Biological Function And Mechanism Of COX-2 In The Development And Progression Of Esophageal Cancer

Background and ObjectiveEsophageal cancer is one kind of the most prevalent digestive system malignancies worldwide with the highest morbidity and mortality.According to histopathological data,Esophageal cancer comprises esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC).Esophageal squamous cell carcinoma mainly occurs in East Asia,while esophageal adenocarcinoma is most prevalent in Western countries.Surgery,radiotherapy,chemotherapy,molecular targeted therapy,and their combination are the main strategies for the current treatment of esophageal cancer.However,due to the insidious onset of esophageal cancer and lack of specificity,the primary diagnosis is mostly in the middle and late stages,leading to its high invasiveness.The prognosis of esophageal cancer is quite poor,with an overall five-year survival rate of less than 20%.Therefore,there is an urgent need to find more biomarkers and therapeutic targets for esophageal cancer.Cyclooxygenase-2(COX-2)is a rate-limiting enzyme in arachidonic acid metabolism and a catalyst for prostaglandin E2(PEG2)prostaglandin biosynthesis.It promotes tumor cell proliferation and inhibits apoptosis.It plays a key role in promoting cell proliferation,inhibiting apoptosis,inhibiting tumor immunity,promoting invasion and metastasis.Growing evidence suggests that COX-2 is the link between inflammation and cancer and is considered a major target for cancer therapy.COX-2 is also associated with poor prognosis in a variety of cancers,especially lymph node metastases in colorectal,gastric,nasopharyngeal,and lung cancers.However,the role and mechanism of COX-2 in the malignant transformation of esophageal squamous cell carcinoma are still unclear.Therefore,the purpose of this study is to analyze the expression level of COX-2 in esophageal cancer from big data,and verify the expression of COX-2 in clinical tissue samples and esophageal cancer cell lines.Collect medical records,and analyze the relationship between COX-2 expression and clinicopathological parameters and prognosis of esophageal cancer.Cell and animal experiments to explore the effects of COX-2 on the function of esophageal cancer cells from in vitro and in vivo.Based on RNA-seq technology to mine the differentially expressed genes of esophageal cancer,and use bioinformatics methods to predict the possible mechanisms of the occurrence and development of esophageal,providing new insight to the prevention and treatment of esophageal cancer.Based on the differential expression of COX-2,exploring the value of new COX-2 fluorescent probes in the early diagnosis of esophageal cancer,and revealing the clinical translation value of molecular markers.Methods(1)The expression of COX-2 in esophageal cancer was analyzed by multiple databases including Oncomine,TCGA,and GEO.The transcriptional and protein levels of COX-2 in esophageal cancer and paracancerous,normal esophageal epithelial cells,and cancer cells were detected by fluorescence quantitative PCR(quantitative real-time PCR,qPCR)and Western blotting(Western blot,WB).(2)108 esophageal cancer tissues and 72 paired adjacent tissues were collected to make tissue microarray.The tissue microarray was used to detect the expression of COX-2 in esophageal cancer and paracancerous normal tissues by immunohistochemical method(IHC).The follow-up data were collected to analyze the relationship between the expression of COX-2 and clinicopathological parameters(TNM stage,grade,survival,Ki67).(3)The lentiviral vector was used to construct the cell models of stable silencing and overexpression of COX-2 in esophageal cancer cell lines(Eca109 and KYSE150).RT-qPCR and WB were used to verify the expression of COX-2 at the RNA and protein levels.The effects of COX-2 expression on cell malignant behavior(proliferation and invasion)were detected by CCK-8 assay,clone formation assay,Ki67 immunofluorescence,flow cytometry,Transwell migration test,Transwell invasion test,and subcutaneous tumor formation test in nude mice.(4)Transcriptional sequencing screened differentially expressed genes in the silenced COX-2 group and negative control group.Bioinformatics methods such as GO analysis,KEGG analysis,and PPI network analysis revealed the possible molecular mechanism of the occurrence and progression of esophageal cancer.(5)Fluorescence imaging of esophageal squamous cell carcinoma and adjacent normal esophageal tissue was performed by a new COX-2 fluorescence probe 2a1.Fluorescence imaging of knockdown and overexpression of COX-2 in esophageal squamous cell carcinoma cells was performed by a new COX-2 fluorescence probe 2a1.Finally,the organoids of human-derived normal esophageal epithelium,human esophageal squamous cell carcinoma epithelium,and precancerous epithelial cells were constructed,and a new type of COX-2 fluorescence probe was used for fluorescence imaging.Results(1)Referring to oncomine,it was found that COX-2 was up-regulated in a variety of cancers including esophageal cancer,and a pan-cancer analysis of the TCGA database found similar results.The sequencing results of esophageal cancer tissue and normal esophageal tissue samples were extracted from TCGA and GTEx databases.Further analysis showed that the expression level of COX-2 in esophageal cancer tissue was drastically upregulated than that in paracancerous tissues and normal tissues,and it had certain diagnostic efficacy.GEO datasets were collected for analysis,which revealed that COX-2 was overexpressed in esophageal cancer tissues compared to adjacent tissues.RT-qPCR and WB showed that the expression of COX-2 RNA and protein in different esophageal cancer cell lines EC9706,KYSE150,Eca109,and TE-1 was higher than that in Het-1A.18 pairs of fresh esophageal cancer and paired paracancerous tissue samples were collected,and RNA and protein were extracted for RT-qPCR and WB analysis,respectively.The results showed that the RNA and protein levels of COX-2 in esophageal carcinoma were significantly higher than those in adjacent normal tissues.