| Objective: Colorectal cancer(CRC)is one of the most common malignant tumors with high morbidity and mortality.The onset of colorectal cancer is relatively insidious,and patients generally have no obvious specific symptoms in the early stage.As the disease progresses,non-specific symptoms appear,which make the disease easy to be ignored.As a result,most patients with middle or late stages when they first see the doctor and lose radical surgery opportunity.If clinical intervention for early CRC is performed,the5-year survival rate of patients is expected to reach 90%,and once the tumor metastasizes,the 5-year survival rate of patients is only 14%.The onset of colorectal cancer goes through a series of processes such as local mucosal infiltration,regional spread,and distant metastasis.This multi-step process often involves the participation of multiple genes and signaling pathways.Therefore,it is particularly important to study the occurrence and development mechanism of colorectal cancer and find important molecular markers for early diagnosis to improve the prognosis of patients with colorectal cancer.CircularRNA(circRNA)is a non-codingRNA that is different from linearRNA.It is connected at the 3’and 5’ends by exon or intron and forms a complete loop structure through covalent bonds.It is not easily degraded by exonuclease,so it is more stable than linearRNA structure.CircRNA has a wide range of biological functions.Because of rich binding sites for miRNA,circRNA can act as molecular sponge for miRNA.Circ-LECRC is derived from the transcription of the YAP1(Yes1 associated transcriptional regulator)gene.There is another circRNA derived from YAP1 gene termed circYAP1 which has been reported in gastric cancer and breast cancer.It can inhibit the proliferation and metastasis of tumor cells and exert a tumor suppressor effect.MiR-135b-5p is a kind of miRNA,showing a significant high expression trend in colorectal cancer.It is reported in the literature that miR-135b-5p can promote the pathological process of colorectal cancer such as proliferation,invasion and metastasis by binding to KLF4 and other target genes.As a transcription factor of eukaryotes,KLF4 participates in the regulation of important physiological processes such as cell proliferation and differentiation.KLF4 was once named as a gastrointestinal enriched Krüppel-like factor.It is considered a tumor suppressor gene in colorectal cancer,and its expression is significantly down-regulated in cancer tissues.Studies have shown that the absence of KLF4 is an independent predictor of the prognosis of colorectal cancer patients.According to bioinformatics websites,miR-135b-5p has binding sites with circ-LECRC and KLF4 3’UTR.Based on the above theory,we speculate that circ-LECRC may bind miR-135b-5p through endogenous competition,thereby enhancing the tumor suppressor role of KLF4.To study the influence of circ-LECRC,miR-135b-5p and KLF4,this project intends to study the expression of circ-LECRC,miR-135b-5p and KLF4 in CRC tissues and cell lines,analyze its relationship with pathological data and prognosis,and study the malignant biology of circ-LECRC on CRC cells in vitro.This research focuses on the study of the relationship between the expression level of circ-LECRC and the pathological changes or prognosis of CRC,and explores its impact on the biological behavior of tumor cells in CRC cell lines,and then reveals the molecular mechanism of the occurrence and development of CRC.Furthermore,this study provides a basis for realizing the early diagnosis and treatment of CRC and improving the treatment effect and prognosis of CRC patients.Methods: 1.CircularRNA verification and stability test: sanger sequencing was used to determine that circ-LECRC was a circularRNA.The totalRNA was processed by RNase R,and the expression was detected by qPCR.The linear and circular stability differences of YAP1 before and after treatment were compared.2.The expression differences of Circ-LECRC in colorectal cancer tissues and cell lines: Collect cancer tissues and adjacent non-cancerous tissue specimens from patients undergoing colorectal cancer surgery in the First Affiliated Hospital of China Medical University,and cultivate human colorectal cancer cell lines: RKO,SW480,HCT116,HT29,SW620,DLD1 cells and normal intestinal mucosal epithelial cells(FHC).In situ hybridization was used to observe the expression of circ-LECRC in paraffin sections of patients with colorectal cancer,and analyze the relationship between its expression and the pathological data and prognosis of patients.The expression difference of circLECRC in fresh colorectal cancer tissues was detected by qPCR,and the relationship between its expression and various pathological data was analyzed.The expression differences between a variety of colon cancer cell lines and normal intestinal epithelium were detected by qPCR.3.Verify the effect of circ-LECRC on the phenotype of intestinal cancer cells: Transfect the plasmid and lentivirus into HCT116 and SW480 cells to construct a stable overexpression and knockdown cell line of circ-LECRC.The proliferation ability of HCT116 and SW480 cells was detected by CCK-8 experiment;the migration and invasion ability of HCT116 and SW480 cells were detected by Transwell experiment;the apoptosis and cycle of HCT116 and SW480 cells were detected