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Title: [The influence of Ras-associated binding protein 23 knockdown on the migration and invasion of esophageal squamous cell carcinoma cells and its mechanism].
Author: Ma G, Liang H, Zhang RP, Sun Y.
Journal: Zhonghua Zhong Liu Za Zhi; 2024 Feb 23; 46(2):108-117. PubMed ID: 38418184.
Abstract:
Objective: To investigate the role and the mechanism of Ras-associated binding protein23 (RAB23) in the migration and invasion of esophageal squamous cell carcinoma (ESCC) cells. Methods: RAB23 mRNA levels were measured in 16 pairs of ESCC and adjacent normal tissues via real-time polymerase chain reactions. RAB23 mRNA levels in the ESCC and adjacent normal tissues of dataset GSE20347 deposited in the Gene Expression Omnibus (GEO) database were also analyzed. Immunohistochemistry (IHC) was used to detect the RAB23 protein expressions in 106 pairs of ESCC and adjacent normal tissues, as well as in the lymph glands and primary tumor tissues of 33 patients with positive lymph nodes and 10 patients with negative lymph nodes. Endogenous RAB23 expression was transiently depleted using siRNAs (si-NC, si-RAB23-1, and si-RAB23-9) or stably reduced using shRNAs (sh-NC and sh-RAB23) in ESCC KYSE30 and KYSE150 cells, and the knockdown efficiency was tested using Western blot assays. Cell counting kit-8 assays and mouse xenograft models were used to test the proliferation of ESCC cells. Transwell assays and tail vein-pulmonary metastasis models in immunocompromised mice were used to examine the migration and invasion of ESCC cells. Cell adhesion assays were used to test the adhesion of ESCC cells. RNA-seq assays were used to analyze how RAB23 knockdown influenced the expression profile of ESCC cells and the implicated signal pathways were confirmed using Western blot assays. Results: The RAB23 mRNA expression in 16 cases of ESCC tissues was 0.009 7±0.008 9, which was markedly higher than that in adjacent normal tissues (0.003 2±0.003 7, P=0.006). GEO analysis on RAB23 expressions in ESCC and adjacent normal tissues showed that the RAB23 mRNA level in ESCC tissues (4.30±0.25) was remarkably increased compared with their normal counterparts (4.10±0.17, P=0.037). Among the 106 pairs of ESCC and tumor-adjacent normal tissues, 51 cases exhibited low expression of RAB23 and 55 cases showed high expression of RAB23, whereas in the paired tumor-adjacent normal tissues 82 cases were stained weakly and 24 strongly for RAB23 protein. These results indicated that RAB23 expression was markedly increased in ESCC tissues (P<0.001). Additionally, only 1 out of 33 primary ESCC tissues with positive lymph nodes showed low RAB23 protein expression. On the other hand, 7 samples of primary ESCC tissues with negative lymph nodes were stained strongly for RAB23 while its level in the other 3 samples was weak. These results showed that RAB23 expression was remarkably increased in primary ESCC