皖南蝮蛇抑瘤组分-Ι通过调控RAI14抑制顺铂耐药胃癌细胞的增殖与侵袭

doi:10.12122/j.issn.1673-4254.2026.01.12

摘要/Abstract

摘要：

目的探讨皖南蝮蛇抑瘤组分-Ι（AHVAC-I）对顺铂耐药胃癌细胞增殖、侵袭能力的影响及其可能的作用机制。方法药物浓度递增法构建顺铂耐药胃癌细胞（MKN-28/DDP），细胞克隆实验和CCK-8实验观察AHVAC-I对MKN-28/DDP增殖能力的影响；将细胞分为对照组（用不含药物的培养基处理）和AHVAC-I处理组（含2、4、8 μg/mL AHVAC-I的培养基）。细胞划痕实验和Transwell实验检测AHVAC-I对MKN-28/DDP侵袭能力的影响；Western blot检测AHVAC-I对MKN-28/DDP上皮-间充质转化的影响；定量PCR与Western blotting实验检测视黄酸诱导蛋白14（RAI14）的表达水平。结果相较于对照组，2、4、8 μg/mL AHVAC-I可显著抑制MKN-28/DDP细胞的增殖能力与侵袭能力，差异有统计学意义（P<0.05）；与MKN-28细胞相比，MKN-28/DDP细胞RAI14的表达水平增加了2.15倍（P<0.01）；与对照组相比，1 μg/mL AHVAC-I处理后RAI14在MKN-28/DDP细胞的表达即降低了25%（P<0.01）；提高RAI14的表达可恢复因AHVAC-I而抑制的MKN-28/DDP细胞的增殖与侵袭。结论 AHVAC-I可通过下调RAI14的表达抑制胃癌顺铂耐药细胞的增殖与侵袭。

关键词: 皖南蝮蛇抑瘤组分-Ι, 胃癌, 顺铂耐药, 增殖, 侵袭, 视黄酸诱导蛋白14

Abstract:

Objective To evaluate the inhibitory effect of antitumor component-Ι in Agkistrodon halys venom (AHVAC-I) on proliferation and migration of cisplatin-resistant gastric cancer cells and explore the underlying mechanism. Methods Cisplatin-resistant MKN-28 (MKN-28/DDP) cells were obtained by continuous exposure of MKN-28 cells to stepwise-increasing concentrations of cisplatin. MKN-28/DDP cells were treated with different concentrations of AHVAC-I, and the changes in proliferation, migration and invasion of the cells were examined with colony-forming assay, CCK-8 assay wound-healing assay, and Transwell assay. Western blotting was performed to examine the effect of AHVAC-I on expressions of epithelial-mesenchymal transition (EMT) markers of MKN-28/DDP cells; the changes in protein and mRNA expression of retinoic acid induced 14 (RAI14) was detected with Western blotting and qRT-PCR. Results Treatment with 2, 4, and 8 μg/mL AHVAC-I significantly inhibited proliferative, migratory and invasion abilities and reduced the expressions of EMT markers in MKN-28/DDP cells. Compared with MKN-28 cells, MKN-28/DP cells showed an increased expression of RAI14, which was significantly lowered after treatment with AHVAC-I. Supplementation with exogenous RAI14 obviously attenuated the inhibitory effect of AHVAC-I on proliferation and migration of MKN-28/DDP cells. Conclusion AHVAC-I decreases proliferation and invasion of MKN-28/DDP cells by downregulating RAI14 expression.

Key words: Agkistrodon halys venom, gastric cancer, cisplatin resistance, invasion, proliferation, retinoic acid induced 14

李艳宇, 李晨, 戴传君, 郭润之, 韩浩宇, 卢林明, 周芳芳, 支慧. 皖南蝮蛇抑瘤组分-Ι通过调控RAI14抑制顺铂耐药胃癌细胞的增殖与侵袭[J]. 南方医科大学学报, 2026, 46(1): 113-121.

Yanyu LI, Chen LI, Chuanjun DAI, Runzhi GUO, Haoyu HAN, Linming LU, Fangfang ZHOU, Hui ZHI. Antitumor component-Ι in Agkistrodon halys venom inhibits proliferation and migration of cisplatin-resistant gastric cancer cells by downregulating RAI14[J]. Journal of Southern Medical University, 2026, 46(1): 113-121.

