Expression And Function Study Of Circ＿cse1l In Colorectal Cancer

Part 1: The expression and significance of Circ＿cse1l in colorectal cancerObjective: To detect the expression difference of circ＿cse1l（hsa＿circ＿0060745）derived from the exon circularization of CSE1 L gene in colorectal cancer tissue and normal mucosa tissue,as well as in the serum of colorectal cancer patients and normal human serum.The relationship between the expression of circ＿cse1l and the clinicopathological parameters of colorectal cancer patients was analyzed.Methods:1.Take 50 cases of colorectal cancer patients’ tumor tissues and normal tissues about 10 cm away from the tumor tissue for HE staining identification.2.Using circBan K and qRT-PCR,agarose electrophoresis technology to screen the circular non-coding RNA derived from the CSE1 L gene.3.The qRT-PCR technology was used to detect the expression of circ＿cse1l in colorectal cancer tissues and normal mucosal tissues,as well as in the serum of colorectal cancer patients and normal human serum.4.Further statistical analysis of the correlation between the expression of circ＿cse1l and clinicopathological parameters of colorectal cancer patients.Results:1.Using qRT-PCR,agarose electrophoresis technology to screen the circular RNA formed by CSE1 L gene,and determine circ＿cse1l（hsa＿circ＿0060745）as the research objectBy searching the circBan K database,it was found that the CSE1 L gene can form a variety of circular RNAs.For 12 kinds of circular RNAs,primers for the cross-circularization site were designed and qRT-PCR detection was performed.It was found that the expression of circ＿cse1l（hsa＿circ＿0060745）was relatively stable and has high expression level.Therefore,it was chosen as the research object.2.The expression of circ＿cse1l in colorectal cancer tissues and serum was significantly reducedThe qRT-PCR test results showed that compared with the control group,the expression of circ＿cse1l in colorectal cancer tissues and serum was significantly decreased,and the expression difference was statistically significant（P<0.05）.3.The expression level of circ＿cse1l is negatively correlated with the depth of tumor invasionThe T test was used to further analyze the relationship between the expression of circ＿cse1l and the clinicopathological data of patients with colorectal cancer.It was found that the expression level of circ＿cse1l was negatively correlated with the depth of tumor invasion（P<0.05）.At the same time,it was found that the expression level of circ＿cse1l had no significant correlation with the patient’s gender,age,tumor size,degree of differentiation,lymph node metastasis,and tumor location.Summary:The expression level of circ＿cse1l in colorectal cancer tissues and serum was significantly decreased,and its expression level was negatively correlated with the depth of tumor invasion,which could be used to evaluate the prognosis of colorectal cancer patients to a certain extent.Part 2: The effect of circ＿cse1l on the biological behavior of colorectal cancer cell linesObjective: By overexpressing circ＿cse1l in tumor cell lines,the effects of circ＿cse1l on the proliferation,migration,invasion of colorectal cancer cells were investigated.Methods:1.Expression of circ＿cse1l in human normal cell lines FHC,as well as colon cancer cell lines HT29,HCT116 and Lo Vo were detected by qRT-PCR.2.The pc DNA3.1 was used to construct the overexpression vector of circ＿cse1l.The HT29 and HCT116 cell lines were transfected with Lipo8000^TM,and the transfection efficiency was detected by qRT-PCR.3.CCK-8,colony formation,transwell and wound healing assays were used to detect the effects of circ＿cse1l overexpression on the proliferation,migration and invasion of colorectal cancer cells.Results:1.The expression of circ＿cse1l in tumor cells is lower than that of normal cellsThe expression of circ＿cse1l in tumor cell lines HT29,HCT116 and Lo Vo was significantly lower than its expression in cell line FHC,and the difference was statistically significant（P<0.05）.2.Successfully overexpressed circ＿cse1l in cell lines HT29 and HCT116After overexpression of circ＿cse1l in HT29 and HCT116 cells,it was found that compared with the control group,the expression of circ＿cse1l in the overexpression group was significantly increased（P<0.05）.3.Overexpression of circ＿cse1l can inhibit the proliferation of colorectal cancer cellsCCK-8 experiment results showed that compared with the control group,the proliferation of HT29 and HCT116 cells was inhibited after overexpression of circ＿cse1l.The plate cloning experiment found that the number of cell clones in the overexpression group was significantly reduced（P<0.05）.The above experiments show that circ＿cse1l can significantly inhibit the proliferation of colorectal cancer cells.4.Overexpression of circ＿cse1l can inhibit the migration and invasion of colorectal cancer cellsThe results of the cell scratch test showed that: compared with the control group,the overexpression group cells grew slowly,and the cell growth and migration ability was inhibited.Transwell experimental results showed that:compared with the control group,overexpression of circ＿cse1l can significantly inhibit the migration and invasion of colorectal cancer cells（P<0.05）.Summary:The expression of circ＿cse1l in tumor cells is lower than that of normal cells.Overexpression of circ＿cse1l can significantly inhibit the proliferation,migration and invasion of colorectal cancer cells.Part 3: Molecular mechanism of Circ＿cse1l inhibiting the proliferation of colorectal cancer cellsObjective: To explore the possible molecular mechanism of circ＿cse1l inhibiting the proliferation of colorectal cancer cells.Methods:1.Western blot was used to detect the expression of proliferation-related protein PCNA in HT29 and HCT116 cell lines after overexpression of circ＿cse1l.2.Use the database Circ Interactome to predict the possible binding proteins of circ＿cse1l.3.RNA-binding protein immunoprecipitation（RIP）and qRT-PCR verified the binding of circ＿cse1l and EIF4A3.4.RNA-binding protein immunoprecipitation（RIP）,qRT-PCR,and agarose electrophoresis were used to detect the expression of PCNA m RNA immunoprecipitated by anti-e IF4A3.Results:1.Overexpression of circ＿cse1l can down-regulate the expression of PCNA proteinThe expression of PCNA in HT29 and HCT116 cells after overexpression of circ＿cse1l was detected by Western blot.The results showed that,compared with the control group,overexpression of circ＿cse1l could down-regulate the protein expression of PCNA（P<0.05）.2.EIF4A3 was the binding protein of circ＿cse1lThrough the prediction of the database Circ Interactome and the verification of RNA-binding protein immunoprecipitation（RIP）,it was found that EIF4A3 was the binding protein of circ＿cse1l.3.Circ＿cse1l downregulated PCNA expression by binding to e IF4A3Compared with the control group,the expression of PCNA m RNA immunoprecipitated by anti-e IF4A3 was reduced by circ＿cse1l overexpression（P<0.05）.Summary:Circ＿cse1l may inhibited the proliferation of colorectal cancer cells by down-regulating the expression of PCNA.In addition,it was confirmed that EIF4A3 was a binding protein of circ＿cse1l,and circ＿cse1l may be combined with EIF4A3,thereby down-regulating the expression of PCNA.Conclusions:1.The expression of circ＿cse11 in colorectal cancer tissues,serum and colorectal cancer cell lines is reduced,and its expression level is negatively correlated with the depth of tumor invasion,which can assess the prognosis of colorectal cancer patients to a certain extent.2.Overexpression of circ＿cse1l can significantly inhibit the proliferation,migration and invasion of colorectal cancer cells.3.Circ＿cse1l may down-regulate the expression of PCNA by binding to EIF4A3,thereby inhibiting the proliferation of colorectal cancer cells.