by flow cytometry.The 4-week-old Balb/c nude mice were injected subcutaneously with SW480 cells overexpressing circ-LECRC and controls to monitor the tumor formation in nude mice.SW480 cells overexpressing circ-LECRC and controls were injected via tail vein of nude mice to observe lung metastasis.4.Conform circ-LECRC/miR-135b-5p/KLF4 regulatory network: Fluorescence in situ hybridization(FISH)was used to determine the location of circ-LECRC in HCT116 and SW480 cells.FISH experiment verified the co-localization relationship between circ-LECRC and miR-135b-5p.The dual-luciferase reporter experiment was used to directly verify the binding relationship between circ-LECRC/miR-135b-5p and miR-135b-5p/KLF4.Ago2 antibody was used forRNA immunoprecipitation(RIP)of SW480 cells to verify the binding relationship between circ-LECRC/miR-135b-5p/KLF4.5.Verify the expression of miR-135b-5p and KLF4 in colorectal cancer and its correlation with circ-LECRC: miR-135b-5p and KLF4 expression were detected in above colorectal cancer tissue used for detecting circ-LECRC.6.Further explore the regulatory relationship of circ-LECRC/miR-135b-5p/KLF4 via RIP experiment.Ago2 antibody was used to enrich circ-LECRC and KLF4 mRNA,and the regulatory changes of circ-LECRC and KLF4 in SW480 cells overexpressing circ-LECRC and NC group were detected.In HCT116 and SW480 cells overexpressing and knocking down circLECRC,Western Blot were used to detect the expression of the target gene KLF4.7.The effect of circ-LECRC/miR-135b-5p/KLF4 axis of action on the biological behavior of colorectal cancer cells: CCK8,Transwell invasion and migration experiments were performed to verify the competitive binding relationship of circ-LECRC/miR-135b-5p/KLF4 axis of action.Results: 1.Through sequencing and comparison,the new circRNA transcript of YAP1 in colorectal cancer was discovered and verified,and the stability of circularRNA was confirmed.2.The expression of circ-LECRC in tissues has no significant relationship with the age,sex,tumor location,tumor size and distant metastasis of colorectal cancer patients.The expression level was negatively correlated with the depth of tumor invasion,lymph node metastasis,and TNM stage.And the expression of circ-LECRC is negatively correlated with the prognosis of patients with colorectal cancer.Compared with normal epithelial FHC,circ-LECRC has lower expression in RKO,HCT116,HT29,SW480 and SW620 cells.3.CCK8 experiment shows that in HCT116 and SW480 cells,overexpression of circ-LECRC can inhibit the proliferation of colorectal cancer cells,and knockdown of circ-LECRC can enhance the proliferation of colorectal cancer cells;Transwell experiments show that in HCT116 and SW480 cells,Overexpression of circ-LECRC can inhibit the migration and ability of colorectal cancer cells.On the contrary,knocking down circ-LECRC can enhance the migration and invasion ability of colorectal cancer cells;also in HCT116 and SW480 cells,overexpression of circ-LECRC can promote colorectal cancer cells apoptosis,and knocking down circ-LECRC can inhibit the apoptosis of colorectal cancer cells.In the nude mouse tumor formation experiment,the tumor growth rate and final weight of the overexpression circ-LECRC group were smaller than those of the control group;in the lung metastasis experiment,the lung metastasis of the overexpression circ-LECRC group was smaller and less.4.FISH experiment verified that circ-LECRC and miR-135b-5p are mainly expressed in the cytoplasm of SW480.And bioinformatics predicted that miR-135b-5p binded to position 9 of circ-LECRC and binds to position924 of KLF4.The dual luciferase reporter experiments verified that miR-135b-5p binds to circ-LECRC and KLF4.The RIP experiment further confirmed that Ago2 antibody can pull down circ-LECRC and KLF4.5.miR-135b-5p is highly expressed in colorectal cancer tissues,and KLF4 is highly expressed in colorectal cancer tissues.And circLECRC was negatively correlated with the expression of miR-135b-5p,miR-135b-5p was negatively correlated with the expression of KLF4,and circ-LECRC was positively correlated with the expression of KLF4.6.Western Blot experiments confirmed that in HCT116 and SW480 cells,over-expression of circ-LECRC increased the expression of KLF4 protein,on the contrary,knocking down circ-LECRC decreased the expression of KLF4 protein.Ago2 antibody was used to enrich circ-LECRC and KLF4 with RIP.In SW480 cells overexpressed with circ-LECRC,the enrichment of Ago2 for circLECRC was significantly increased.In contrast,the enrichment of Ago2 for KLF4 was decreased.7.CCK8 and Transwell migration and invasion experiments showed that circ-LECRC regulates the expression of KLF4 by competitively binding miR-135b-5p,thereby affecting the biological characteristics of colorectal cancer cells.Conclusion: For the first time,a variant circularRNA transcript derived from YAP1gene(circ-LECRC)was found in colorectal cancer.It is confirmed that circ-LECRC is low expressed in colorectal cancer tissues,and the low expression level is closely related to the depth of tumor invasion,lymph node metastasis,TNM staging and prognosis.Circ-LECRC regulates the expression of KLF4 by competitively binding miR-135b-5p,thus inhibiting tumor cell proliferation,invasion and migration,and promotes cell apoptosis. |
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