RgpB通过抑制自噬小体与溶酶体融合避免Cx43降解参与食管癌化疗耐药

doi:10.12122/j.issn.1673-4254.2024.09.06

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (9): 1670-1676.doi: 10.12122/j.issn.1673-4254.2024.09.06

RgpB通过抑制自噬小体与溶酶体融合避免Cx43降解参与食管癌化疗耐药

杜越¹^,²(), 张秀森¹^,², 周克旭¹^,², 金星¹^,², 原翔¹^,²(), 高社干¹^,²()

^1.河南省微生态与食管癌防治重点实验室//河南省肿瘤表观遗传重点实验室，河南洛阳 471003
^2.河南科技大学第一附属医院//河南科技大学临床医学院，河南洛阳 471003

收稿日期:2024-05-28 出版日期:2024-09-20 发布日期:2024-09-30
通讯作者: 原翔,高社干 E-mail:duyue97612@163.com;yuanxiangdrive@163.com;gsg112258@163.com
作者简介:杜越，医师，在读硕士研究生，E-mail: duyue97612@163.com
基金资助:
国家自然科学基金(82073029);河南省科技攻关项目(242102310146)

RgpB contributes to chemoresistance in esophageal squamous cell carcinoma by preventing Cx43 degradation via inhibiting autophagosome-lysosome fusion

Yue DU¹^,²(), Xiusen ZHANG¹^,², Kexu ZHOU¹^,², Xing JIN¹^,², Xiang YUAN¹^,²(), Shegan GAO¹^,²()

^1.Henan Provincial Key Laboratory of Microecology and Esophageal Cancer Prevention and Treatment, Henan Provincial Key Laboratory of Tumor Epigenetics, Luoyang 471003, China
^2.First Affiliated Hospital/ School of Clinical Medicine, Henan University of Science and Technology, Luoyang 471003, China

Received:2024-05-28 Online:2024-09-20 Published:2024-09-30
Contact: Xiang YUAN, Shegan GAO E-mail:duyue97612@163.com;yuanxiangdrive@163.com;gsg112258@163.com
Supported by:
National Natural Science Foundation of China(82073029)

摘要/Abstract

摘要：

目的探讨牙龈卟啉单胞菌（Pg）的毒力因子RgpB在食管鳞癌化疗耐药中的作用机制。方法采用免疫沉淀-质谱联用技术筛选与Pg的毒力因子RgpB互作的自噬调节因子；运用免疫共沉淀实验探索RgpB和自噬调节因子TBC1D5之间的相互作用；采用蛋白免疫印迹法测定Pg感染对食管癌细胞膜受体分子Cx43表达的影响；使用细胞免疫荧光检测Lamp1、Cx43与TBC1D5的关系；利用自噬双荧光观察Pg感染对自噬小体与溶酶体融合的影响；使用平板克隆及CCK-8法检测Pg感染及Cx43抑制剂在使用化疗药后对食管癌细胞系增殖作用的影响。结果免疫沉淀-质谱联用技术筛选出与RgpB互作的自噬调节因子TBC1D5；免疫共沉淀结果显示，Pg分泌的毒力因子 RgpB可与自噬调节因子TBC1D5直接结合并相互作用；蛋白免疫印迹法结果显示，感染Pg后食管癌细胞膜Cx43表达量高于未感染组（P<0.05）；细胞免疫荧光结果显示，在感染Pg后，Cx43在细胞膜表面的表达量增加（P<0.05）；stubRFP-sensGFP-LC3自噬双荧光结果显示，在Pg感染时，自噬体与溶酶体融合受到阻断；平板克隆及CCK-8法结果显示，使用化疗药后，Cx43抑制剂能够逆转Pg感染对食管癌细胞系的促进作用。结论 Pg入侵食管癌，其毒力因子RgpB阻碍自噬小体与溶酶体融合，从而使膜受体分子Cx43免受溶酶体降解，导致食管癌化疗耐药。

关键词: 食管鳞状细胞癌, 牙龈卟啉单胞菌, RgpB, Cx43, 化疗耐药

Abstract:

