The Role And Mechanism Of Pentraxin 3 (PTX3) In Esophageal Squamous Cell Carcinoma

Esophageal cancer has always been one of the major malignant tumor that threatens the health of Chinese residents.Henan Province is the high incidence areas of esophageal cancer.In recent years,the incidence and mortality of esophageal cancer have decreased,but the mortality rate of esophageal cancer in China is still the highest in the world.Esophageal cancer has its own characteristics,and the squamous cell carcinoma is the main pathological type,accounting for more than 90% of all types of esophageal cancer in China.The esophageal cancer is insidious,and the early symptoms are generally not obvious.Once the symptoms appeared,the patients are mostly in the advanced stage and lose the opportunity for surgery.Only comprehensive treatments such as radiotherapy and chemotherapy can be adopted,and the survival rate is very poor.The exact pathogenesis of esophageal cancer is still unclear,and the available treatment options are limited.Only by thoroughly exploring its pathogenesis and looking for new therapeutic targets can the patient's prognosis be effectively improved.In recent years,studies have generally suggested a potential link between cancer and inflammation,but the specific relationship needs further elaboration.PTX3 is a member of pentraxin family who is a typical acute phase protein expressed by hematopoietic cells and stromal cells in response to primary pro-inflammatory stimuli.It is one of the essential components of humoral immunity in innate immunity who is involved in the innate resistance to pathogens,regulation of inflammation and tissue remodeling.Studies have shown that PTX3 plays an important role in tumor progression,including tumorigenesis,angiogenesis,metastatic spread,and tumor immune regulation.However,the study of PTX3 in esophageal cancer,especially esophageal squamous cell carcinoma,has rarely been reported.Its specific role in esophageal squamous cell carcinoma and its related mechanisms need further exploration.We first conducted a systematic review and meta-analysis(Meta-analysis)of the association between PTX3 expression and survival outcomes in carcinoma to understand the relationship between PTX3 expression and overall survival(OS)and disease-free survival(DFS)in cancer patients.Then,we examined the protein expression of PTX3 in esophageal squamous cell carcinoma by immunohistochemical staining,and analyzed the relationship between PTX3 expression and clinicopathological parameters and prognosis.Then,we conducted an in-depth study on the role of PTX3 gene in esophageal squamous cell carcinoma.We used CRISPR/Cas9-mediated gene editing technology and p CDH-CMV-PTX3-EF1-cop GFP lentiviral vector to obtain the PTX3 knockout and overexpressing esophageal squamous cell carcinoma cell line KYSE450,and explored the effects of PTX3 gene on the biological characteristics and function of esophageal squamous cell carcinoma cells and related mechanisms.Finally,we performed a nude mouse tumor formation experiment to verify the role and related mechanisms of the PTX3 gene in vivo.Part I: Correlation between PTX3 expression in esophageal squamous cell carcinoma and clinicopathological parameters and prognosis Methods 1.Meta-analysis of the association between PTX3 expression and survival outcomes in cancer by searching MEDLINE,EMBASE,Cochrane CENTRAL and Web of Science databases.2.The protein expression of PTX3 in 294 esophageal squamous cell carcinoma tissues were detected by immunohistochemistry,and the relationship between PTX3 expression and clinicopathological parameters and survival time were analyzed.3.Statistical analysis was performed by SPSS 22.0 software.The correlation between PTX3 protein expression and clinical variables were analyzed by Chi-square or Fisher`s exact test.Patients' survival time was evaluated by Kaplan-Meier.Log-rank test was used to compare differences between groups,and COX regression was used to detect the effect of each indicator on prognosis.Results 1.The Meta-analysis proved that PTX3 was significantly related to prognosis in carcinoma regarding OS and DFS.Prospective studies are