Relationship Between Serine Protease HtrA3 And Colorectal Cancer And Its Influence On Colorectal Cancer Phenotype

ObjectiveColorectal cancer is a common malignant tumor of digestive system.Its occurrence and development involve multiple genes,and there are still no efficient and specific clinical markers.In this study,serine protease HtrA3 has been selected based on proteomics technology.We determined HtrA3 expression in the tissue specimens and performed cell function experiments to investigate the effect of HtrA3 expression on the phenotype of colorectal cancer cells.We aimed to provide potential protein marker for early diagnosis and prognostic treatment of colorectal cancer.Methods1.(1)Totally 25 and 24 pairs of colorectal cancer tissues and corresponding normal tissues were selected according to the inclusion criteria.HtrA3 m RNA and protein expression level were detected by qPCR and western blot,respectively.(2)According to the inclusion criteria,75 pairs of colorectal cancer tissues and corresponding normal tissues were selected to verify HtrA3 protein expression by immunohistochemistry,and the effect of HtrA3 expression on patients’ survival was investigated.Meanwhile,EORTC QLQ-C30 and QLQ-CR38 follow-up survey scale were used to analyze the postoperative quality of life of patients using time to deterioration model.2.Using bioinformatics analysis,TCGA,GEO and CPTAC databases were selected to analyze the carcinogenic effect of HtrA3 for pan-cancer analysis.The association between HtrA3 and common digestive system tumors was explored in terms of gene expression,survival prognosis,genetic alternation,immune infiltration and related mechanistic pathways.3.(1)Western blot was used to investigate HtrA3 expression in five colorectal cancer cell lines;(2)HCT116 cell with stable overexpression of HtrA3 and SW480 cell with transient silencing of HtrA3 were constructed by lentiviral infection of colorectal cancer cells.Western blot was used to verify the expression of HtrA3;(3)CCK-8 assay,wound-healing assay,transwell cell migration assay and transwell cell invasion assay were used to detect the effects of HtrA3 overexpression and silencing on the proliferation,vertical migration and invasion of colorectal cancer cells.Results1.(1)QPCR and western blot suggested that both transcriptional and protein expression levels of HtrA3 were higher expression in colorectal cancer tissues than those in corresponding normal tissues;(2)Immunohistochemistry further verified that HtrA3 expression was high expressed in colorectal cancer tissues,but no correlation was found between HtrA3 expression and patient prognosis and survival.2.(1)For common digestive system tumors,only in colon adenocarcinoma,the expression level of HtrA3 was higher than that in the corresponding normal tissues.Besides,high HtrA3 expression was correlated with short overall survival of patients;(2)HtrA3 genetic alterations were not associated with patient survival,and HtrA3 expression was positively correlated with infiltration values of tumor-associated fibroblasts.Besides,the role of HtrA3 in tumor pathogenesis may be related to "PI3K-Akt signaling pathway","focal adhesion","protein digestion and absorption",and "ECM-receptor interaction".3.(1)HCT116 cell line with stable overexpression of HtrA3 and SW480 cell line with transient silencing of HtrA3 were constructed.The expression level of HtrA3 was verified by western blot;(2)HtrA3 over-expression could promote the proliferation,vertical migration and invasion of HCT116 cell.When HtrA3 was knocked down,the proliferation,vertical migration and invasion of SW480 cell were inhibited.Conclusion HtrA3 is up-regulated in colorectal cancer tissues and can promote the proliferation,vertical migration as well as invasion of colorectal cancer cells,and high expression of HtrA3 may promote the occurrence and development of colorectal cancer.