研究背景与目的

研究显示GPX4在肝癌、肺癌、胶质瘤、前列腺癌、鼻咽癌等多种肿瘤组织中呈高表达，与肿瘤发生发展等具有相关性，是部分肿瘤预后不良的独立影响因素。GPX4在结肠腺癌中的表达及其表达对增殖、迁移等生物学行为的影响及作用机制尚不明确。本研究拟通过比较GPX4蛋白在结肠腺癌组织及结肠粘膜组织中的表达情况，探索GPX4蛋白表达与临床病理参数及生存预后的相关性；通过功能实验探索GPX4对增殖、侵袭、迁移等特性的影响，初步探讨GPX4蛋白在结肠腺癌细胞中可能参与的生物学过程及代谢通路。

研究方法

1.利用公共数据库探索GPX4蛋白在结肠腺癌组织中表达情况及其与生存预后的关系。

2.采用Western Blot法及免疫组织化学染色法检测GPX4蛋白在结肠腺癌组织及结肠粘膜中的表达情况，分析其与临床病理特征及生存预后的相关性。整合相关变量绘制预测生存率的列线图（Nomogram分析）。

3.采用Western Blot法检测GPX4在结肠腺癌细胞株中表达；采用Lipofectamine 2000转染GPX4-siRNA，Real time PCR及Western Blot法筛选干扰效果较好的siRNA。

4.CCK-8法及软琼脂克隆形成实验检测GPX4对HCT116/LOVO细胞增殖的影响；划痕实验及Transwell小室实验检测GPX4对HCT116/LOVO细胞迁移、侵袭能力的影响；Western Blot法检测GPX4对细胞周期及EMT相关蛋白的影响。

5.在LOVO细胞株中干扰GPX4表达后进行转录组测序，基于转录组测序结果进行差异表达基因的筛选、聚类分析、KEGG通路富集分析及GO分析。

研究结果

1、TCGA数据库分析显示GPX4 mRNA在结肠腺癌组织中表达高于正常结肠粘膜组织，差异有统计学意义（P＜0.001）。GPX4高表达的结肠腺癌患者预后较差。

2.Western Blot法显示GPX4蛋白在结肠腺癌组织中的表达水平增高。

3.免疫组织化学染色法结果GPX4蛋白在结肠腺癌组织中表达水平高于正常结肠粘膜组织。GPX蛋白表达与肿瘤部位及淋巴结转移相关（P＜0.05）。

4.GPX4蛋白高表达组患者的5年OS率低于GPX4蛋白低表达组患者（25.7%比57.9%，x²=9.051，P＜0.05）。

5.COX风险比例回归分析显示GPX4高表达是结肠腺癌患者不良预后的独立危险因素（HR = 2.783， 95% CI   1.598~4.848， P ＜ 0.001）。

6．降低GPX4蛋白表达后，结肠腺癌细胞增殖、迁移及侵袭能力降低；增殖相关蛋白Cyclin D1和C-myc表达降低；迁移相关蛋白E钙黏蛋白表达增加，Vimentin和β-catenin表达降低。

7.RNA-seq测序数据检测到共有107个基因表达发现显著变化，其中上调基因52个，下调基因57个。GO富集分析显差异表达基因主要参与脂质代谢、类固醇的生物合成及代谢等生物过程；与氧化还原酶的活性、离子通道活动等分子功能相关。KEGG Pathway富集分析显示GPX4与活性氧的化学致癌作用、T细胞受体信号通路、脂质和胆固醇代谢等密切相关。

研究结论

1.结肠腺癌组织中GPX4 mRNA及GPX4蛋白表达水平高于正常结肠粘膜组织。

2.结肠腺癌组织中GPX4蛋白表达与淋巴结转移及肿瘤部位呈相关性。

3.GPX4高表达组患者5年OS率较低，其高表达是结肠腺癌患者不良预后的独立危险因素。

4.降低GPX4表达可以调控HCT116细胞及LOVO细胞的增殖、侵袭、迁移能力，其机制可能是通过下调细胞周期及EMT相关蛋白来实现。

5.GPX4可能通过脂质代谢、活性氧的化学致癌作用等多种生物学过程，影响肿瘤细胞的增殖、侵袭、迁移等表型。

关键词      结直肠癌；GPX4；增殖；预后；侵袭迁移；  

Background and Purpose

Studies have shown that GPX4 is highly expressed in liver cancer, lung cancer,  glioma, prostate cancer, nasopharyngeal cancer and other malignant tumors, which is correlated with tumor development and treatment response, and is an influential factor for poor prognosis of some tumors. At present, the expression of GPX4 in colorectal adenocarcinoma and its effects on the proliferation and migration of colorectal adenocarcinoma cells as well as the mechanism of action remain unclear. Our study aimed to explore the correlation between GPX4 protein expression and clinicopathological parameters and survival prognosis by comparing the expression of GPX4 in colorectal adenocarcinoma tissues and normal colon mucosal tissues. The effects of GPX4 on the proliferation, invasion and migration of colorectal adenocarcinoma cells were investigated by functional experiments, and the possible biological processes and metabolic pathways of GPX4 in colorectal adenocarcinoma cells were explored.