warranted to examine the association between PTX3 expression and survival outcomes in cancer.2.In 294 cases of esophageal squamous cell carcinoma tissue specimens,123/155(79.35%)of patients who were died within one year showed positive expression of PTX3 protein,and 74/139(53.24%)of patients who had survived for more than five years showed positive expression of PTX3 protein.The expression of PTX3 protein was negatively correlated with survival time(p=0.000),and positively correlated with TNM stage(p=0.020)and clinical stage(p=0.003).The expression of PTX3 was negatively correlated with overall patient survival.3.Multivariate analysis showed that PTX3 expression,age,tumor size,TNM stage and lymph node metastasis could be used as predictors of overall survival rate in patients with esophageal squamous cell carcinoma Part ?:Construction and functional analysis of PTX3 gene knockout and overexpression esophageal squamous cell carcinoma cell lines Methods 1.RT-PCR and Western Blot were used to verify the expression of PTX3 in esophageal immortalized cell lines and esophageal squamous cell carcinoma cell lines.2.The CRISPR/Cas9-mediated gene editing technology was used to knock out the PTX3 gene in the esophageal squamous cell carcinoma cell line KYSE450,a single clone was selected and then a stable PTX3 knockout cell line was established.At the same time,a PTX3 overexpressing KYSE450 cell line was established by constructed p CDH-CMV-PTX3-EF1-cop GFP lentiviral vector.3.The cell proliferative capacity was detected by Incucyte ZOOM system and plate cloning experiments.Flow cytometry was used to examine cell cycle and cell apoptosis.Cell migration capacity was determined by transwell experiments and scratch assays.The sensitivity of cell chemotherapy and radiotherapy were detected by Incucyte ZOOM system and colony formation experiments.Seahorse XFe96 was performed to detect cell energy metabolism,through which can reflect the changes of glycolytic and oxidative phosphorylation patterns of esophageal squamous cell carcinoma cells after PTX3 gene knockout.The intracellular ROS content was detected by flow cytometry.The angiogenesis experiment simulated the angiogenesis of cells.The ability of cell self-renewal was observed by the ball-forming experiment.The ultrastructural changes of cells were observed under electron microscope.Results 1.The gene and protein expression of PTX3 in different cell lines showed that the expression of PTX3 was highest in KYSE450 cell line,and the expression of PTX3 in esophageal squamous cell carcinoma cell line(KYSE140,EC109 and KYSE450)were higher than that in immortalized esophageal epithelial cell line Het1 A.2.KYSE450 cell line model with stable knockout of PTX3 gene was successfully constructed and named as KYSE450 PTX3-/-.A stable cell line overexpressing PTX3 gene was named as KYSE450 PTX3-OV.3.After PTX3 gene was knockout,proliferation of cells was slowed down,cell cycle was inhibited,cell apoptosis increased,migration ability was weakened,chemotherapy/radiotherapy sensitivity was higher,glycolysis metabolism was decreased and oxidative phosphorylation was enhanced;ROS production was reduced,the ability to angiogenesis and self-renewal was weakened,protein synthesis and mitochondrial division activity were diminished.Conversely,PTX3 overexpressing cells promoted cell proliferation,accelerated cell cycle,reduced apoptosis,enhanced migration,and reduced sensitivity to chemotherapy/radiation.Part ?:Mechanism of action of PTX3 in esophageal squamous cell carcinoma Methods 1.The upstream regulatory site of PTX3 was predicted.CHIP and EMSA were performed to verify whether SOX9 directly regulates the transcriptional expression of PTX3.2.Cells were transfected to decrease the expression of SOX9,and cell proliferation,cycle and apoptosis were observed after SOX9 silencing.3.The expression of SOX9 in human esophageal squamous cell carcinoma was detected by immunohistochemistry,and correlated with PTX3 expression.The co-localization of SOX9 and PTX3 in esophageal squamous cell carcinoma was detected by immunofluorescence.4.The transcriptome sequencing of KYSE450 and KYSE450 PTX3-/-cells were