cells were divided into the low, the medium and the high dose groups. The lncRNA PAX8-AS1 overexpression or knockdown plasmid and miR-494-3p mimic were transfected into 40 mg/L hypericin-treated SUNE1 cells respectively. The activity, clonal formation, migration and invasion of SUNE1 cells were detected by CCK-8 assays, plate cloning experiment and Transwell assays. Western blot assay was used to analyze the expression of matrix metalloproteinase (MMP) -2 and MMP-9 proteins. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of long non-coding RNA (lncRNA) paired-box gene 8 antisense RNA 1 (PAX8-AS1) and miR-494-3p. Dual luciferase report experiment and RT-qPCR were used to determine the targeting relationship between lncRNA PAX8-AS1 and miR-494-3p. Results Low, medium and high dose-hyperoside significantly reduced the cell viability, migration number and invasion number of SUNE1 cells, and down-regulated expression levels of MMP-2 protein, MMP-9 protein and miR-494-3p, and up-regulated the expression level of lncRNA PAX8-AS1 (P<0.05). lncRNA PAX8-AS1 negatively regulated miR-494-3p expression. lncRNA PAX8-AS1 overexpression significantly enhanced inhibitory effects of 40 mg/L hyperoside on SUNE1 cell viability, migration and invasion, and MMP-2 and MMP-9 protein expression (P<0.05). Knockdown of lncRNA PAX8-AS1 and overexpression of miR-494-3p significantly reduced inhibitory effects of 40 mg/L hyperoside on SUNE1 cell viability, migration, invasion and MMP-2 and MMP-9 protein expression (P<0.05). Conclusion Hyperoside may inhibit the proliferation and migration, and invasion of nasopharyngeal carcinoma cells by up-regulating the lncRNA PAX8-AS1 /miR-494-3p axis.

Key words: nasopharyngeal neoplasms, molecular mechanisms of pharmacological action, genes, tumor suppressor, cell proliferation, neoplasm invasiveness, hyperoside, lncRNA PAX8-AS1, miR-494-3p

中图分类号:

R739.6

图/表 15

表1 不同浓度金丝桃苷对SUNE1细胞增殖的影响（n=9，$\bar{x}±s$）

Tab.1 Effects of different concentrations of hyperin on SUNE1 cell proliferation

组别	OD₄₅₀	细胞克隆数（个）
NC组	1.15±0.11	124.00±8.53
低剂量组	0.90±0.07^a	87.00±6.12^a
中剂量组	0.61±0.05^ab	62.00±4.57^ab
高剂量组	0.47±0.04^abc	47.00±2.88^abc
F	157.123^＊＊	292.522^＊＊

图1 细胞克隆形成实验检测细胞增殖活力

Fig.1 Cell proliferation activity detected by cell clonogenesis assay

图2 不同浓度金丝桃苷对SUNE1细胞迁移和侵袭的影响（结晶紫染色，×200）

Fig.2 Effects of hyperin at different concentrations on migration and invasion of SUNE1 cells (crystal violet staining, ×200)

图3 不同浓度金丝桃苷对SUNE1细胞相关蛋白表达的影响

Fig.3 Effects of hyperin at different concentrations on expression of related proteins in SUNE1 cells

表2 不同浓度金丝桃苷对SUNE1细胞迁移和侵袭的影响（n=9，$\bar{x}±s$）

Tab.2 Effects of hyperin at different concentrations on migration and invasion of SUNE1 cells

组别	细胞迁移数量（个/视野）	细胞侵袭数量（个/视野）	MMP-2	MMP-9
NC组	234.00±16.73	164.00±9.17	0.81±0.07	0.78±0.07
低剂量组	189.00±12.37^a	131.00±8.02^a	0.68±0.06^a	0.65±0.05^a
中剂量组	138.00±9.47^ab	82.00±6.11^ab	0.50±0.05^ab	0.43±0.03^ab
高剂量组	91.00±7.54^abc	68.00±4.21^abc	0.42±0.03^abc	0.31±0.02^abc
F	238.698^＊＊	347.897^＊＊	93.655^＊＊	185.759^＊＊

表3 不同浓度金丝桃苷对lncRNA PAX8-AS1和miR-494-3p表达的影响（n=9，$\bar{x}±s$）

Tab.3 Effects of different concentrations hyperin on expression levels of lncRNA PAX8-AS1 and miR-494-3p

组别	lncRNA PAX8-AS1	miR-494-3p
NC组	1.00±0.09	1.00±0.08
低剂量组	1.67±0.12^a	0.79±0.07^a
中剂量组	2.34±0.18^ab	0.58±0.04^ab
高剂量组	2.71±0.22^abc	0.40±0.03^abc
F	198.529^＊＊	175.891^＊＊

图4 starbase对lncRNA PAX8-AS1和miR-494-3p结合进行预测示意图 A：网站预测图；B：野生序列与突变序列对比图。

Fig.4 Schematic diagram of Starbase's prediction of lncRNA PAX8-AS1 and miR-494-3p binding

