背景:已发现CALD1在多种恶性肿瘤中异常表达,包括胃癌(GC),并与肿瘤进展和免疫浸润有关;然而,CALD1在GC上皮-间质转化(EMT)中的作用和机制尚不清楚。
目的:探讨CALD1在GC进展中的作用及机制。入侵,和移民。
方法:在本研究中,CALD1和GC之间的关系,以及CALD1可能的网络调控机制,通过生物信息学研究和实验验证。合成CALD1-siRNA并用于转染GC细胞。使用CCK-8方法测量细胞活性,使用伤口愈合试验和Transwell试验测量细胞迁移和侵袭能力,采用qRT-PCR和Westernblot检测各组细胞中相关基因和蛋白的表达水平。建立GC细胞异种移植模型以验证体外实验结果。
结果:生物信息学结果显示CALD1在GC组织中高表达,并且CALD1在EMT型GC组织中明显高于其他类型GC组织。CALD1高表达患者的预后较低表达患者差,建立并评价预后模型。实验结果与生物信息学分析结果一致。CALD1在GC细胞系中的表达水平均高于胃上皮细胞系GES-1,其中在AGS和MKN45细胞中的表达最强。CALD1-siRNA转染AGS和MKN45细胞后,细胞活性显著降低。CALD1-siRNA转染后,AGS和MKN45细胞的迁移和侵袭能力降低,相关的mRNA和蛋白表达发生改变。根据GC样品中的生物信息学发现,CALD1基因与PI3K-AKT-mTOR信号通路和EMT信号通路成员的表达显著相关,与PI3K-Akt信号通路密切相关。实验验证表明,CALD1的上调增加了PI3K的表达,p-AKT,和p-mTOR,PI3K-Akt通路的成员,PTEN的表达降低;PI3K-Akt抑制剂处理降低PI3K的表达,p-AKT,过表达CALD1的细胞中p-mTOR(仍高于正常组),但PTEN表达增加(仍低于正常组)。CCK-8结果表明,添加抑制剂可降低CALD1对肿瘤细胞活性的影响。Scratch和Transwell实验表明,添加PI3K-Akt抑制剂可减弱CALD1对肿瘤细胞迁移和侵袭的影响。CALD1的过表达极大地改变了AGS和MKN45细胞中EMT相关基因的mRNA和蛋白水平,而CALD1过表达的作用则显著减弱了PI3K-Akt抑制剂的加入。动物实验表明,CALD1抑制后肿瘤生长缓慢,一些PI3K-Akt和EMT途径蛋白的表达发生了改变。
结论:CALD1的表达增加是进展的关键因素,入侵,和GC的转移,这可能与调节PI3K-Akt途径促进EMT有关。
BACKGROUND: CALD1 has been discovered to be abnormally expressed in a variety of malignant tumors, including gastric cancer (GC), and is associated with tumor progression and immune infiltration; however, the roles and mechanisms of CALD1 in epithelial-mesenchymal transition (EMT) in GC are unknown.
OBJECTIVE: To investigate the role and mechanism of CALD1 in GC progression, invasion, and migration.
METHODS: In this study, the relationship between CALD1 and GC, as well as the possible network regulatory mechanisms of CALD1, was investigated by bioinformatics and validated by experiments. CALD1-siRNA was synthesized and used to transfect GC cells. Cell activity was measured using the CCK-8 method, cell migration and invasive ability were measured using wound healing assay and Transwell assay, and the expression levels of relevant genes and proteins in each group of cells were measured using qRT-PCR and Western blot. A GC cell xenograft model was established to verify the results of in vitro experiments.
RESULTS: Bioinformatics results showed that CALD1 was highly expressed in GC tissues, and CALD1 was significantly higher in EMT-type GC tissues than in tissues of other types of GC. The prognosis of patients with high expression of CALD1 was worse than that of patients with low expression, and a prognostic model was constructed and evaluated. The experimental results were consistent with the results of the bioinformatics analysis. The expression level of CALD1 in GC cell lines was all higher than that in gastric epithelial cell line GES-1, with the strongest expression found in AGS and MKN45 cells. Cell activity was significantly reduced after CALD1-siRNA transfection of AGS and MKN45 cells. The ability of AGS and MKN45 cells to migrate and invade was reduced after CALD1-siRNA transfection, and the related mRNA and protein expression was altered. According to bioinformatics findings in GC samples, the CALD1 gene was significantly associated with the expression of members of the PI3K-AKT-mTOR signaling pathway as well as the EMT signaling pathway, and was closely related to the PI3K-Akt signaling pathway. Experimental validation revealed that upregulation of CALD1 increased the expression of PI3K, p-AKT, and p-mTOR, members of the PI3K-Akt pathway,while decreasing the expression of PTEN; PI3K-Akt inhibitor treatment decreased the expression of PI3K, p-AKT, and p-mTOR in cells overexpressing CALD1 (still higher than that in the normal group), but increased the expression of PTEN (still lower than that in the normal group). CCK-8 results revealed that the effect of CALD1 on tumor cell activity was decreased by the addition of the inhibitor. Scratch and Transwell experiments showed that the effect of CALD1 on tumor cell migration and invasion was weakened by the addition of the PI3K-Akt inhibitor. The mRNA and protein levels of EMT-related genes in AGS and MKN45 cells were greatly altered by the overexpression of CALD1, whereas the effect of overexpression of CALD1 was significantly weakened by the addition of the PI3K-Akt inhibitor. Animal experiments showed that tumour growth was slow after inhibition of CALD1, and the expression of some PI3K-Akt and EMT pathway proteins was altered.
CONCLUSIONS: Increased expression of CALD1 is a key factor in the progression, invasion, and metastasis of GC, which may be associated with regulating the PI3K-Akt pathway to promote EMT.