Validation,Functional Study And Detection System Of RAD23B And TSP-1 For Colorectal And Esophageal Cancer Markers

Digestive tract tumors are the most common tumors in China,accounting for about 50% of the total cancer morbidity and mortality in China,among which colorectal cancer and esophageal cancer rank in the top five.At present,the common feature of the clinical application of markers for digestive tract tumors is low sensitivity,and itis not suitable for early diagnosis of digestive tract tumors.As a result,when the digestive tract tumors were found,that is usually in the middle and late stage.Therefore,finding new markers for digestive tract tumors is an urgent scientific problem facing oncologists.Firstly,Chips and Dot blot technique is used to measure RAD23B in colorectal carcinoma tissues and urine of colorectal cancer.we found that compared with the tissue adjacent to carcinoma,colorectal cancer RAD23B expression level increased obviously.The increased cytoplasmic RAD23B of colorectal cancer tissues was related to colorectal cancer pathology classification,AJCC staging and colorectal cancer liver metastasis.Kapan-Meier survival analysis showed that the increased cytoplasmic RAD23B of colorectal cancer tissues was related to colorectal cancer patients' overall survival(OS)shorten.RAD23B in the urine of colorectal cancer patients was measured by Dot blot,we found that the expression of RAD23B was significantly highercompared with healthy controls,and it was correlated with colorectal cancer liver metastasis.When the urine RAD23B was used todiagnose CRC and CRC-LM,the area under the ROC curve was 0.623 and 0.637,respectively.These findings suggest that RAD23B may be a new biomarker for olorectal cancer liver metastasis.Next,we investigated the biological function of RAD23B in colorectal cancer in vivo and in vitro.Firstly,the RAD23Bof colorectal cancer SW480 and HCT8 cells was knocked down by molecular biology techniques in vitro,which lead to reducethe ability of proliferation,invasion and metastasis of colorectal cancer.The proliferation and liver metastasis of colorectal cancer was also inhibited in vivo when RAD23B was knockdown.Then,using RNA-seq technology to explore the mechanism,we found that Integrin signaling pathway was suppressed when we koockdown the expression of RAD23B.Western blot confirm Talin1/2/Integrin/FAK/RhoA signalling pathways were significantly inhibited.We further analyzed Rad23B binding proteins in colorectal cancer using IP MASS technique,and found that RAD23B interacts with COROIC.Immunoprecipitation and immunofluorescence confirmed the interaction and co-localization of RAD23B and CORO1c at protein,cell and tissue levels in colorectal cancer.Overexpressed RAD23B promoted the co-localization of CORO1C with RAC1 and Cortactin at the cell margin,and promoted the formation of invadopodia,while silencedd RAD23B could inhibit CORO1C aggregating to the cell margin.So we speculated that RAD23 regulation invasion and metastasis of colorectal cancer by Talin1/2/integrin/FAK/RhoA/RAC1/CORO1C axis.Finally,previous laboratory studies have shown that the expression of TSP-1 peptide is significantly increased in the plasma of esophageal patients,which is a potential marker for early diagnosis of esophageal cancer.Thromboreactive protein-1 belongs to the platelet clotting protein family and has a variety of biological functions and plays a role in cell adhesion,migration,proliferation and angiogenesis.In tumors,TSP-1 plays different roles,either promoting or inhibiting tumor progression,depending on the tumor.Studies have shown that TSP-1 in esophageal cancer tissues and plasma was significantly higher compared with paracancerous tissues and healthy controls,which is a potential biomarker for early esophageal cancer.In this study,hybridoma technique was used to prepare monoclonal antibodies against the N-terminus and C-terminus of the TSP-1 peptide,and the plasma TSP-1 chemiluminescence detection system was established and initially verified.we found that TSP-1 increased accompanied with the progression of esophageal dysplasia.When the esophageal disease progressed to the stage of esophageal cancer,the plasma TSP-1 level decreased,but it was still significantly higher than that of healthy controls.When theTSP-1 was used todiagnose esophageal atypical hyperplasia and esophageal cancer,the area under the ROC curve was 0.938 and 0.66,respectively.The results suggestedthat TSP-1 could be used to ascreen esophageal cancer.