羽扇豆醇调控miR-100-5p对非小细胞肺癌NCI-H1299细胞生物学行为的影响

doi:10.11958/20220899

摘要/Abstract

摘要：

目的探讨羽扇豆醇调控miR-100-5p对非小细胞肺癌（NSCLC）NCI-H1299细胞增殖、迁移和侵袭的影响。方法将体外培养的NCI-H1299细胞分为正常对照（NC）组（未处理）、低剂量组（12.5 μmol/L羽扇豆醇）、中剂量组（25 μmol/L羽扇豆醇）、高剂量组（50 μmol/L羽扇豆醇）、miR-NC组（转染miR-NC）、miR-100-5p组（转染miR-100-5p）、anti-miR-NC+高剂量组（转染anti-miR-NC后用50 μmol/L羽扇豆醇处理）、anti-miR-100-5p+高剂量组（转染anti-miR-100-5p后用50 μmol/L羽扇豆醇处理）。CCK-8和克隆形成实验检测NCI-H1299细胞增殖；Transwell法检测NCI-H1299细胞迁移和侵袭。荧光定量PCR（qPCR）检测miR-100-5p表达；Western blot检测细胞周期蛋白D1（CyclinD1）、基质金属蛋白酶（MMP）2和MMP9蛋白的表达。结果与NC组比较，低、中、高剂量组NCI-H1299细胞活力、细胞克隆数、迁移数、侵袭数、CyclinD1、MMP2和MMP9蛋白表达降低（P<0.05），miR-100-5p表达逐渐升高（P<0.05）。与miR-NC组比较，miR-100-5p组miR-100-5p表达升高，NCI-H1299细胞活力、细胞克隆数、迁移数、侵袭数、CyclinD1、MMP2和MMP9蛋白表达降低（P<0.05）；与anti-miR-NC+高剂量组比较，anti-miR-100-5p+高剂量组miR-100-5p表达下降，NCI-H1299细胞活力、细胞克隆数、迁移数、侵袭数、CyclinD1、MMP2和MMP9蛋白表达升高（P<0.05）。结论羽扇豆醇通过上调miR-100-5p表达抑制NSCLC细胞NCI-H1299增殖、迁移和侵袭。

关键词: 癌，非小细胞肺, 羽扇豆醇, 微RNAs, 细胞增殖, 细胞运动, 细胞周期蛋白D1, 基质金属蛋白酶类, miR-100-5p

Abstract:

Objective To investigate the effect of lupeol on the proliferation, migration and invasion of non-small cell lung cancer (NSCLC) NCI-H1299 cells by regulating miR-100-5p. Methods NCI-H1299 cells cultured in vitro were divided into the normal control (NC) group (untreated), the low-dose group (12.5 μmol/L lupeol), the medium-dose group (25 μmol/L lupeol), the high-dose group (50 μmol/L lupeol), the miR-NC group (transfected with miR-NC), the miR-100-5p group (transfected with miR-100-5p), the anti-miR-NC+high-dose group (the cells were treated with 50 μmol/L lupeol after transfected with anti-miR-NC), and the anti-miR-100-5p+high-dose group (the cells were treated with 50 μmol/L lupeol after transfected with anti-miR-100-5p). CCK-8 and colony formation assays were performed to evaluate the proliferation of NCI-H1299 cells. Transwell method was applied to detect the migration and invasion of NCI-H1299 cells. RT-qPCR was used to detect the expression of miR-100-5p. Western blot assay was used to detect the expression of CyclinD1, matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) proteins. Results Compared with the NC group, NCI-H1299 cell viability, colony formation number, migration number, invasion number, the expression levels of CyclinD1, MMP2 and MMP9 proteins were decreased in the low, medium and high dose groups (P<0.05), and the expression of miR-100-5p was significantly increased (P<0.05). Compared with the miR-NC group, the expression of miR-100-5p was increased, the NCI-H1299 cell viability, colony formation number, migration number, invasion number, the expression of CyclinD1, MMP2 and MMP9 proteins decreased in the miR-100-5p group (P<0.05). Compared with the anti-miR-NC+ high-dose group, the expression of miR-100-5p was decreased, NCI-H1299 cell viability, colony formation number, migration number, invasion number, the expression of CyclinD1, MMP2 and MMP9 proteins increased in the anti-miR-100-5p+ high-dose group (P<0.05). Conclusion Lupeol inhibits the proliferation, migration and invasion of NSCLC cells NCI-H1299 by up-regulating miR-100-5p.

