| 张钰杰,尹玉施,何翔,等.分子伴侣介导的自噬通过降解KRT6B蛋白促进结肠癌细胞辐射抗性研究[J].中华放射医学与防护杂志,2026,46(2):176-182.Zhang Yujie,Yin Yushi,He Xiang,et al.Mechanisms of chaperon-mediated autophagy to promote radioresistance of colon cancer cells by degrading protein KRT6B[J].Chin J Radiol Med Prot,2026,46(2):176-182 |
| 分子伴侣介导的自噬通过降解KRT6B蛋白促进结肠癌细胞辐射抗性研究 |
| Mechanisms of chaperon-mediated autophagy to promote radioresistance of colon cancer cells by degrading protein KRT6B |
| 投稿时间：2025-09-25 |
| DOI：10.3760/cma.j.cn112271-20250925-00344 |
| 中文关键词: 自噬 辐射抵抗 结肠癌 KRT6B |
| 英文关键词:Autophagy Radioresistance Colon cancer KRT6B |
| 基金项目:国家自然科学基金(82574034);浙江省自然科学基金(LMS25H220002) |
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| 中文摘要: |
| 目的 探讨分子伴侣介导的自噬调控结肠癌辐射敏感性的机制。方法 使用结肠癌SW480细胞系,在8 Gy γ射线照射4、8、12、24、48 h后,利用免疫印迹法检测细胞中CMA活性蛋白LAMP2、HSPA8的表达变化;蛋白质谱分析筛选CMA潜在的调控蛋白KRT6B;分别转染LAMP2、HSPA8及KRT6B过表达质粒,检测过表达对照Vector组、过表达LAMP2、HSPA8组后KRT6B的表达水平;台盼蓝染色法检测细胞死亡率;采用克隆形成实验检测细胞辐射敏感性;免疫共沉淀(CO-IP)检测KRT6B与HSPA8、LAMP2的相互作用及结合位点。结果 8 Gy电离辐射促进SW480细胞中CMA关键蛋白LAMP2表达(t=3.15~3.72,P < 0.05),HSPA8表达未见显著变化;蛋白质组学分析显示,KRT6B在沉默LAMP2后表达上调最显著,且在8 Gy电离辐射后表达降低;与NC组15.8%相比,过表达KRT6B显著增加电离辐射诱导的细胞死亡至32.5%(t=9.42,P < 0.05),沉默KRT6B则减少细胞死亡至9.86%(t=3.73,P< 0.05),并被LAMP2的沉默部分逆转至16.39%(t=6.18,P< 0.05)。克隆形成实验进一步证实,过表达KRT6B能够显著提高细胞的辐射敏感性(F=179.70,P<0.001);经CO-IP实验证实,KRT6B可与LAMP2、HSPA8发生直接相互作用,且电离辐射进一步加强这种结合;血清饥饿处理或过表达LAMP2、HSPA8均可显著抑制KRT6B的蛋白表达。此外,构建KRT6B与HSPA8结合位点的突变体质粒并进行CO-IP分析,表明HSPA8与KRT6B的结合位点位于204~208位氨基酸残基区域。结论 电离辐射促进结肠癌细胞中CMA的活性增加,导致KRT6B蛋白被降解,进而降低SW480结肠癌细胞的辐射敏感性。 |
| 英文摘要: |
| Objective To explore the mechanisms by which chaperone-mediated autophagy (CMA) regulates the radiosensitivity of colon cancer cells. Methods Colon cancer cell line SW480 was selected and exposed to γ-ray irradiation at a dose of 8 Gy. At 4, 8, 12, 24, and 48 h post-irradiation, changes in the expression of CMA activity proteins, i.e., lysosome-associated membrane protein type 2A (LAMP2) and heat shock 70 kDa protein 8 (HSPA8), were detected using the Western blotting technique. Then, keratin 6B (KRT6B), a potential regulatory protein of CMA, was selected using proteomic mass spectrometry. Following the transfection of the overexpression plasmids of LAMP2, HSPA8, and KRT6B, the expression levels of KRT6B in the Vector control group, LAMP2-overexpression group, and HSPA8-overexpression group were detected. The mortality rate of cells was determined through trypan blue staining, and their radiosensitivity was detected using colony formation assays (CFAs). The interaction and binding sites of KRT6B with HSPA8 and LAMP2 were examined through co-immunoprecipitation (CO-IP) experiments. Results Ionizing radiation at a dose of 8 Gy promoted the expression of LAMP2, a key CMA protein in cells SW480 (t=3.15-3.72, P<0.05), while no significant changes were observed in the expression of HSPA8. Proteomic analysis indicates that KRT6B was up-regulated the most significantly following LAMP2 silencing and was down-regulated after ionizing radiation. Compared to the normal control (NC) group(15.8%), the ionizing radiation-induced cell mortality rate was significantly increased to 32.5% (t=9.42, P<0.05) by KRT6B overexpression. In contrast, the KRT6B silencing significantly reduced the cell mortality rate to 9.86% (t=3.73, P<0.05), which was partially reversed to 16.39% (t=6.18, P<0.05) by LAMP2 silencing. CFAs further confirmed that the KRT6B overexpression significantly increased the radiosensitivity of cells SW480 (F=179.70, P<0.001). CO-IP experiments verified that KRT6B could directly interact with LAMP2 and HSPA8, with ionizing radiation further enhancing their interactions. Both serum starvation and the LAMP2 and HSPA8 overexpression significantly inhibited the KRT6B expression. In addition, CO-IP analysis of mutant plasmids suggested that the binding sites of HSPA8 and KRT6B were located in the amino acid residues 204-208. Conclusions Ionizing radiation can enhance the CMA activity in colon cancer cells, leading to the degradation of protein KRT6B and further reducing the radiosensitivity of colon cancer cells SW480. |
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