Regulation Of GPX3 Gene Expression And Its Combined Transcription-metabolomic Analysis In Esophageal Squamous Cell Carcinoma

Background:Esophageal carcinoma is one of the most common malignancies of the gastrointestinal tract,with the death rate ranking the sixth in the world^[1-3].In our country,about 350,000 people die of esophageal carcinoma every year^[4],of which about 90%belong to esophageal squamous cell carcinoma^[5,6].Esophageal squamous cell carcinoma has a poor prognosis and high mortality^[7].Therefore,it is an important research direction to search for biological markers of esophageal squamous cell carcinoma.In recent years,epigenetic silencing of multiple tumor suppressor genes has been found in ESCC,which indicates that epigenetic silencing of tumor suppressor genes is one of the major molecular changes in the carcinogenesis process of ESCC.Previous studies have identified a new methylation silencing gene GPX3 in ESCC.The silencing of GPX3may impair the defense against endogenous and exogenous genotoxic compounds,thus increasing the gene mutation rate^[13].GPX3 plays a crucial role in eliminating hydrogen peroxide produced in the body and catalyzing the conversion of reduced glutathione（GSH）to oxidized glutathione（GSSG）^[6,7].In addition to being present in cytoplasm and mitochondria,it also circulates in plasma^[9].Therefore,the level of GPX3 can be measured with plasma,making it possible to become a useful prognostic and diagnostic biomarker.In this study,by detecting the methylation status and transcription level of GPX3promoter region in esophageal squamous cell carcinoma,we explored the effect of GPX3promoter region methylation level on its expression.At the same time,we constructed esophageal squamous cell carcinoma cell lines with GPX3 overexpression,detected the changes of downstream genes when GPX3 expression increased by using transcriptomics,and then detected the effect of GPX3 on the metabolites of esophageal squamous cell carcinoma by using metabonomics to explore the mechanism of action of GPX3.Methods:The subjects were 78 patients with esophageal carcinoma treated by surgery from Liaoning Cancer Hospital from January 2018 to January 2021,and collected esophageal cancer focus and NATs.The methylation of GPX3 promoter region was detected by the matrix assisted laser desorption ionization time of flight mass spectrometry（MALDI-TOF MS）after sulfite PCR,and the expression of GPX3 mRNA was measured by q PCR.The esophageal squamous cell carcinoma cell line with stable overexpression of GPX3 and the corresponding empty body control group cell line were constructed.The infection efficiency was detected by q PCR and Western Blot,and then the RNA-seq and non-targeted metabolites were detected.Results:The expression of GPX3 mRNA was decreased in esophageal squamous cell carcinoma（0.0138±0.0105 vs 0.0031±0.004,p<0.05）,and the expression of GPX3was significantly positively correlated with nest-like,lump-like growth and S100expression（P<0.05）,and negatively correlated with nerve invasion（P<0.05）.A total of 24readable CpG sites were detected in the GPX3 promoter region.The average methylation rate of cancer tissue was significantly higher than that of the control tissue（0.1（0.2）vs0.05（0.12）,p<0.01）.All but sites 1,14,and 25 showed statistically significant differences in methylation rates between cancer and normal samples（p<0.05）.In addition,the methylation rate of GPX3 was negatively correlated with tumor volume and tumor length and diameter（P<0.05）,but positively correlated with vascular tumor thrombus（P<0.01）.The CpG site in its promoter region was correlated with age,smoking,tumor location and other clinicopathologic parameters.Construction of esophageal squamous cell carcinoma cells with GPX3 overexpression.The results of non-targeted metabonomics showed that GPX3 mainly affected the metabolism of organic acids and their derivatives,nucleotides,nucleotides and their analogues,lipids and lipid molecules,and organic heterocyclic compounds in esophageal squamous cell carcinoma.Transcriptome sequencing results showed that 675 genes encoding proteins were obtained,of which 439 were up-regulated and 218 were down-regulated.Conjoint analysis of metabolome-transcriptome results found that GPX3 not only participates in the regulation of glutathione metabolic pathway,but also may affect the production of methionine and other metabolites by down-regulating FOSL1,thus leading to the occurrence of esophageal squamous cell carcinoma.Conclusion:1.The expression of GPX3 gene in esophageal squamous cell carcinoma is down-regulated by the specific hypermethylation of CpG in the promoter region;2.GPX3 may affect the carcinogenesis of esophageal squamous cell carcinoma by down-regulating the expression of FOSL1 and affecting the metabolism of 5’-Methylthioadenosine and so on.