Key words: carcinoma, non-small-cell lung, lupeol, microRNAs, cell proliferation, cell movement, cyclin D1, matrix metalloproteinases, miR-100-5p

中图分类号:

R734.2

图/表 14

图1 羽扇豆醇对NCI-H1299细胞克隆的影响

Fig.1 Effects of lupeol on NCI-H1299 cell clone

图2 Western blot检测CyclinD1蛋白的表达

Fig.2 The expression of CyclinD1 protein detected by Western blot assay

表1 各组NCI-H1299细胞增殖和CyclinD1蛋白表达水平比较（n=9，$\bar{x}±s$）

Tab.1 Comparison of proliferation and expression level of CyclinD1 protein in NCI-H1299 cells between four groups

组别	OD₄₅₀	细胞克隆数（个）	CyclinD1
NC组	1.01±0.10	113.00±8.26	0.85±0.09
低剂量组	0.90±0.09^a	84.00±7.15^a	0.73±0.07^a
中剂量组	0.73±0.07^ab	61.00±5.13^ab	0.53±0.05^ab
高剂量组	0.51±0.05^abc	50.00±4.02^abc	0.41±0.04^abc
F	67.047^＊＊	172.777^＊＊	81.965^＊＊

图3 Transwell检测羽扇豆醇对NCI-H1299细胞迁移、侵袭的影响

Fig.3 Effects of lupeol on migration and invasion of NCI-H1299 cells detected by Transwell assay

图4 Western blot检测MMP2和MMP9蛋白的表达

Fig.4 Expressions of MMP2 and MMP9 proteins detected by Western blot assay

表2 各组NCI-H1299细胞迁移、侵袭及MMP2、MMP9蛋白表达水平比较（n=9，$\bar{x}±s$）

Tab.2 Comparison of migration and invasion of NCI-H1299 cells and the expression levels of MMP2 and MMP9 proteins between four groups

组别	细胞迁移数（个）	细胞侵袭数（个）	MMP2	MMP9
NC组	154.00±12.25	136.00±13.32	0.83±0.08	0.75±0.06
低剂量组	127.00±10.13^a	101.00±10.03^a	0.70±0.07^a	0.62±0.06^a
中剂量组	100.00±9.24^ab	88.00±8.02^ab	0.57±0.05^ab	0.48±0.04^ab
高剂量组	62.00±5.16^abc	51.00±4.01^abc	0.42±0.04^abc	0.37±0.03^abc
F	152.245^＊＊	123.809^＊＊	72.156^＊＊	101.567^＊＊

图5 miR-100-5p对NCI-H1299细胞克隆的影响

Fig.5 Effects of miR-100-5p on NCI-H1299 cell clone

图6 Transwell检测miR-100-5p对NCI-H1299细胞迁移、侵袭的影响

Fig.6 Effects of miR-100-5p on migration and invasion of NCI-H1299 cells detected by Transwell assay

图7 Western blot检测CyclinD1、MMP2和MMP9蛋白的表达

Fig.7 Expression of CyclinD1, MMP2 and MMP9 proteins detected by Western blot assay

表3 过表达miR-100-5p后2组NCI-H1299细胞增殖、迁移和侵袭及CyclinD1、MMP2、MMP9蛋白表达水平比较（n=9，$\bar{x}±s$）

Tab.3 Comparison of proliferation, migration, invasion and the expression levels of CyclinD1, MMP2 and MMP9 proteins in NCI-H1299 cells between two groups after overexpression of miR-100-5p

组别	miR-100-5p	OD值	细胞克隆数（个）	细胞迁移数（个）	细胞侵袭数（个）	CyclinD1	MMP2	MMP9
miR-NC组	1.01±0.11	1.09±0.08	109.00±11.21	152.00±13.01	145.00±13.12	0.79±0.04	0.82±0.07	0.72±0.08
miR-100-5p组	2.68±0.34	0.69±0.08	56.00±4.12	85.00±6.33	68.00±5.11	0.52±0.06	0.56±0.07	0.45±0.06
t	14.020^＊＊	10.607^＊＊	13.313^＊＊	13.893^＊＊	16.406^＊＊	11.233^＊＊	7.879^＊＊	8.100^＊＊

图8 抑制miR-100-5p表达可逆转羽扇豆醇对NCI-H1299细胞克隆的影响

Fig.8 Inhibition of miR-100-5p expression reversed the effect of lupeol on NCI-H1299 cell clone

图9 Transwell检测抑制miR-100-5p表达对羽扇豆醇干预后NCI-H1299细胞迁移、侵袭的影响

Fig.9 Effects of inhibition of miR-100-5p expression on migration and invasion of NCI-H1299 cells after lupeol intervention detected by Transwell

图10 Western blot检测CyclinD1、MMP2和MMP9蛋白的表达

Fig.10 Expression of CyclinD1, MMP2 and MMP9 proteins detected by Western blot Assay

表4 抑制miR-100-5p表达后2组NCI-H1299细胞增殖、迁移和侵袭及CyclinD1、MMP2、MMP9蛋白表达水平比较（n=9，$\bar{x}±s$）

Tab.4 Comparison of proliferation, migration and invasion of NCI-H1299 cells and the expression levels of CyclinD1, MMP2, MMP9 proteins between two groups after inhibition of miR-100-5p

组别	miR-100-5p	OD值	细胞克隆数（个）	细胞迁移数（个）	细胞侵袭数（个）	CyclinD1	MMP2	MMP9
anti-miR-NC+高剂量组	1.00±0.09	0.58±0.05	64.00±5.34	47.00±4.34	44.00±3.83	0.42±0.04	0.40±0.03	0.35±0.03
anti-miR-100-5p+高剂量组	0.45±0.04	1.12±0.10	132.00±9.14	77.00±6.54	70.00±5.28	0.87±0.07	0.82±0.07	0.76±0.07
t	16.753^＊＊	14.490^＊＊	19.271^＊＊	11.466^＊＊	11.958^＊＊	16.745^＊＊	16.545^＊＊	16.151^＊＊

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