performed to detect differential gene expression and changes in differential gene-related GO and KEGG pathways.Results 1.CHIP and EMSA experiments demonstrated that SOX9 located in the promoter region at the upstream of PTX3 and directly regulated the transcriptional expression of PTX3.2.Decreased expression of SOX9 caused a decrease in the expression of PTX3,which slowed cell proliferation,increased cell cycle inhibition and cell apoptosis.3.Immunohistochemical detection showed that the expression of SOX9 and PTX3 was positively correlated,and the tissues with high SOX9 expression,PTX3 expression were also high,and vice versa.The result of immunofluorescence assay showed that the colocalization expression between SOX9 and PTX3 was positively correlated in patients with esophageal squamous cell carcinoma.All these results indicated a positive correlation between SOX9 and PTX3 in esophageal squamous cell carcinoma.4.Sequencing detection analysis showed that the human leukocyte antigen(HLA)system-related genes of KYSE450 PTX3-/-cells were significantly up-regulated,and the results of RT-PCR were consistent with those of transcriptome sequencing.The staining results of HLA system-related proteins in esophageal squamous cell carcinoma demonstrated that HLA Class I ABC,HLA DR+DP+DQ and CD74 expression were correlated with the survival time of patients with esophageal squamous cell carcinoma,showing a significant positive correlation.Part IV:Effect of PTX3 on tumorigenic ability in vivo and related mechanisms Methods 1.Nude mouse tumor formation experiments were done to observe the changes in tumor cell formation ability,and sensitivity to docetaxel.2.HE staining was used to observe the histopathological structure.Immunohistochemistry was used to detect the expression of PTX3 and HLA-related proteins in nude mice.Results 1.KYSE450 PTX3-/-cells grew slowly in vivo,and the formed tumors were small in size and sensitive to docetaxel.Conversely,KYSE450 PTX3-OV cells grew faster than the control group and were not sensitive to docetaxel.2.The expression of PTX3 in KYSE450 PTX3-/-group was significantly down-regulated in nude mice,and only a small number of cells were weakly positively expressed.In KYSE450 PTX3-/-administration group,PTX3 expression was basically negative.The expression of HLA Class I ABC,HLA DR+DP+DQ and CD74 of KYSE450 PTX3-/-group was significantly higher than that in KYSE450 group.Conclusions 1.PTX3 was significantly related to prognosis in carcinoma regarding OS and DFS.Prospective studies are warranted to examine the association between PTX3 expression and survival outcomes in carcinoma.In human esophageal squamous cell carcinoma,the protein expression of PTX3 was negatively correlated with survival time,and positively correlated with TNM stage and clinical stage.The high expression of PTX3 was significantly correlated with the overall survival of patients,indicating that PTX3 plays an important role in the development process of esophageal squamous cell carcinoma.2.PTX3 affected cell proliferation,cycle,apoptosis,migration and sensitivity of chemotherapy/radiotherapy.KYSE450 PTX3-/-cells had reduced glycolysis,enhanced oxidative phosphorylation,decreased ROS production,decreased angiogenic capacity and self-renewal capacity.Protein synthesis and mitochondrial division activity were attenuated.All the results suggested that PTX3 can be used to assess the malignancy of esophageal squamous cell carcinoma and a potential therapeutic target.3.The expression of SOX9 was positively correlated with the expression of PTX3 in esophageal squamous cell carcinoma.SOX9 directly regulated the expression of PTX3.After PTX3 was knockout,the HLA system related genes were up-regulated,indicating that the role of PTX3 in esophageal squamous cell carcinoma may be due to the regulation of SOX9,which in turn affects the HLA system.4.PTX3 affected the tumorigenic ability of cells and the sensitivity to docetaxel.The expression of HLA Class I ABC,HLA DR+DP+DQ and CD74 in KYSE450 PTX3-/-group were higher which verified the results of in vitro experiment.The role of PTX3 in esophageal squamous cell carcinoma was further illustrated.