图/表 7

表1 AHVAC-I对MKN-28/DDP细胞的毒性作用

Tab.1 Toxic effects of AHVAC-I on MKN-28/DDP cells (Mean±SD, %)

Group	AHVAC-I (μg/mL)
Group	1	2	4	8	16	32
Cell viability	104.37±2.68	100.8±5.45	85.65±4.67	85.65±4.67	46.23±4.74	3.01±1.87
t	3.09	0.28	5.82	8.26	23.07	89.64
P	>0.05	>0.05	<0.05	<0.01	<0.001	<0.0001

图1 CCK8检测不同浓度AHVAC-I作用MKN-28/DDP细胞72 h后细胞增殖率变化

Fig.1 Cytotoxicity of AHVAC-I in MKN-28/DDP cells after treatment for 72 h. A: Cells viability assessed by CCK8 assay. B: Colony formation assay of MKN-28/DDP cells at 3 weeks after AHVAC-I treatment. C: Quantitative analysis of colonys in each group. Con: Blank control. n=5, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Control.

图2 AHVAC-I抑制MKN-28/DDP细胞迁移与侵袭

Fig.2 Inhibitory effects of AHVAC-I on migration and invasion of MKN-28/DDP cells. A: Wounding-healing assay for assessing migration ability of MKN-28/DDP cells (Original magnification:×40). B: Quantitative analysis of migration ability of MKN-28/DDP cells in each group. C: Transwell assay for assessing invasion ability of MKN-28/DDP cells (×100). D: Quantitative analysis of invading cells in each group, n=5, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Control.

图3 AHVAC-I抑制MKN-28/DDP细胞上皮间质化

Fig.3 AHVAC-I inhibits epithelial-mesenchymal transition (EMT) of MKN-28/DDP cells. A: Immunoblots of E-cad, N-cad, vimentin and snail in MKN-28/DDP cells treated with different concentrations of AHVAC-I. B-E: Protein expression levels of E-cad, N-cad, vimentin and snail, n=3, **P<0.01, ***P<0.001, ****P<0.0001 vs Control.

图4 AHVAC-I抑制MKN-28/DDP细胞RAI14表达

Fig.4 AHVAC-I down-regulates RAI14 expression in MKN-28/DDP cells. A: qRT-PCR for detecting RAI14 mRNA expression in MKN-28 cells and MKN-28/DDP cells (n=6, **P<0.01 vs MKN-28 cells). B: qRT-PCR of RAI14 expression in MKN-28/DDP cells treated with different concentrations of AHVAC-I (n=6, **P<0.01, ***P<0.001 vs blank Control). C: Immunoblots of RAI14, AKT and AKT-p in MKN-28/DDP cells treated with different concentrations of AHVAC-I. D: Protein expression levels of RAI14 in different groups (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Control.

图5 增加RAI14可有效抑制AHVAC-I对 MKN-28/DDP细胞增殖能力的抑制效果

Fig.5 RAI14 supplementation promotes proliferation of AHVAC-I-treated MKN-20/DDP (MKN-20/DDPAHVAC-I) cells. A: Immunoblots of AKT and AKT-p in MKN-20/DDPAHVAC-I cells treated with RAI14 (n=3). B, C: Colony-forming assay showing restored proliferation ability of MKN-20/DDPAHVAC-I cells after RAI14 treatment (n=5). **P<0.01 vs AHVAC-1.

图6 增加RAI14可有效抑制AHVAC-I对 MKN-28/DDP细胞迁移、侵袭能力的抑制效果

Fig.6 RAI14 supplementation promotes migration and invasion ability of MKN-20/DDPAHVAC-I cells. A: Migration ability of MKN-28/DDP cells analyzed by wounding-healing assay (×40). B: Quantitative analysis of migration ability of MKN-28/DDP cells. C: Invasion ability of MKN-28/DDP cells was analyzed by Transwell assay (×100). D: Quantitative analysis of number of invaded cells.n=5, *P<0.05, **P<0.01, ***P<0.001 vs AHVAC-I.

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