沉默KRT17抑制人食管鳞癌细胞系KYSE-150增殖

doi:10.16352/j.issn.1001-6325.2025.12.1548

摘要/Abstract

摘要： 目的探究角蛋白17(KRT17)对人食管鳞癌细胞系KYSE-150增殖的作用和机制。方法用GEPIA2网站分析KRT17表达水平与食管鳞癌分期和患者生存期相关性。将人食管鳞癌细胞系KYSE-150分为对照组、si-NC组、si-KRT17组和激活剂组。将si-NC和si-KRT17分别转染至si-NC组和si-KRT17组细胞,激活剂组细胞转染si-KRT17且培养基中加入终浓度为30 μmol/L的PI3K/AKT/mTOR通路激活剂740 Y-P处理。用CCK-8法检测细胞增殖,集落形成实验检测细胞集落形成。用TUNEL染色检测细胞凋亡。用流式细胞术检测细胞周期。用Transwell实验检测细胞侵袭。用试剂盒检测细胞上清液中葡萄糖、乳酸和丙酮酸含量。用qRT-PCR检测KRT17基因表达。用Western blot检测KRT17、GLUT1、PDK1和LDHA、p-PI3K,PI3K,p-AKT,AKT,p-mTOR和mTOR蛋白质表达。结果 KRT17在食管鳞癌组织中表达量高于正常组织(P＜0.05),KRT17的表达水平与食管鳞癌分期相关(P＜0.05),KRT17低表达患者的生存预后好于KRT17高表达患者(P＜0.05)。与对照组或si-NC组比较,si-KRT17组KRT17 mRNA和蛋白表达水平均降低(P＜0.05),细胞增殖活力、集落形成数、迁移和侵袭细胞数均降低(P＜0.05),乳酸含量、GLUT1、PDK1和LDHA蛋白表达水平均降低(P＜0.05),p-PI3K/PI3K、p-AKT/AKT和p-mTOR/mTOR值均降低(P＜0.05),G₀/G₁期细胞比例、TUNEL阳性率、葡萄糖和丙酮酸含量均升高(P＜0.05)。与si-KRT17组比较,激活剂组KRT17 mRNA和蛋白表达水平均升高(P＜0.05),细胞增殖、集落形成、迁移和侵袭细胞数量均升高(P＜0.05),乳酸含量、GLUT1、PDK1和LDHA蛋白表达水平均升高(P＜0.05),p-PI3K/PI3K、p-AKT/AKT和p-mTOR/mTOR值均升高(P＜0.05),G₀/G₁期细胞比例、TUNEL阳性率、葡萄糖和丙酮酸含量均降低(P＜0.05)。结论 KRT17在人食管鳞癌组织和细胞系KYSE-150中高表达,沉默KRT17抑制人食管鳞癌细胞系KYSE-150增殖、迁移和侵袭,并促进细胞凋亡和细胞周期阻滞。

关键词: 角蛋白17(KRT17), 食管鳞癌, PI3K/AKT/mTOR通路, 糖酵解

Abstract: Objective To explore the effect and mechanism of keratin 17 (KRT17) on proliferation of human esophageal squamous cell line KYSE-150. Methods The correlation of KRT17 expression with the disease stage and survival of patients with esophageal squamous cell carcinoma was analyzed by GEPIA2 website. Human esophageal squamous cell line KYSE-150 was divided into control group,si-NC group,si-KRT17 group and activator group. Small interfering RNA of si-NC and si-KRT17 were transfected into cells of si-NC group and si-KRT17 group,respectively. Cells in the activator group were transfected with si-KRT17 and treated with 740 Y-P(PI3K/AKT/mTOR pathway activator) with a final concentration of 30 μmol/L in the medium. The cell proliferation was detected by CCK-8 assay. The clonal formation was detected by clonal formation experiment. The apoptosis was detected by TUNEL staining. The cell cycle was detected by flow cytometry. The cell migration and invasion were detected by Transwell assay. The contents of glucose, lactic acid and pyruvate in cell supernatant were detected by commercially available kits. The expression of KRT17 mRNA was detected by qRT-PCR. And the expression of KRT17,GLUT1,PDK1 and LDHA,p-PI3K,PI3K,p-AKT,AKT,p-mTOR and mTOR protein were detected by Western blot. Results The expression level of KRT17 in esophageal squamous cell carcinoma tissues was significantly higher than that in normal tissues(P＜0.05). There was a statistically significant correlation between the expression level of KRT17 and the stage of esophageal squamous cell carcinoma (P＜0.05). The survival prognosis of patients with low KRT17 expression was better than that of patients with high KRT17 expression (P＜0.05). Compared with control group or si-NC group,the mRNA and protein expression of KRT17 in si-KRT17 group were decreased (P＜0.05),and the cell proliferation activity,number of clone formation,migration and invasion cells were decreased (P＜0.05). And the lactic acid content,protein expression levels of GLUT1,PDK1 and LDHA were decreased (P＜0.05),values of p-PI3K/PI3K,p-Akt/AKT and p-mTOR/mTOR were decreased (P＜0.05). The proportion of cells in G₀/G₁ phase,TUNEL positive rate,and contents of glucose and pyruvate were increased(P＜0.05). Compared with si-KRT17 group,the mRNA and protein expression levels of KRT17 in activator group were increased (P＜0.05),and the cell proliferation activity,number of clone formation,migration and invasion cells were increased (P＜0.05). And the lactic acid content,protein expression levels of GLUT1,PDK1 and LDHA were increased (P＜0.05),the values of p-PI3K/PI3K,p-Akt/AKT and p-mTOR/mTOR were increased (P＜0.05). The proportion of cells in G₀/G₁ phase,TUNEL positive rate,and contents of glucose and pyruvate were decreased (P＜0.05). Conclusions KRT17 is highly expressed in esophageal squamous cell carcinoma tissues and cells. Silencing KRT17 inhibits proliferation,migration and invasion of human esophageal squamous cell line KYSE-150,and promotes apoptosis and cell cycle arrest.

Key words: keratin 17 (KRT17), esophageal squamous cell carcinoma, PI3K/AKT/mTOR pathway, glycolysis

中图分类号:

R735.1

张卉, 甘世保, 李慧, 周嘉逊, 赵梦琪. 沉默KRT17抑制人食管鳞癌细胞系KYSE-150增殖[J]. 基础医学与临床, 2025, 45(12): 1548-1556.

ZHANG Hui, GAN Shibao, LI Hui, ZHOU Jiaxun, ZHAO Mengqi. Silencing KRT17 inhibits proliferation of human esophageal squamous cell line KYSE-150[J]. Basic & Clinical Medicine, 2025, 45(12): 1548-1556.

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