表4 2组双荧光素酶活性检测（n=9，$\bar{x}±s$）

Tab.4 Double luciferase activity detection between the two groups

组别	WT-lncRNA PAX8-AS1	MUT-lncRNA PAX8-AS1
miR-NC组	1.00±0.10	1.00±0.11
miR-494-3p mimics组	0.46±0.03	1.03±0.09
t	15.517^＊＊	0.633

表5 各组RT-qPCR检测miR-494-3p的表达比较（n=9，$\bar{x}±s$）

Tab.5 Comparison of expression of miR-494-3p detected by RT-qPCR in each group

组别	lncRNA PAX8-AS1	miR-494-3p
pcDNA组	1.00±0.11	1.00±0.09
pcDNA-lncRNA PAX8-AS1组	2.13±0.17^a	0.43±0.04^a
si-NC组	1.02±0.08	0.98±0.09
si-lncRNA PAX8-AS1组	0.45±0.03^b	2.16±0.18^b
F	370.137^＊＊	380.002^＊＊

表6 过表达或敲低lncRNA PAX8-AS1对金丝桃苷处理的SUNE1细胞增殖、迁移和侵袭的影响（n=9，$\bar{x}±s$）

Tab.6 Effects of overexpression or knockdown of lncRNA PAX8-AS1 on proliferation, migration and invasion of hypericin-treated SUNE1 cells

组别	lncRNA PAX8-AS1	MMP-2	MMP-9	OD₄₅₀	细胞克隆数（个）	细胞迁移数量（个/视野）	细胞侵袭数量（个/视野）
NC组	1.00±0.06	0.84±0.09	0.80±0.05	1.16±0.13	128.00±10.62	241.00±19.85	161.00±13.08
pcDNA+高剂量组	2.73±0.24^a	0.43±0.04^a	0.33±0.02^a	0.48±0.04^a	46.00±3.65^a	94.00±7.14^a	63.00±5.02^a
pcDNA-lncRNA PAX8-AS1+高剂量组	4.32±0.38^b	0.14±0.01^b	0.10±0.01^b	0.21±0.02^b	21.00±1.35^b	59.00±3.66^b	41.00±2.84^b
si-NC+高剂量组	2.69±0.21^a	0.45±0.03^a	0.35±0.03^a	0.48±0.05^a	44.00±3.67^a	92.00±7.16^a	61.00±5.04^a
si-lncRNA PAX8-AS1+高剂量组	1.43±0.12^c	0.65±0.05^c	0.59±0.08^c	0.90±0.09^c	97.00±8.54^c	189.00±14.68^c	132.00±11.89^c
F	288.578^＊＊	234.443^＊＊	314.257^＊＊	218.868^＊＊	404.524^＊＊	363.920^＊＊	326.267^＊＊

图5 过表达或敲低lncRNA PAX8-AS1对金丝桃苷处理的SUNE1细胞克隆形成、迁移、侵袭的影响

Fig.5 Effects of overexpression or knockdown of lncRNA PAX8-AS1 on cloning formation, migration and invasion of hypericin-treated SUNE1 cells

图6 过表达或敲低lncRNA PAX8-AS1对金丝桃苷处理的SUNE1细胞MMP-2、MMP-9表达的影响 A：NC组；B：pcDNA+高剂量组；C：pcDNA-lncRNA PAX8-AS1+高剂量组；D：si-NC+高剂量组；E：si-lncRNA PAX8-AS1+高剂量组。

Fig.6 Effects of overexpression or knockdown of lncRNA PAX8-AS1 on related MMP-2, MMP-9 protein expression in hypericin-treated SUNE1 cells

表7 过表达miR-494-3p对金丝桃苷处理的SUNE1细胞增殖、迁移和侵袭的影响（n=9，$\bar{x}±s$）

Tab.7 Effects of overexpression of miR-494-3p on proliferation, migration and invasion of hypericin-treated SUNE1 cells

组别	miR-494-3p	MMP-2	MMP-9	OD₄₅₀	细胞克隆数（个）	细胞迁移数量（个/视野）	细胞侵袭数量（个/视野）
miR-NC+高剂量组	1.00±0.10	0.41±0.04	0.30±0.02	0.47±0.04	43.00±3.25	94.00±6.17	63.00±5.09
miR-494-3p+高剂量组	1.95±0.16	0.91±0.03	0.72±0.06	1.09±0.08	98.00±7.35	183.00±15.01	147.00±11.31
t	15.105^＊＊	30.000^＊＊	19.922^＊＊	20.795^＊＊	20.531^＊＊	16.452^＊＊	20.318^＊＊

图7 过表达miR-494-3p对金丝桃苷处理的SUNE1细胞克隆、迁移和侵袭的影响

Fig.7 Effects of overexpression of miR-494-3p on cloning, migration and invasion of hypericin-treated SUNE1 cells

图8 过表达miR-494-3p对金丝桃苷处理的SUNE1细胞MMP-2、MMP-9蛋白表达水平的影响 A：miR-NC+高剂量组；B：miR-494-3p+高剂量组。

Fig.8 Effects of overexpression of miR-494-3p on MMP-2, MMP-9 protein expression levels of hypericin-treated SUNE1 cells

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