异牡荆素通过miR-339-5p/HSPA8轴调节胰腺癌细胞的生物学行为

doi:10.11958/20242056

天津医药 ›› 2025, Vol. 53 ›› Issue (3): 230-235.doi: 10.11958/20242056

异牡荆素通过miR-339-5p/HSPA8轴调节胰腺癌细胞的生物学行为

闫玲新(), 李森, 郭改莉, 孟婉秋, 徐超()

邯郸市中心医院消化内科一病区（邮编056000）

收稿日期:2024-12-02 修回日期:2025-01-20 出版日期:2025-03-15 发布日期:2025-03-31
通讯作者: △E-mail：xzgibrca@163.com
作者简介:闫玲新（1988），女，主治医师，主要从事幽门螺杆菌感染、胃炎、胰腺炎等消化系统疾病的诊治方面研究。E-mail：x96onx@163.com
基金资助:
邯郸市科学技术研究与发展计划项目(23422083233)

Isovitexin regulates proliferation, migration and invasion of pancreatic cancer cells via the miR-339-5p/HSPA8 axis

YAN Lingxin(), LI Sen, GUO Gaili, MENG Wanqiu, XU Chao()

First Ward of Gastroenterology Department, Handan Central Hospital, Handan 056000, China

Received:2024-12-02 Revised:2025-01-20 Published:2025-03-15 Online:2025-03-31
Contact: △E-mail：xzgibrca@163.com

闫玲新, 李森, 郭改莉, 孟婉秋, 徐超. 异牡荆素通过miR-339-5p/HSPA8轴调节胰腺癌细胞的生物学行为[J]. 天津医药, 2025, 53(3): 230-235.

YAN Lingxin, LI Sen, GUO Gaili, MENG Wanqiu, XU Chao. Isovitexin regulates proliferation, migration and invasion of pancreatic cancer cells via the miR-339-5p/HSPA8 axis[J]. Tianjin Medical Journal, 2025, 53(3): 230-235.

摘要/Abstract

摘要：

目的探究异牡荆素（Isov）对胰腺癌细胞的生物学行为的影响及作用机制。方法采用Isov处理人正常胰腺导管上皮细胞HPDE和PC细胞系；CCK-8检测细胞存活率并计算半数抑制浓度（IC₅₀）。将PC细胞系PANC-1细胞依次分为Control组、Isov组、Isov+in-miR-NC组、Isov+in-miR-339-5p组、Isov+in-miR-339-5p+si-NC组和Isov+in-miR-339-5p+si-HSPA8组。CCK-8、划痕愈合实验和Transwell实验检测PANC-1细胞存活、迁移和侵袭情况；实时荧光定量PCR检测PANC-1细胞中miR-339-5p和热休克蛋白家族A成员8（HSPA8）mRNA表达情况；Western blot法检测各组细胞中蛋白HSPA8的表达情况；双萤光素酶报告基因检测miR-339-5p与HSPA8的靶向作用；异种移植裸鼠模型验证Isov的体内抗癌作用。结果 Isov可抑制PC细胞增殖，但对HPDE细胞几乎没有细胞毒性。Isov可显著降低PANC-1细胞存活率和划痕愈合率，减少侵袭细胞数量，上调miR-339-5p表达，下调HSPA8 mRNA和蛋白水平（P<0.05），而这种作用可被下调miR-339-5p阻断（P<0.05）。HSPA8是miR-339-5p的靶标基因，敲低HSPA8后逆转了Isov对PANC-1细胞恶性生物学行为的调控作用（P<0.05）。体内研究证实，Isov治疗后，裸鼠肿瘤体积和质量减小，且miR-339-5p表达升高，HSPA8 mRNA表达降低（P<0.05）。结论 Isov可能通过miR-339-5p/HSPA8轴抑制PC细胞的增殖、迁移和侵袭。

关键词: 牡荆素, 胰腺肿瘤, 细胞增殖, HSPA8, miR-339-5p

Abstract:

Objective To explore the biological behavior and mechanism of isovitexin (Isov) on pancreatic cancer cells. Methods Isov was used to treat the human normal pancreatic ductal epithelial cells HPDE and PC cell lines, and CCK-8 was used to detect the cell proliferation and calculate the half inhibitory concentration (IC₅₀). The PC cell line PANC-1 cells were grouped into the control group, the Isov group, the Isov+in-miR-NC group, the Isov+in-miR-339-5p group, the Isov+in-miR-339-5p+si-NC group and the Isov+in-miR-339-5p+si-HSPA8 group. The survival, migration and invasion of PANC-1 cells were detected by CCK-8, scratch healing assay and Transwell assay. Real time fluorescence quantitative PCR was used to detect the mRNA expression of miR-339-5p and heat shock protein family A member 8 (HSPA8) in PANC-1 cells. Western blot assay was used to detect protein HSPA8 expression in various groups of cells. Dual luciferase reporter gene was used to detect the targeting effect of miR-339-5p and HSPA8. A xenograft nude mouse model was used to determine the in vivo anticancer effects of Isov. Results Isov inhibited PC cell proliferation but had little cytotoxicity to HPDE cells. Isov could obviously reduce the survival rate and scratch healing rate of PANC-1 cells, reduce the number of invasive cells, up-regulate miR-339-5p expression and down-regulate HSPA8 mRNA and protein levels (P<0.05), while these effects were blocked by down-regulated miR-339-5p (P<0.05). In addition, HSPA8 was the target gene of miR-339-5p, and knockdown of HSPA8 reversed the regulatory effect of Isov on the malignant biological behavior of PANC-1 cells. In vivo studies confirmed that after Isov treatment, the tumor volume and weight of nude mice decreased, the expression of miR-339-5p was increased and the expression of HSPA8 mRNA was decreased (P<0.05). Conclusion Isov may inhibit the proliferation, migration and invasion of PC cells through the miR-339-5p/HSPA8 axis.

