蔓荆子黄素调控miR-378/PRRX1轴对胃癌生物学行为的影响

doi:10.11958/20222053

摘要/Abstract

摘要：

目的探究蔓荆子黄素（Cas）对胃癌（GC）SGC-7901细胞增殖、侵袭、迁移及微小RNA-378（miR-378）/配对相关同源框1（PRRX1）轴的影响。方法不同浓度Cas（0、5、10、20、40、80 μmol/L）处理SGC-7901细胞48 h。将对数生长期的SGC-7901细胞分为：对照组、Cas低浓度组（10 μmol/L）、Cas中浓度组（20 μmol/L）、Cas高浓度组（40 μmol/L）、Cas高浓度+miR-378小干扰RNA（siRNA）组（40 μmol/L Cas+miR-378 siRNA）、Cas高浓度+PRRX1组（40 μmol/L Cas+PRRX1过表达质粒）、Cas高浓度+miR-378 siRNA+PRRX1组（40 μmol/L Cas+miR-378 siRNA+PRRX1过表达质粒）。MTT法、集落形成实验、流式细胞术、Transwell小室、划痕实验分别测定各组SGC-7901细胞活力、集落形成数量、凋亡率、侵袭及迁移能力；qPCR、蛋白印迹实验分别检测SGC-7901细胞miR-378、PRRX1蛋白表达水平；双萤光素酶实验验证miR-378和PRRX1靶向关系；体内异种移植SGC-7901细胞建立裸鼠移植瘤模型，验证Cas的抗癌活性及对miR-378表达的影响。结果 Cas 10、20、40、80 μmol/L组SGC-7901细胞活力呈浓度依赖性降低（P<0.05），选取10、20、40 μmol/L的Cas作为后续研究浓度。与对照组相比，Cas低、中、高浓度组SGC-7901细胞活力、集落形成数量、侵袭细胞数量、划痕愈合率、PRRX1蛋白表达水平依次降低（P<0.05），细胞凋亡率和miR-378表达水平依次升高（P<0.05）；与Cas高浓度组相比，Cas高浓度+miR-378 siRNA组和Cas高浓度+PRRX1组SGC-7901细胞活力、集落形成数量、侵袭细胞数量、划痕愈合率、PRRX1蛋白表达水平升高，细胞凋亡率降低（P<0.05），Cas高浓度+miR-378 siRNA组SGC-7901细胞miR-378表达水平降低（P<0.05）；与Cas高浓度+miR-378 siRNA组相比，Cas高浓度+PRRX1组SGC-7901细胞miR-378表达水平升高（P<0.05）；与Cas高浓度+miR-378 siRNA组、Cas高浓度+PRRX1组相比，Cas高浓度+miR-378 siRNA+PRRX1组SGC-7901细胞活力、集落形成数量、侵袭细胞数量、划痕愈合率、PRRX1蛋白表达水平升高（P<0.05），细胞凋亡率降低（P<0.05）。在SGC-7901细胞中，miR-378可靶向调控PRRX1表达；Cas可抑制体内裸鼠移植瘤生长并促进miR-378表达（P<0.05）。结论 Cas可能通过调控miR-378/PRRX1轴抑制SGC-7901细胞增殖、侵袭和迁移。

关键词: 蔓荆子, 胃肿瘤, 微RNAs, 细胞增殖, 细胞运动, 肿瘤浸润, 同源盒结构域蛋白质类

Abstract:

