Rps3 Attenuates Gastric Precancerous Lesions by Promoting Dendritic Cells Maturation via AKT/β-Catenin Pathway

This study aimed to investigate the dysregulated proteins
and the
underlying mechanisms of gastric precancerous lesions. Proteomic and
phosphoproteomic methods were used to characterize the proteome and
phosphoproteome profiles of N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-induced gastric
precancerous lesions. The hub differentially expressed proteins (DEPs)
and phosphoproteins (DEPPs) were identified by using differential
expression and protein–protein interaction network analyses.
Western blot assay, quantitative reverse transcription (qRT)-PCR,
and CCK-8 assays detected the expression of Rps3, N-cadherin, E-cadherin, AKT, p-AKT,
and β-catenin and verified the roles of Rps3 on the MNNG-induced
human gastric epithelial cell line (GES-1) cells. Hub DEPs and phosphoproteins
Rps3, Akt1, and Ctnnb1 were significantly correlated with five dendritic
cells (DCs) pathways, and Akt1 and Ctnnb1 were significantly negatively
correlated with Rps3. MNNG administration markedly reduced the Rps3
mRNA and protein expression levels (all P < 0.05).
Overexpression of Rps3 significantly inhibited tumorigenesis of MNNG-induced
GES-1 cells (all P < 0.01) and changed the protein
levels of N-cadherin, E-cadherin,
AKT, p-AKT, and β-catenin. Similarly, SC79
treatment substantially increased the expression of interleukin (IL)-6, IL-10, and vascular endothelial
growth factor (all P < 0.05). Rps3 was poorly
expressed in precancerous gastric lesions. Correspondingly, overexpression
of Rps3 promoted DC maturation via the AKT/β-catenin pathway,
inhibiting the progression of gastric precancerous lesions.

本研究旨在探究胃癌前病变的异常表达蛋白及其潜在作用机制。采用蛋白质组学与磷酸化蛋白质组学方法，对N-甲基-N'-硝基-N-亚硝基胍（N-methyl-N-nitro-N-nitrosoguanidine, MNNG）诱导的胃癌前病变的蛋白质组与磷酸化蛋白质组谱进行表征。通过差异表达分析及蛋白质-蛋白质相互作用网络分析，筛选鉴定得到核心差异表达蛋白（differentially expressed proteins, DEPs）与差异表达磷酸化蛋白（differentially expressed phosphoproteins, DEPPs）。采用蛋白质免疫印迹（Western blot）、定量反转录聚合酶链反应（quantitative reverse transcription PCR, qRT-PCR）及细胞计数试剂盒-8（Cell Counting Kit-8, CCK-8）实验，检测Rps3、N-钙黏蛋白（N-cadherin）、E-钙黏蛋白（E-cadherin）、AKT、磷酸化AKT（p-AKT）与β-连环蛋白（β-catenin）的表达水平，并验证Rps3在MNNG诱导的人胃上皮细胞系（GES-1）中的作用。核心DEPs与DEPPs Rps3、Akt1及Ctnnb1与五种树突状细胞（dendritic cell, DC）通路显著相关，且Akt1、Ctnnb1与Rps3呈显著负相关。MNNG处理可显著降低Rps3的mRNA与蛋白表达水平（均P<0.05）。过表达Rps3可显著抑制MNNG诱导的GES-1细胞的成瘤能力（均P<0.01），并改变N-钙黏蛋白、E-钙黏蛋白、AKT、磷酸化AKT及β-连环蛋白的蛋白水平。同样，SC79处理可显著升高白细胞介素-6（interleukin-6, IL-6）、白细胞介素-10（IL-10）与血管内皮生长因子的表达水平（均P<0.05）。Rps3在胃癌前病变中呈低表达。相应地，过表达Rps3可通过AKT/β-连环蛋白通路促进树突状细胞成熟，进而抑制胃癌前病变的进展。