Key words: vitexin, pancreatic neoplasms, cell proliferation, HSPA8, miR-339-5p

中图分类号:

R735.9

图/表 9

表1 HPDE和各组PC细胞在不同浓度Isov下的存活率比较（n=3，%，$\bar{x} \pm s$）

Tab.1 Comparison of survival rate of HPDE and PC cells between different Isov concentration groups

细胞类型	0 mmol/L	5 mmol/L	10 mmol/L	20 mmol/L	40 mmol/L	F
HPDE	100.00±0.00	108.36±9.79	104.37±8.76	98.65±7.65	92.32±8.15	1.853
AsPC-1	100.00±0.00	87.65±7.12	72.24±5.67^ABe	59.23±4.69^ABCe	43.12±4.62^ABCDe	60.181^＊＊
PANC-1	100.00±0.00	83.32±8.24^A	67.15±7.05^ABe	52.12±5.08^ABCe	35.18±4.13^ABCDe	60.480^＊＊
SW1990	100.00±0.00	89.17±8.25	75.24±6.80^Ae	62.13±5.62^ABe	47.12±4.23^ABCe	40.493^＊＊
F		5.237^＊＊	16.440^＊＊	37.790^＊＊	64.791^＊＊

图1 划痕愈合实验检测Isov对PC细胞迁移的影响（×200） A—F分别为Control组、Isov组、Isov+in-miR-NC组、Isov+in-miR-339-5p组、Isov+in-miR-339-5p+si-NC组、Isov+in-miR-339-5p+si-HSPA8组。

Fig.1 Scratch healing experiment detecting the effect of Isov on PC cell migration (×200)

图2 Transwell检测Isov对PC细胞侵袭的影响（结晶紫染色，×200）

Fig.2 Transwell detection of the effect of Isov on PC cell invasion (crystal violet staining, ×200)

图3 各组PC细胞中HSPA8蛋白表达 A—F依次为Control组、Isov组、Isov+in-miR-NC组、Isov+in-miR-339-5p组、Isov+in-miR-339-5p+si-NC组和Isov+in-miR-339-5p+si-HSPA8组。

Fig.3 HSPA8 protein expression in PC cells of each group

表2 Isov对PC细胞增殖、迁移和侵袭的影响（n=3，$\bar{x} \pm s$）

Tab.2 Effects of Isov on proliferation, migration and invasion of PC cells

组别	细胞存活率/%	划痕愈合率/%	侵袭细胞数量/个	miR-339-5p	HSPA8 mRNA	HSPA8蛋白
Control组	100.00±0.00	68.54±6.25	159.65±12.36	1.02±0.05	1.03±0.04	0.85±0.05
Isov组	54.32±5.23^a	21.15±2.45^a	56.26±5.64^a	2.39±0.36^a	0.32±0.03^a	0.20±0.03^a
Isov+in-miR-NC组	51.36±4.62	23.36±3.05	61.23±6.28	2.05±0.30	0.30±0.02	0.25±0.04
Isov+in-miR-339-5p组	75.23±7.36^b	48.36±4.12^b	97.54±10.13^b	1.62±0.14^b	0.68±0.04^b	0.62±0.05^b
Isov+in-miR-339-5p+si-NC组	80.13±6.25	51.23±5.03	108.65±9.54	1.70±0.15	0.70±0.06	0.65±0.04
Isov+in-miR-339-5p+si-HSPA8组	63.12±5.45^c	37.54±3.25^c	81.36±7.65^c	1.73±0.16	0.50±0.08^c	0.40±0.05^c
F	34.980^＊＊	54.791^＊＊	54.402^＊＊	13.070^＊＊	94.071^＊＊	99.850^＊＊

图4 生物信息学预测miR-339-5p与HSPA8之间的结合序列图

Fig.4 Bioinformatics prediction of the binding sequence between miR-339-5p and HSPA8

表3 2组双萤光素酶活性结果比较（n=3，$\bar{x} \pm s$）

Tab.3 Comparison of results of double luciferase activity between two groups

组别	HSPA8-WT	HSPA8-MUT
miR-NC组	1.00±0.06	1.02±0.05
miR-339-5p组	0.38±0.04	0.95±0.07
t	14.891^＊＊	1.409

图5 2组裸鼠异种移植肿瘤生长情况

Fig.5 Xenograft tumor growth in 2 groups of nude mice

表4 Isov对裸鼠肿瘤生长的影响（n=5，$\bar{x} \pm s$）

Tab.4 Effects of Isov on tumor growth in nude mice

组别	肿瘤质量/g	肿瘤体积/mm³	miR-339-5p	HSPA8 mRNA
Control组	2.15±0.20	1 450.36±150.23	1.03±0.09	1.02±0.07
Isov组	1.24±0.13	682.36±68.25	2.15±0.24	0.45±0.05
t	6.608^＊＊	8.062^＊＊	7.568^＊＊	11.480^*＊＊

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异牡荆素通过miR-339-5p/HSPA8轴调节胰腺癌细胞的生物学行为

Isovitexin regulates proliferation, migration and invasion of pancreatic cancer cells via the miR-339-5p/HSPA8 axis

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