Yang Shibin, Deng Liang, Lai Yuanhui, Liu Zhaoguo
Department of General Surgery, The First Affiliated Hospital, Sun Yat-sen University Guangzhou, Guangdong, China.
Department of General Thoracic Surgery, The First Affiliated Hospital, Sun Yat-sen University Guangzhou, Guangdong, China.
Int J Clin Exp Pathol. 2018 Jul 1;11(7):3426-3435. eCollection 2018.
Beclin-1 and GPR30, both very important proteins, have been associated with tumor development. In our pre-experiment, the co-expression of GPR30 and Beclin-1 was observed in esophageal squamous cell carcinoma (ESCC), an observation not reported in other studies. The aim of our research was to investigate the relationship of these two proteins in the further progression of ESCC.
The over expression of GPR30 and Beclin-1 proteins was observed and confirmed by immunohistochemistry and immunofluorescence arrays. By interfering with GPR30 and p38 MAPK expression in EC-109, KYSE510, and KYSE3 cell lines, MTT and a scratch wound healing assay were used to investigate the impact of the GPR30 protein on the proliferative and migrative abilities of ESCC cells. A co-immunoprecipitation assay was used to observe the interaction between the p38 MAPK and Beclin-1 proteins; meanwhile, at a different time, in each group, the GPR30, MAPK, p ERK1/2, p38 MAPK, and Beclin-1 proteins were analyzed. The correlation between GPR30, Beclin-1 expression levels, and the clinical characteristics were evaluated by Mann-Whitney and chi-square tests. Using Kaplan-Meier plots and the Cox proportional hazard model analysis, we determined overall survival (OS) and progression free survival (PFS).
GPR30 and Beclin-1 were over expressed significantly in ESCC (both p=0.0000) and were distributed into cytoplasms the most (the former p=0.0223, latter p=0.0018). In contrast to the non-agonist group, the abilities of GPR30 in promoting cell proliferation and metastasis were observed in the agonist group, and the effects could be blocked by p38MAPK inhibitors. In further assays, GPR30 agonists, via binding to GPR30, up-regulated Beclin-1, MAPK, and p38 MAPK expression, and Beclin-1 expression was reversed in the p38MAPK inhibitor group. In the GPR30 agonist group, an interaction between p38MARK and Beclin-1 was observed, but no similar results were observed in the non-agonist group. The high expression of both GPR30 and Beclin-1 was observed with p-stage and pT advancing (both p<0.0001). In a Kaplan-Meier analysis, compared to GPR30's negative expression, high expression identified a group of patients with the shortest overall survival (OS, p=0.0072) and progression free survival (PFS, p=0.0074). The Cox proportional hazard models revealed that they predicted a short time to OS (p=0.0125).
Over expression of GPR30 up-regulated Beclin-1 expression and indicated a poor prognosis and may promote ESCC development via p38 MAPK in ESCC progression.
Beclin-1和GPR30这两种非常重要的蛋白质都与肿瘤发展相关。在我们的前期实验中，观察到食管鳞状细胞癌（ESCC）中存在GPR30和Beclin-1的共表达，其他研究未报道过这一现象。我们研究的目的是在ESCC的进一步发展过程中探究这两种蛋白质之间的关系。
通过免疫组织化学和免疫荧光阵列观察并证实GPR30和Beclin-1蛋白的过表达。通过干扰EC-109、KYSE510和KYSE3细胞系中GPR30和p38 MAPK的表达，采用MTT和划痕伤口愈合试验来研究GPR30蛋白对ESCC细胞增殖和迁移能力的影响。采用免疫共沉淀试验观察p38 MAPK与Beclin-1蛋白之间的相互作用；同时，在不同时间对每组中的GPR30、MAPK、p ERK1/2、p38 MAPK和Beclin-1蛋白进行分析。通过Mann-Whitney和卡方检验评估GPR30、Beclin-1表达水平与临床特征之间的相关性。使用Kaplan-Meier曲线和Cox比例风险模型分析，我们确定了总生存期（OS）和无进展生存期（PFS）。
GPR30和Beclin-1在ESCC中均显著过表达（两者p = 0.0000），且大多分布于细胞质中（前者p = 0.0223，后者p = 0.0018）。与非激动剂组相比，在激动剂组中观察到GPR30具有促进细胞增殖和转移的能力，且这些作用可被p38MAPK抑制剂阻断。在进一步的试验中，GPR30激动剂通过与GPR30结合，上调了Beclin-1、MAPK和p38 MAPK的表达，而在p38MAPK抑制剂组中Beclin-1的表达则发生了逆转。在GPR30激动剂组中，观察到p38MARK与Beclin-1之间存在相互作用，但在非激动剂组中未观察到类似结果。随着p分期和pT进展，观察到GPR30和Beclin-1均高表达（两者p < 0.0001）。在Kaplan-Meier分析中，与GPR30的阴性表达相比，高表达确定了一组总生存期（OS，p = 0.0072）和无进展生存期（PFS，p = 0.0074）最短的患者。Cox比例风险模型显示，它们预测OS时间较短（p = 0.0125）。
GPR30的过表达上调了Beclin-1的表达，提示预后不良，并且在ESCC进展过程中可能通过p38 MAPK促进ESCC的发展。
