Ge Jing, Liu Sheng-Lu, Zheng Jing-Xiu, Shi Yu, Shao Ying, Duan Yu-Jing, Huang Rui, Yang Li-Jun, Yang Tao
Department of Biochemistry & Molecular Biology, Shanxi Medical University, Taiyuan, Shanxi 030001, China.
Department of Pharmacology, Shanxi Medical University, Taiyuan, Shanxi 030001, China.
Transl Oncol. 2023 Aug;34:101683. doi: 10.1016/j.tranon.2023.101683. Epub 2023 May 22.
ALKBH5 belongs to the ALKB family consists of a Fe (II) and a-ketoglutarate-dependent dioxygenase. ALKBH5 directly catalyzes the oxidative demethylation of m6A-methylated adenosine. ALKBH5 involves in tumorigenesis and tumor progression, and is often dysregulated in a wide range of cancers, including colorectal cancer. Emerging evidence indicates that the expression of ALKBH5 is associated with the abundance of infiltrating immune cells in the microenvironment. However, how ALKBH5 affects immune cell infiltration in the microenvironment in colorectal cancer (CRC) has not been reported. The aim of this study was to identify how the expression of ALKBH5 affects the biological behaviors of CRC cell lines and regulates the effects on infiltrating CD8 T cells in CRC microenvironment with its specific mechanism.
Firstly, the transcriptional expression profiles of CRC were downloaded from TCGA database and integrated via R software (4.1.2). Between CRC and normal colorectal tissues, ALKBH5 mRNA expressions were compared (Wilcoxon rank-sum). We further identified the expression levels of ALKBH5 in CRC tissues and cell lines through quantitative PCR, western blot, and immunohistochemistry. Then, how ALKBH5 affects the biological behaviors of CRC cells were confirmed by gain- and loss-of-function analysis. Furthermore, the relationship between ALKBH5 level and 22 tumor-infiltrating immune cells was examined through CIBERSORT in R software. Furthermore, we explored the correlation between ALKBH5 expression and tumor-infiltrated CD8, CD4 and regulatory T cells by utilizing the TIMER database. Finally, the association between chemokines and CD8 T cells infiltration in CRC was analyzed using GEPIA online database. qRT-PCR, WB and IHC were used to further determine the effect of ALKBH5 on NF-κB-CCL5 signaling axis and CD8 T cells infiltration.
Clinically, ALKBH5 expression was downregulated in CRC and low levels of ALKBH5 expression were correlated with poor overall survival (OS). Functionally, overexpression of ALKBH5 reduced the proliferation, migration and invasion of CRC cells, and vice versa. Overexpression of ALKBH5 suppresses NF-κB pathway, thus reduces CCL5 expression and promotes CD8 T cells infiltration in CRC microenvironment.
ALKBH5 is poorly expressed in CRC, and overexpression of ALKBH5 attenuates CRC malignant progression by inhibiting CRC cell proliferation, migration, invasion and promoting CD8 T cells infiltration in the tumor microenvironment through NF-κB-CCL5 axis.
ALKBH5属于ALKB家族，由一种依赖铁（II）和α-酮戊二酸的双加氧酶组成。ALKBH5直接催化m6A甲基化腺苷的氧化去甲基化。ALKBH5参与肿瘤发生和肿瘤进展，并且在包括结直肠癌在内的多种癌症中常常失调。新出现的证据表明，ALKBH5的表达与微环境中浸润免疫细胞的丰度相关。然而，ALKBH5如何影响结直肠癌（CRC）微环境中的免疫细胞浸润尚未见报道。本研究的目的是确定ALKBH5的表达如何影响CRC细胞系的生物学行为，并通过其特定机制调节对CRC微环境中浸润性CD8 T细胞的影响。
首先，从TCGA数据库下载CRC的转录表达谱，并通过R软件（4.1.2）进行整合。比较CRC组织与正常结直肠组织之间ALKBH5 mRNA的表达情况（Wilcoxon秩和检验）。我们通过定量PCR、蛋白质免疫印迹和免疫组织化学进一步确定ALKBH5在CRC组织和细胞系中的表达水平。然后，通过功能获得和功能缺失分析证实ALKBH5如何影响CRC细胞的生物学行为。此外，利用R软件中的CIBERSORT检测ALKBH5水平与22种肿瘤浸润免疫细胞之间的关系。此外，我们利用TIMER数据库探索ALKBH5表达与肿瘤浸润性CD8、CD4和调节性T细胞之间的相关性。最后，使用GEPIA在线数据库分析CRC中趋化因子与CD8 T细胞浸润之间的关联。采用qRT-PCR、WB和IHC进一步确定ALKBH5对NF-κB-CCL5信号轴和CD8 T细胞浸润的影响。
临床上，CRC中ALKBH5表达下调，且ALKBH5低表达与总体生存期（OS）差相关。在功能上，ALKBH5的过表达降低了CRC细胞的增殖、迁移和侵袭能力，反之亦然。ALKBH5的过表达抑制NF-κB通路，从而降低CCL5表达并促进CD8 T细胞在CRC微环境中的浸润。
ALKBH5在CRC中表达较低，ALKBH5的过表达通过抑制CRC细胞增殖、迁移、侵袭，并通过NF-κB-CCL5轴促进肿瘤微环境中CD8 T细胞浸润，从而减弱CRC的恶性进展。