Methods

1. To explore the expression of GPX4 protein in colon adenocarcinoma tissues and its relationship with survival and prognosis by using public databases.

2. Immunohistochemical staining was used to detect the expression of GPX4 protein in colorectal adenocarcinoma tissues and paired normal tissues, and its correlation with clinicopathological features and survival prognosis was analyzed. A Nomogram analysis was performed to predict survival by integrating relevant variables.

3. Western Blot was used to detect the expression of GPX4 in colorectal adenocarcinoma cells. Gpx4-sirna was transfected with Lipofectamine 2000, and siRNA with good interference effect was screened by Real time PCR and Western Blot.

4. The effects of GPX4 on the proliferation of colorectal adenocarcinoma cell lines were detected by CCK-8 assay and clonogenesis assay. The effects of GPX4 on migration and invasion of cell lines were detected by scratch test and Transwell cell assay. Western Blot was used to detect the effect of GPX4 on cell cycle and EMT-related proteins.

5. Transcriptome sequencing was performed after GPX4 expression was interfered in LOVO cells. Differential expression genes were screened based on transcriptome sequencing, and cluster analysis, GO function analysis and KEGG pathway enrichment analysis were performed for differential expression genes.

Results

1. TCGA database analysis showed that GPX4 mRNA expression in colorectal adenocarcinoma tissues was higher than that in normal colon mucosa tissues, and the difference was statistically significant. Patients with colorectal adenocarcinoma with high GPX4 expression have poor prognosis.

2.Western Blot showed that the expression level of GPX4 protein in colorectal adenocarcinoma tissues was increased.

3. Immunohistochemical staining results showed that the expression level of GPX4 protein in colorectal adenocarcinoma tissues was higher than that in normal colon mucosa tissues. The expression of GPX protein was correlated with lymph node metastasis and tumor site (P < 0.05).

4. The 5-year OS rate of patients with high GPX4 expression group was lower than that of patients with low GPX4 expression group (25.7% vs 57.9%), and the difference between the two groups was statistically significant (X2 =9.051, P < 0.05).

5. COX proportional resgression analyses showed that high GPX4 expression was an independent risk factor for poor prognosis in patients with colorectal adenocarcinoma (HR = 2.783, 95%CI 1.598~4.848, P < 0.001).

6. Decreased GPX4 protein expression reduced cell proliferation, migration and invasion. The expression of Cyclin D1 and C-myC was decreased. The expression of transfer-related protein E cadherin increased, and Vimentin and β-catenin decreased.

7. Rna-seq sequencing data detected significant changes in the expression of 107 genes, including 52 up-regulated genes and 57 down-regulated genes. GO enrichment analysis showed that differentially expressed genes were mainly involved in lipid metabolism, steroid biosynthesis and metabolism, phospholipid metabolism and other biological processes, and were related to molecular functions such as REDOX enzyme activity and ion channel activity. KEGG Path way enrichment analysis showed that GPX4 may be closely related to the chemical carcinogenesis of reactive oxygen species, T cell receptor signaling pathway, lipid and cholesterol metabolism, bile acid metabolism, primary immune deficiency, and steroid biosynthesis and metabolism.

Conclusions

1. The expression levels of GPX4 mrna and GPX4 protein in colorectal adenocarcinoma tissues were higher than those in normal mucosal tissues.

2. GPX4 protein expression in colorectal adenocarcinoma was correlated with lymph node metastasis and tumor site.

3. The 5-year OS rate in patients with high GPX4 expression group was lower, and its high expression was an independent risk factor for poor prognosis in patients with colorectal adenocarcinoma.

4. Decreased GPX4 expression can regulate the proliferation, invasion and migration of HCT116 and LOVO cells, possibly through down-regulation of cell cycle and EMT-related proteins.

5.GPX4 may affect tumor cell proliferation, invasion, migration and other phenotypes through various biological processes such as lipid metabolism and chemical carcinogenesis of reactive oxygen species.

Keywords    Colorectal Cancer; GPX4;  Proliferation; Prognosis;  Invasion migration;