Objective To explore effects of Casticin (Cas) on the proliferation, invasion, migration and microRNA-378 (miR-378)/paired related homeobox 1 (PRRX1) axis of gastric cancer (GC) SGC-7901 cells. Methods SGC-7901 cells were treated with Cas at different concentrations (0, 5, 10, 20, 40 and 80 μmol/L) for 48 h, and the cell viability of SGC-7901 was detected by tetrazole salt (MTT) method. SGC-7901 cells at logarithmic growth stage were divided into 7 groups: the control group, the Cas low concentration group (10 μmol/L), the Cas medium concentration group (20 μmol/L), the Cas high concentration group (40 μmol/L), the Cas high concentration+miR-378 small interfering RNA (siRNA) group (40 μmol/L Cas+miR-378 siRNA), the Cas high concentration+PRRX1 group (40 μmol/L Cas+PRRX1 overexpressed plasmid) and the Cas high concentration+miR-378 siRNA+PRRX1 group (40 μmol/L Cas+miR-378 siRNA+PRRX1 overexpression plasmid). Cell activity, colony formation number, apoptosis rate, invasion and migration ability of SGC-7901 cells were determined by MTT assay, colony formation assay, flow cytometry, Transwell chamber assay and scratch assay, respectively. Expression levels of miR-378 and PRRX1 protein in SGC-7901 cells were detected by fluorescence quantitative PCR and Western blot assay. Double luciferase assay verified the targeting relationship between miR-378 and PRRX1. Xenografted SGC-7901 cells were xenografted into nude mice to verify the anticancer activity of Cas and its effect on the expression of miR-378. Results The cell viability of SGC-7901 cells in Cas 10, 20, 40 and 80 μmol/L groups decreased in a concentration-dependent manner (P<0.05), and 10, 20 and 40 μmol/L Cas were selected as the follow-up study concentration. Compared with the control group, the cell viability, colony formation number, invasion cell number, scratch healing rate and PRRX1 protein expression level of SGC-7901 cells in the Cas low, medium and high concentration groups were decreased successively (P<0.05), while the apoptosis rate and the expression level of miR-378 were increased successively (P<0.05). Compared with Cas high concentration group, the cell viability, colony formation number, invasion cell number, scratch healing rate and PRRX1 protein expression levels were increased in the Cas high concentration+miR-378 siRNA group and the Cas high concentration+PRRX1 group, and the apoptosis rate was decreased (P<0.05). The expression level of miR-378 in SGC-7901 cells was decreased in the Cas high concentration+miR-378 siRNA group (P<0.05). Compared with the Cas high concentration+miR-378 siRNA group, the expression level of miR-378 in SGC-7901 cells was increased in the Cas high concentration+PRRX1 group (P<0.05). Compared with the Cas high concentration+miR-378 siRNA group and the Cas high concentration+PRRX1 group, the cell viability, colony formation number, invasion cell number, scratch healing rate and PRRX1 protein expression levels were increased in the Cas high concentration+miR-378 siRNA+PRRX1 group (P<0.05), and the apoptosis rate was decreased (P<0.05). In SGC-7901 cells, miR-378 could target the expression of PRRX1. Cas inhibited the growth of transplanted tumor and promoted the expression of miR-378 in nude mice (P<0.05). Conclusion Cas may inhibit the proliferation, invasion and migration of SGC-7901 cells by regulating the miR-378/PRRX1 axis.

Key words: viticis fructus, stomach neoplasms, microRNAs, cell proliferation, cell movement, tumor infiltration, homeodomain proteins

中图分类号:

R285.5，R735.2

图/表 12

图1 各组SGC-7901细胞集落形成情况（瑞氏-吉姆萨染色，×100） A：对照组；B：Cas低浓度组；C：Cas中浓度组；D：Cas高浓度组；E：Cas高浓度+miR-378 siRNA组；F：Cas高浓度+PRRX1组；G：Cas高浓度+miR-378 siRNA+PRRX1组。

Fig.1 Colony formation of SGC-7901 cells in each group (Reich-Giemsa staining, ×100)

图2 各组SGC-7901细胞凋亡流式图 A：对照组；B：Cas低浓度组；C：Cas中浓度组；D：Cas高浓度组；E：Cas高浓度+miR-378 siRNA组；F：Cas高浓度+PRRX1组；G：Cas高浓度+miR-378 siRNA+PRRX1组。

Fig.2 Flow chart of SGC-7901 cell apoptosis in each group

表1 各组SGC-7901细胞活力、集落形成数量、细胞凋亡率比较 (n=6, $\bar{x}±s$)

Tab.1 Comparison of SGC-7901 cell viability, colony formation number and cell apoptosis rate between the seven groups

组别	细胞活力/%	集落形成数量/个	细胞凋亡率/%
对照组	100.00±0.00	124.56±10.25	4.01±0.24
Cas低浓度组	81.12±7.62^a	88.26±7.06^a	8.20±0.85^a
Cas中浓度组	68.24±6.53^ab	74.15±6.85^ab	15.83±1.77^ab
Cas高浓度组	52.03±5.07^abc	60.24±5.12^abc	26.57±2.19^abc
Cas高浓度+miR- 378 siRNA组	77.04±6.80^d	86.35±7.68^d	8.64±0.93^d
Cas高浓度+ PRRX1组	80.28±7.71^d	90.36±9.34^d	9.02±1.05^d
Cas高浓度+miR- 378 siRNA+ PRRX1组	93.40±8.55^ef	115.28±10.17^ef	5.10±0.47^ef
F	34.436^＊＊	43.591^＊＊	235.574^＊＊

表2 各组SGC-7901细胞侵袭细胞数量和划痕愈合率比较 (n=6, $\bar{x}±s$)