tissues with positive lymph nodes compared with those with negative lymph nodes (P=0.024). Further tests showed that 32 out of 33 positive lymph nodes were stained strongly for RAB23, whereas no negative lymph nodes (n=10) exhibited high expression of RAB23 (P<0.001). Both transient and stable knockdown of endogenous RAB23 expression failed to cause detectable changes in the proliferation of KYSE30 cells in vitro and in vivo, but attenuated the migration and invasion of KYSE30 cells as well as the invasion of KYSE150 cells. RAB23 knockdown was found to significantly decrease the number of adhesive KYSE30 cells in the sh-RAB23 group (313.75±89.34) compared with control cells in the sh-NC group (1 030.75±134.29, P<0.001). RAB23 knockdown was also found to significantly decrease the number of adhesive KYSE150 cells in the sh-RAB23 group (710.5±31.74) compared with the number of control cells in the sh-NC group (1 005.75±61.09, P<0.001). RNA-seq assays demonstrated that RAB23 knockdown using two siRNAs targeting RAB23 mRNA markedly impaired focal adhesion-related signal pathways, and decreased the levels of phosphorylated FAK (p-FAK) and phosphorylated paxillin (p-paxillin) in KYSE30 and KYSE150 cells. Conclusions: Significantly increased RAB23 in ESCC tissues positively correlates with lymph node metastasis. Depleted RAB23 expression attenuates focal adhesion-related signal pathways, thus impairing the invasion, metastasis, and adhesion of ESCC cells. 目的： 探讨Ras相关结合蛋白23（RAB23）在食管鳞状细胞癌（简称食管鳞癌）细胞侵袭和迁移中的作用和机制。 方法： 采用实时荧光定量聚合酶链反应检测16例配对食管鳞癌及癌旁正常组织中RAB23 mRNA的表达。比较基因表达综合（GEO）数据库的GSE20347数据集中食管鳞癌和配对癌旁正常组织RAB23 mRNA的表达水平。免疫组织化学检测106例配对食管鳞癌和癌旁正常组织、33例淋巴结阳性与10例淋巴结阴性患者原发灶与淋巴结组织中RAB23蛋白含量。在食管鳞癌KYSE30和KYSE150细胞中瞬时敲降RAB23表达（转染si-RAB23-1和si-RAB23-9）或者稳定敲降RAB23表达（转染sh-RAB23），采用Western blot法验证RAB23敲降效率，采用细胞计数试剂盒8法和裸鼠皮下成瘤实验检测食管鳞癌细胞的增殖能力，采用Transwell实验和裸鼠尾静脉-肺转移实验检测食管鳞癌细胞的侵袭和迁移能力，采用细胞黏附实验检测食管鳞癌细胞的黏附能力，采用转录组测序技术分析RAB23敲降后对细胞转录谱的影响，并通过Western blot检测验证相关信号通路。 结果： 16例食管鳞癌组织中RAB23 mRNA表达水平为0.009 7±0.008 9，高于癌旁正常组织［0.003 2±0.003 7，P=0.006］。对GEO数据库GSE20347数据集中食管鳞癌和配对癌旁正常组织表达谱的生信分析显示，食管鳞癌组织中RAB23 mRNA表达水平为4.30±0.25，高于癌旁正常组织（4.10±0.17，P=0.037）。106例食管鳞癌组织中，51例RAB23低表达，55例RAB23高表达；而配对癌旁正常组织中，82例RAB23低表达，24例RAB23高表达，食管鳞癌组织中RAB23表达水平高于配对癌旁正常组织（P＜0.001）。33例淋巴结阳性食管鳞癌组织中，1例RAB23低表达，32例RAB23高表达；10例淋巴结阴性的食管鳞癌组织中，3例RAB23低表达，7例RAB23高表达。淋巴结阳性食管鳞癌组织中RAB23表达水平高于淋巴结阴性食管鳞癌组织（P=0.024）。33例阳性淋巴结组织中，1例RAB23低表达，32例RAB23高表达，而10例阴性淋巴结组织中均为RAB23低表达。阳性淋巴结组织中RAB23表达水平高于阴性淋巴结组织（P＜0.001）。瞬时或稳定敲降RAB23后，KYSE30细胞体外和体内的增殖能力无明显变化，KYSE30细胞的细胞侵袭和迁移能力下降，而KYSE150细胞仅侵袭能力下降，迁移能力无明显变化。sh-RAB23组KYSE30细胞黏附细胞的数量为（313.75±89.34）个，少于sh-NC组［（1 030.75±134.29）个，P＜0.001］。sh-RAB23组KYSE150细胞的黏附细胞数为（710.5±31.74）个，也少于sh-NC组［（1 005.75±61.09）个，P＜0.001］。转录组测序分析显示，敲降RAB23后，si-RAB23-1组和si-RAB23-9组KYSE30细胞中黏着斑相关信号通路均被削弱，sh-RAB23组KYSE30和KYSE150细胞中p-FAK和p-paxillin表达水平降低。 结论： RAB23在食管鳞癌组织中高表达，与淋巴结转移有关。敲低RAB23表达后能够削弱黏着斑相关信号通路，进而抑制食管鳞癌细胞的侵袭、迁移和黏附。.

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