(2)The results of IHC showed that the expression level of COX-2 in esophageal carcinoma was significantly higher than that in adjacent tissues(P<0.001).The high expression of COX-2 was closely related to tumor size,pathological differentiation,depth of tumor invasion,lymph node metastasis,TNM stage,and Ki67 expression.However,there was no significant correlation with the age and sex of the patients.Esophageal cancer patients with high expression of COX-2 have a short survival time,which can be used as an independent risk factor for the prognosis of esophageal cancer.(3)The cell models of stable silencing and overexpression of COX-2 were constructed,and the validity was identified by RT-PCR and WB at the RNA and protein levels.CCK8 assay showed that silencing COX-2 significantly inhibited the growth of esophageal cancer cells in vitro,while overexpression of COX-2 significantly promoted the growth of esophageal cancer cells.Clone formation assay also showed that silencing COX-2 significantly inhibited the colony formation ability of esophageal cancer cells,while overexpression of COX-2 significantly promoted the colony formation ability of esophageal cancer cells.Ki67 immunofluorescence staining showed that the number of Ki67 positive cells in silenced COX-2 Eca109 and KYSE150 decreased,while the number of Ki67 positive cells in Eca109 and KYSE150 overexpressing COX-2 increased.Flow cytometry analysis of cell cycle distribution showed that COX-2 silencing could inhibit the cell cycle progression of Eca109 and KYSE150,while overexpression of COX-2 could promote the cell cycle progression of Eca109 and KYSE150.Transwell migration and invasion assay showed that silencing COX-2 inhibited the migration and invasion of Eca109 and KYSE150 esophageal cancer cells,and overexpression of COX-2 promoted the migration and invasion of esophageal cancer cells.The subcutaneous tumor formation experiment of nude mice showed that the proliferation rate of KYSE150 esophageal cancer cells in the silenced COX-2 group was lower than that in the control group,and the proliferation rate of esophageal cells in the overexpression COX-2 group was significantly faster than that in the control group.(4)Based on the RNA-seq technique,342 differentially expressed genes in esophageal cancer were found after COX-2 knockdown,including 237 up-regulated genes and 105 down-regulated DEGs.The most significant pathways of gene enrichment include the PI3K-AKT signal pathway,TNF signal pathway,cell cycle,fatty acid metabolism,DNA repair,and so on.SERPINE1,ITGA5,EFCAB14,STEAP1,RASSF6,MMP3,SENP3EIF4A1,SERPINE2,CD177,and IL1R2 genes were down-regulated by COX-2 knockdown,and their expression levels in esophageal cancer tissues were higher than those in para-esophageal cancer tissues and normal tissues through TCGA database,and their expression was positively correlated with COX-2 expression.The result indicated that COX-2 may participate in the malignant process of esophageal cancer through these downstream genes.(5)The value of novel COX-2 fluorescent probe in early diagnosis of esophageal carcinoma.COX-2 new fluorescent molecular probe 2a1 was used for fluorescence imaging of esophageal cancer and paracancerous tissue sections.Excitedly,after incubation with 2a1,bright red fluorescence was observed in ESCC tissue,but not in adjacent tissue,in which COX-2 expression is lower than that of cancer tissue.Compared with the respective negative virus control groups,medium-intensity fluorescence was observed.The COX-2 knockdown group had only weak fluorescence,while the overexpression COX-2 group showed brighter fluorescence.Compared with the negative virus control group,moderate fluorescence was observed in the stable transformant strain by 2a1.There was only weak fluorescence in the low COX2 group and bright red fluorescence in the overexpression COX-2 group.The fluorescence signal was mainly distributed in the cytoplasm of esophageal cancer cells,but no fluorescence signal was found in the extracellular and nuclear cells.As a research model,3D organoids are cell complexes that provide a compromise between 2D cell lines and living animals.For this purpose,the ESCC tissue,adjacent esophageal tissue,and normal esophageal tissue were collected and cultured to 3D esophageal organoids(EOs).As we expected,bright red fluorescence of 2a1 was observed in human ESCC EOs,compared to the adjacent esophageal EOs and normal esophageal EOs,in which only slight and no fluorescence was detected,respectively.These results strongly suggested that 2a1 can be developed to visualize COX-2 overexpressing cancer tissues in patients.Conclusion(1)COX-2 was upregulated in esophageal cancer tissues compared to paracancerous tissues.The patients with high expression of COX-2 had unfavorable overall survival.COX-2 was one of the independent prognostic factors for esophageal cancer patients.(2)Regulating the expression of COX-2 in esophageal cancer cells can affect the proliferation,migration,and invasion of in vitro and in vivo.(3)The expression of COX-2 is most closely related to signaling pathways such as proliferation,migration,and invasion phenotype of esophageal cancer cells.(4)COX-2 specific fluorescent probe can effectively distinguish esophageal cancer tissues from non-cancerous tissues and esophageal organs from different sources by targeting COX-2 protein,indicating that 2a1 has potential application value in early diagnosis of esophageal squamous cell carcinoma.