钾电压门控通道亚家族D成员1对胰腺癌放射抵抗的影响及其机制

The effect of potassium voltage-gated channel subfamily D member 1 on radioresistance in pancreatic cancer and its underlying mechanisms

摘要

目的: 探究钾离子通道蛋白钾电压门控通道亚家族D成员1(potassium voltage-gated channel subfamily D member 1,KCND1)在胰腺癌放射抵抗中的作用及其潜在机制。方法: 体外培养人胰腺癌PANC1细胞系,将其分为对照组(不照射)和X线照射组(8 Gy),照射48 h,采用转录组测序(RNA-seq)和实时荧光定量PCR检测细胞中钾离子通道相关mRNA表达水平;采用免疫荧光检测KCND1蛋白表达水平。利用CRISPR/Cas9技术敲除PANC1细胞中KCND1,将细胞分为sgControl、sgKCND1、sgControl+X线照射(8 Gy)、sgKCND1+X线照射(8 Gy)组,照射48 h,采用蛋白免疫印迹检测细胞内caspase-3和Bax蛋白表达;采用流式细胞术检测细胞内凋亡和活性氧水平;采用蛋白质免疫印迹和免疫荧光技术检测E-钙黏蛋白和N-钙黏蛋白表达水平;采用划痕迁移实验检测细胞迁移能力。取24只6~8周龄C57BL/6小鼠,于右侧背部皮下接种小鼠胰腺癌PANC02细胞,2周后随机分为4组,分别为对照组、X线照射组(5 Gy)、丙米嗪组(20 mg/kg)和X线照射(5 Gy)+丙米嗪组(20 mg/kg),每组6只;检测小鼠瘤体体积并比较;采用蛋白免疫印迹检测小鼠皮下瘤组织中抗凋亡蛋白和促凋亡蛋白以及E-钙黏蛋白和N-钙黏蛋白表达水平。结果: 与对照组相比,X线照射组细胞钾离子通道KCNN4、KCND1、KCNA7、KCNA5、KCNMA1、KCNJ14等mRNA表达水平显著升高(P<0.05或P<0.01),其中KCND1 mRNA最显著。与X线照射组相比,sgKCND1+X线照射组caspase-3和Bax蛋白表达水平显著升高(P<0.05或P<0.01),细胞凋亡比例明显升高(P<0.05或P<0.01),N-钙黏蛋白表达水平显著降低(P<0.01),E-钙黏蛋白表达水平明显升高(P<0.05),细胞划痕愈合率明显降低(P<0.01)。与X线照射组相比,丙米嗪+X线照射组皮下瘤体积显著减小,肿瘤重量明显减少(P<0.05)。蛋白免疫印迹结果显示,与X线照射组相比,X线照射+丙米嗪组抗凋亡蛋白Bcl-2表达水平显著降低(P<0.05),而促凋亡蛋白切割型caspase-3和Bax表达量则显著升高(P<0.05);此外,E-钙黏蛋白表达水平显著升高(P<0.01),N-钙黏蛋白表达水平显著降低(P<0.05)。结论: 钾离子通道KCND1可能通过促进胰腺癌细胞上皮间充质转化和凋亡抵抗特性,从而导致胰腺癌产生放射抵抗。

Abstract

Objective: To investigate the role of potassium voltage-gated channel subfamily D member 1 (KCND1) in pancreatic cancer radioresistance and its underlying mechanisms. Methods: Human pancreatic cancer cell line PANC1 was cultured in vitro and divided into control group (no irradiation) and X-ray irradiation group (8 Gy). After 48 hours of irradiation, mRNA expression levels of potassium channel-related genes were analyzed by RNA sequencing (RNA-seq) and real-time quantitative PCR; KCND1 protein expression was detected by immunofluorescence. CRISPR/Cas9 technology was used to knockout KCND1 in PANC1 cells, which were then divided into four groups: sgControl, sgKCND1, sgControl + X-ray (8 Gy), and sgKCND1 + X-ray (8 Gy). After 48 hours of irradiation, caspase-3 and Bax protein expression were assessed by Western blotting; apoptosis and reactive oxygen species (ROS) levels were measured by flow cytometry; E-cadherin and N-cadherin expression levels were evaluated using Western blotting and immunofluorescence; and cell migration ability was determined by scratch wound healing assay. Twenty-four 6-8-week-old C57BL/6 mice were subcutaneously inoculated with PANC02 cells on the right back. Two weeks later, mice were randomly assigned to four groups: control group, X-ray irradiation (5 Gy) group, imipramine (20 mg/kg) group, and X-ray irradiation (5 Gy) + imipramine (20 mg/kg) group, with six mice per group. Tumor volume and weight were measured and compared. Expression levels of anti-apoptotic and pro-apoptotic proteins, as well as E-cadherin and N-cadherin, were analyzed in subcutaneous tumor tissues by Western blotting. Results: Compared with the control group, mRNA expression levels of potassium channels including KCNN4, KCND1, KCNA7, KCNA5, KCNMA1, and KCNJ14 were significantly upregulated in the X-ray irradiation group (P<0.05 or P<0.01), among which KCND1 showed the most prominent upregulation. Compared with the X-ray irradiation group, the sgKCND1 + X-ray group showed significantly higher expression of caspase-3 and Bax proteins (P<0.05 or P<0.01), significantly reduced N-cadherin expression (P<0.01), markedly elevated E-cadherin expression (P<0.05), and a significantly lower scratch wound healing rate (P<0.01). Compared with the X-ray irradiation group, the X-ray + imipramine group exhibited significantly smaller tumor volumes and reduced tumor weights (P<0.05). Western blotting results showed that, compared with the X-ray group, the X-ray + imipramine group had significantly lower expression of the anti-apoptotic protein Bcl-2 (P<0.05), while pro-apoptotic proteins cleaved caspase-3 and Bax were significantly upregulated (P<0.05); additionally, E-cadherin expression was significantly increased (P<0.01), while N-cadherin expression was significantly decreased (P<0.05). Conclusion: The potassium channel KCND1 may contribute to radioresistance in pancreatic cancer by promoting epithelial-mesenchymal transition and enhancing apoptosis resistance in pancreatic cancer cells.

关键词

钾电压门控通道亚家族D成员1 / 胰腺癌 / 放射 / 钾离子 / 肿瘤微环境

Key words

potassium voltage-gated channel subfamily D member 1 / pancreatic cancer / radiotherapy / potassium ions / tumor microenvironment

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基金资助

镇江市科技计划社会发展项目(SH2024007)