Objective To investigate the mechanism through which RgpB, a virulence factor of Porphyromonas gingivalis (Pg), induces chemoresistance in esophageal squamous carcinoma. Methods The autophagy-regulating factors that interact with RgpB were screened by immunoprecipitation-mass spectrometry. The interaction between RgpB and the autophagy regulator TBC1D5 was investigated using co-immunoprecipitation. The impact of Pg infection on the expression of esophageal cancer cell membrane receptor molecule Cx43 was assessed using Western blotting. Immunofluorescence assay was used to analyze the relationship among Lamp1, Cx43 and TBC1D5. The effect of Pg infection on autophagosome-lysosome fusion was evaluated using autophagy double fluorescence technique. The effects of Pg infection and a Cx43 inhibitor on proliferation of esophageal cancer cells after chemotherapy were examined with plate cloning assay and CCK-8 method. Results Immunoprecipitation-mass spectrometry identified TBC1D5 as an autophagy regulator interacting with RgpB, and co-immunoprecipitation suggested that RgpB could directly bind to TBC1D5. In Pg-infected esophageal cancer cells, the expression of Cx43 on the cell membrane was significantly higher than that in non-infected cells. Immunofluorescence assay showed that the expression of Cx43 on the membrane of esophageal cancer cells increased significantly after Pg infection, which blocked autophagosome-lysosome fusion as shown by stubRFP-sensGFP-LC3 lentivirus study. Plate cloning assay and CCK-8 assay showed that the Cx43 inhibitor significantly attenuated the effect of Pg infection for promoting proliferation of esophageal cancer cells after chemotherapy. Conclusion Pg infection in esophageal cancer blocked autophagosome-lysosome fusion in the tumor cells, thereby preventing Cx43 from lysosomal degradation and leading to chemoresistance of esophageal cancer.

Key words: esophageal squamous cell carcinoma, Porphyromonas gingivalis, RgpB, Cx43, chemotherapy resistance

杜越, 张秀森, 周克旭, 金星, 原翔, 高社干. RgpB通过抑制自噬小体与溶酶体融合避免Cx43降解参与食管癌化疗耐药[J]. 南方医科大学学报, 2024, 44(9): 1670-1676.

Yue DU, Xiusen ZHANG, Kexu ZHOU, Xing JIN, Xiang YUAN, Shegan GAO. RgpB contributes to chemoresistance in esophageal squamous cell carcinoma by preventing Cx43 degradation via inhibiting autophagosome-lysosome fusion[J]. Journal of Southern Medical University, 2024, 44(9): 1670-1676.

图/表 8

图1 RgpB免疫沉淀的银染结果

Fig.1 Silver staining results of RgpB immunoprecipitation.

图2 RgpB相关蛋白质互作网络图

Fig.2 Diagram of RgpB-related protein interaction network.

图3 RgpB与TBC1D5之间的相互作用

Fig.3 Interaction between RgpB and TBC1D5. A, B: Co-IP experiments demonstrating an interaction between both RgpB and TBC1D5.

图4 Western blotting检测Cx43免疫沉淀结果

Fig.4 Immunoprecipitation results of Cx43 detected by Western blotting. A: Effect of Pg infection and TBC1D5 knockdown on Cx43 protein expression in KYSE150 cells. B: Quantitative analysis of Western blotting results. ***P<0.001.

图5 免疫细胞荧光检测Pg感染的KYSE150细胞Lamp1、Cx43与TBC1D5表达变化情况

Fig.5 Changes in Lamp1, Cx43 and TBC1D5 expressions in Pg-infected KYSE150 cells detected by immunocytofluorescence staining (Scale bar=10 μm).

图6 Pg感染对自噬小体与溶酶体融合的作用

Fig.6 Effect of Pg infection on fusion of autophagosomes and lysosomes in KYSE150 cells. A: Transfection efficiency of stubRFP-sensGFP-LC3 in KYSE150 cells by fluorescence detection. B: Changes in autophagosome-lysosome fusion after Pg infection in stubRFP-sensGFP-LC3-KYSE150 cells detected by laser confocal microscopy (Scale bar=10 μm).

图7 平板克隆实验检测Pg感染及加入Cx43抑制剂对KYSE150细胞增殖的影响

Fig.7 Effects of Pg infection and addition of Cx43 inhibitor on proliferation of KYSE150 cells examined by plate cloning assay.

图8 Pg感染对KYSE150细胞增殖的影响及Cx43抑制剂的逆转作用

Fig.8 Effect of Pg infection and Cx43 inhibitor on the proliferation of KYSE150 cells. ***P<0.001.

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RgpB通过抑制自噬小体与溶酶体融合避免Cx43降解参与食管癌化疗耐药

RgpB contributes to chemoresistance in esophageal squamous cell carcinoma by preventing Cx43 degradation via inhibiting autophagosome-lysosome fusion

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