Tan Caixia, Li Chen, Ge Ruihan, Zhang Wei, Wu Ziyi, Wang Shengpeng, Cui Haotian, Wang Xinmin, Zhang Le
Department of Pathophysiology, Shihezi University School of Medicine, Shihezi, Xinjiang, China.
Xinjiang Key Laboratory of Endemic and Ethnic Diseases, Shihezi University School of Medicine, Shihezi, Xinjiang, China.
Cancer Cell Int. 2025 Feb 15;25(1):48. doi: 10.1186/s12935-025-03676-3.
Bacillus Calmette-Guérin (BCG) is the primary method of postoperative perfusion treatment for bladder cancer. The myeloid cell leukemia gene-1 (Mcl-1) is closely associated with the development of malignant tumors. Previous research by our group has demonstrated that downregulating Mcl-1 using shRNA can enhance the efficacy of BCG treatment in bladder cancer. This study aims to investigate the impact of Mcl-1 downregulation in combination with BCG treatment on bladder cancer, macrophage polarization, and the underlying mechanism of action, with the goal of reducing recurrence and metastasis in bladder cancer.
The GSE190529 dataset was analyzed to identify differential genes for enrichment analysis. The WGCNA algorithm was then employed to pinpoint gene modules closely associated with the Mcl-1 gene. The overlapping genes between these modules and the differentially expressed genes were subjected to enrichment analysis in GO and KEGG pathways to unveil crucial signaling pathways. In vitro experiments involved the co-culture of Raw264.7 macrophages and MB49 to establish a tumor microenvironment model, while in vivo experiments utilized an MNU-induced rat bladder cancer model. Various methods including Enzyme-Linked Immunosorbent Assay (ELISA), Western blot, immunofluorescence, HE staining, etc. were utilized to assess macrophage polarization and the expression of proteins linked to the ASK1/MKK7/JNK/cJUN signaling pathway.
Bioinformatics analysis indicates that the therapeutic mechanism of Mcl-1 in BCG treatment for bladder cancer may be linked to the Mitogen-Activated Protein Kinase (MAPK) signaling pathway. Both in vivo and in vitro experiments have demonstrated that the combination of BCG treatment and Mcl-1shRNA intervention results in elevated expression of M1 markers (TNF-α, CD86, INOS) and reduced expression of M2 markers (IL-10, CD206, Arg-1). Moreover, there was a notable increase in protein levels of P-ASK1, P-MKK7, P-JNK, P-cJUN, and CX43, leading to a significant rise in the apoptosis rate of bladder cancer cells and diminished proliferation, migration, and invasion capabilities. The expression of these markers can be reversed by employing the JNK signaling pathway inhibitor SP600125.
Down-regulation of Mcl-1 promotes the polarization of macrophages towards the M1 type through activation of the ASK1/MKK7/JNK signaling pathway. This enhances intercellular communication and improves the efficacy of BCG in bladder cancer treatment.
卡介苗（BCG）是膀胱癌术后灌注治疗的主要方法。髓样细胞白血病基因1（Mcl-1）与恶性肿瘤的发生发展密切相关。我们团队之前的研究表明，使用短发夹RNA（shRNA）下调Mcl-1可增强BCG治疗膀胱癌的疗效。本研究旨在探讨下调Mcl-1联合BCG治疗对膀胱癌、巨噬细胞极化及其潜在作用机制的影响，以降低膀胱癌的复发和转移。
分析GSE190529数据集以鉴定用于富集分析的差异基因。然后采用加权基因共表达网络分析（WGCNA）算法确定与Mcl-1基因密切相关的基因模块。对这些模块与差异表达基因之间的重叠基因进行基因本体论（GO）和京都基因与基因组百科全书（KEGG）通路的富集分析，以揭示关键信号通路。体外实验采用Raw264.7巨噬细胞与MB49共培养建立肿瘤微环境模型，体内实验采用N-甲基-N-亚硝基脲（MNU）诱导的大鼠膀胱癌模型。采用酶联免疫吸附测定（ELISA）、蛋白质免疫印迹法、免疫荧光、苏木精-伊红（HE）染色等多种方法评估巨噬细胞极化以及与凋亡信号调节激酶1（ASK1）/丝裂原活化蛋白激酶激酶7（MKK7）/c-Jun氨基末端激酶（JNK）/c-Jun信号通路相关蛋白的表达。
生物信息学分析表明，Mcl-1在BCG治疗膀胱癌中的作用机制可能与丝裂原活化蛋白激酶（MAPK）信号通路有关。体内和体外实验均表明，BCG治疗与Mcl-1 shRNA干预相结合可导致M1标志物（肿瘤坏死因子-α、CD86、诱导型一氧化氮合酶）表达升高，M2标志物（白细胞介素-10、CD206、精氨酸酶-1）表达降低。此外，P-ASK1、P-MKK7、P-JNK、P-c-Jun和连接蛋白43（CX43）的蛋白水平显著升高，导致膀胱癌细胞凋亡率显著上升，增殖、迁移和侵袭能力减弱。使用JNK信号通路抑制剂SP600125可使这些标志物的表达逆转。
下调Mcl-1通过激活ASK1/MKK7/JNK信号通路促进巨噬细胞向M1型极化。这增强了细胞间通讯并提高了BCG治疗膀胱癌的疗效。
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