Tab.2 Comparison of the number of invasive cells and scratch healing rate of SGC-7901 cells between the seven groups

组别	侵袭细胞数量/ （个/视野）	划痕愈合率/%
对照组	240.15±22.15	65.34±5.89
Cas低浓度组	191.34±18.79^a	42.29±4.05^a
Cas中浓度组	151.62±13.49^ab	34.80±3.15^ab
Cas高浓度组	114.39±10.36^abc	24.33±2.08^abc
Cas高浓度+miR-378 siRNA组	163.69±15.21^d	40.12±4.08^d
Cas高浓度+PRRX1组	169.62±17.05^d	41.61±5.07^d
Cas高浓度+miR-378 siRNA+ PRRX1组	228.75±20.34^ef	57.35±5.64^ef
F	39.274^＊＊	56.610^＊＊

图3 各组Transwell小室下室中SGC-7901细胞侵袭图（结晶紫染色，×200） A：对照组；B：Cas低浓度组；C：Cas中浓度组；D：Cas高浓度组；E：Cas高浓度+miR-378 siRNA组；F：Cas高浓度+PRRX1组；G：Cas高浓度+miR-378 siRNA+PRRX1组。

Fig.3 Invasion map of SGC-7901 cells in the lower compartment of Transwell cell in each group (crystal violet staining, ×200)

图4 各组SGC-7901细胞迁移图（×100） A：对照组；B：Cas低浓度组；C：Cas中浓度组；D：Cas高浓度组；E：Cas高浓度+miR-378 siRNA组；F：Cas高浓度+PRRX1组；G：Cas高浓度+miR-378 siRNA+PRRX1组。

Fig.4 SGC-7901 cell migration diagram of each group (×100)

图5 各组SGC-7901细胞PRRX1蛋白印迹图 A：对照组；B：Cas低浓度组；C：Cas中浓度组；D：Cas高浓度组；E：Cas高浓度+miR-378 siRNA组；F：Cas高浓度+PRRX1组；G：Cas高浓度+miR-378 siRNA+PRRX1组。

Fig.5 PRRX1 protein blotting of SGC-7901 cells in each group

表3 各组SGC-7901细胞miR-378和PRRX1蛋白表达水平比较 (n=6, $\bar{x}±s$)

Tab.3 Comparison of miR-378 and PRRX1 protein expression levels between the seven groups of SGC-7901 cells

组别	miR-378	PRRX1蛋白（/GAPDH）
对照组	1.02±0.05	0.85±0.07
Cas低浓度组	1.58±0.10^a	0.68±0.06^a
Cas中浓度组	1.96±0.18^ab	0.57±0.06^ab
Cas高浓度组	2.34±0.21^abc	0.46±0.04^abc
Cas高浓度+miR-378 siRNA组	1.56±0.14^d	0.58±0.06^d
Cas高浓度+PRRX1组	2.31±0.20^e	0.62±0.07^d
Cas高浓度+miR-378 siRNA+ PRRX1组	1.54±0.16^f	0.79±0.06^ef
F	54.139^＊＊	29.519^＊＊

图6 Starbase预测的PRRX1 3'-UTR与miR-378互补结合的核苷酸序列

Fig.6 Nucleotide sequence of complementary binding of PRRX1 3'-UTR and miR-378 predicted by Starbase

表4 各组SGC-7901细胞荧光素酶相对活性比较(n=6, $\bar{x}±s$)

Tab.4 Comparison of relative activity of luciferase between the two groups of SGC-7901 cells

组别	PRRX1-WT	PRRX1-MUT
miR-NC mimics	1.02±0.05	0.99±0.07
miR-378 mimics	0.46±0.04	0.95±0.06
t	22.840^＊＊	1.079

图7 各组异种移植裸鼠模型中形成的肿瘤组织代表性图片

Fig.7 Representative pictures of tumor tissue formed in xenotransplantation nude mouse models in each group

表5 各组异种移植裸鼠模型中肿瘤体积、肿瘤质量及肿瘤组织中miR-378表达水平比较 (n=5,$\bar{x}±s$)

Tab.5 Comparison of tumor volume, tumor weight and miR-378 expression level in tumor tissue of xenotransplantation nude mice between the three groups

组别	肿瘤体积/mm³	肿瘤质量/g	miR-378
模型组	1 225.23±145.36	0.89±0.06	1.02±0.04
Cas组	352.78±42.26^a	0.28±0.03^a	1.87±0.15^a
Cas+miR-378 siRNA组	754.12±70.86^b	0.60±0.05^b	1.29±0.11^b
F	102.392^＊＊	199.500^＊＊	78.163^＊＊

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