The Efffect Of Hsa_circ₀009361 During The Progression Of Colorectal Cancer And Its Mechanism

Colorectal cancer?CRC?is one of the most common malignancies in the world with high morbidity and mortality.Recently,although the clinical treatment technology of CRC has made great progress,the prognosis of patients with advanced disease is still very poor.Local invasion and distant metastasis are the main causes of death.Therefore,it is necessary to find the driving factors of CRC growth and metastasis,explore the mechanism of CRC progression,and find new targets for treatment.The inactivation of the adenomatous polyposis coli?APC?and its homologous product APC2 or the oncogenic mutation of ?-catenin is the rate-limiting event of most sporadic CRC.?-catenin exhibits a high signal in tumor cells locating at the invasion front or migrating to adjacent matrices.This differential Wnt signaling activity reflects the different proliferation and epithelial-mesenchymal transition?EMT?capabilities of cells,leading to malignant behaviors,such as tumor growth,invasion and metastasis.Circular RNA?circRNA?is a special RNA with a circular closed structure,which can indirectly regulate the expression of target genes by competitively binding to corresponding microRNAs?miRNAs?,and plays an important role in post-transcriptional regulation,also known as the competitive endogenous RNA?ceRNA?mechanism.Some circRNAs are found to be abnormally expressed in CRC and are closely related to tumor size,clinical stage and prognosis.However,as a ceRNA,the competitive regulation pattern between circRNA and miRNA and its downstream target genes in the development of CRC,especially in EMT-related invasion and metastasis is not clear,which needs further investigation.This study consists of three parts.Part I screened the differentially expressed circRNAs between CRC and paracancerous tissues by circRNA chip,and detected the expression level of hsa_circ₀009361 in CRC tissues and cells.Part ? clarifies the biological function of hsa_circ₀009361 in CRC by in vitro and in vivo experiments.Part III clarified the mechanism of the ceRNA network "hsa_circ 0009361/miR-582/APC2"involved in CRC progression through bioinformatics methods and cell experiments.Part I the expression characteristics of hsa circ 0009361 in CRC tissues and cellsObjective:To clarify the expression profile of circRNAs in CRC tissues and paracancerous tissues,and screen for the most differentially expressed circRNA.Methods:The expression profiles of circRNAs in 10 pairs of CRC tissues and adjacent non-tumor tissues was analyzed by circRNA microarray.qRT-PCR was used to verify the circRNAs expression in 20 cases of fresh CRC tissues.The most differentially expressed circRNA was selected and its expression level was detected in CRC cell lines by qRT-PCR.Results:A total of 48 circRNAs with significantly different expression between cancer and paracancerous tissues were screened from 15,095 circRNAs?Fold Change>2,P<0.05?,of which 40 were up-regulated and eight were down-regulated in CRC tissues.The top five up-regulated circRNAs?hsa_circ₀000423,hsa_circ₀000512,hsa_circ₀000511,hsa_circ₀000467 and hsa_circ₀000231?and the top five down-regulated circRNAs?hsa_circ₀009361,hsa_circ₀012185,hsa_circ₀001455,hsa_circ₀000775 and hsa circ 0001669?were verified by qRT-PCR.We found that hsa_circ₀009361 was the most down-regulared circRNA in CRC tissues?Fold Change=31.25,P<0.001?.Compared with human normal colonic epithelial cells NCM460,hsa_circ₀009361 was significantly down-regulated in CRC cells HT29,SW480,HCT-116,Lovo,SW48,DLD-1 and Caco-2.Conclusion:There are more abnormally up-regulated circRNAs than down-regulated circRNAs in CRC tissues.Hsa_circ₀009361 is the most down-regulated circRNA in CRC tissues,and its expression level in most CRC cell lines is significantly lower than that in normal colonic epithelial cells.Part 11 the biological function of hsa_circ₀009361 in CRCObjective:To clarify the biological function of hsa_circ₀009361 in CRC cells and animal models.Method:The hsa_circ₀009361 overexpression vector and the siRNA covering the reverse splicing region of hsa_circ₀009361 were transfected into HCT-116 cells and Lovo cells,respectively,and HCT-116 cells with hsa_circ₀009361 overexpression and Lovo cells with hsa_circ₀009361 silenced were established.Cell proliferation,apoptosis,migration and invasion were detected by CCK-8 proliferation assay,flow cytometry,wound healing assay and Transwell invasion assay,respectively.HCT-116 cells transfected with hsa_circ₀009361 overexpression vector,or empty vector or without transfection were subcutaneously inoculated into the back of BALB/c nude mice to establish a xenograft model,and the tumor volume and weight were measured periodically.The above three types of HCT-116 cells were injected into BALB/c nude mice through the tail vein respectively to establish a metastasis model,and the number of lung metastases was observed and compared.The expression levels of APC2,E-cadherin and ?-catenin in mice tumor tissues were detected by immunohistochemistry?IHC?.Results:Overexpression of hsa_circ₀009361 in HCT-116 cells inhibited cell proliferation,promoted apoptosis,and significantly reduced cell migration and invasion.Silencing hsa_circ₀009361 in Lovo cells promoted cell migration and invasion,but had no significant effect on cell proliferation and apoptosis.In addition,overexpression of hsa_circ₀009361 significantly increased the expression of E-cadherin and decreased the vimentin level in HCT-116 cells,while silencing hsa_circ₀009361 significantly decreased the expression of E-cadherin and increased the vimentin level in Lovo cells.In animal experiments,the mean volume and weight of tumors,as well as the number of lung metastases in the hsa_circ₀009361 overexpression group were significantly lower than those in the control groups.IHC showed that the expression levels of APC2 and E-cadherin in tumor tissues with hsa_circ₀009361 overexpressed were increased compared with the controls,while the expression level of ?-catenin was decreased.Conclusion:Hsa_circ₀009361 can inhibit the proliferation and promote the apoptosis of CRC cells.It positively and negatively regulates the expression of E-cadherin and vimentin,respectively:by inhibiting EMT,it significantly reduces the migration and invasion capabilities of CRC cells.In vivo,hsa_circ₀009361 also significantly inhibits the growth and metastasis of CRC,possibly related to Wnt/?-catenin pathway activity.Part III the regulation mechanism of the ceRNA network"hsa circ 0009361/miR-582/APC2”involved in CRC progressionObjective:To identify the miRNA adsorbed by hsa circ 0009361 and the downstream target gene,and elucidate the regulation mechanism of hsa_circ₀009361 in the progression of CRC.Method:The candidate miRNAs with potential binding to hsa_circ₀009361 were screened by bioinformatics methods,and their expression levels in CRC tissues were detected by qRT-PCR.Overexpressing or silencing hsa_circ₀009361 in CRC cells,and the changes in target miRNA expression were observed.Fluorescence in situ hybridization?FISH?was used to observe the colocalization of hsa circ 0009361 with the target miRNA in HCT-116 cells.RNA immunoprecipitation?RIP?and dual luciferase reporter assays were used to verify the binding of hsa_circ₀009361 to the target miRNA.The target genes that may be regulated by the target miRNA were screened by bioinformatics methods,and RIP and dual luciferase reporter assays were used to verify binding of the target miRNA to the target gene.Regulating the expression of hsa_circ₀009361 and the target miRNA in HCT-116 cells,the expression of the target gene was detected by Western blotting,and the activity of Wnt/?-catenin pathway was evaluated by TOP/FOP Flash dual luciferase reporter assay.Rescue experiments were conducted to verify the above results.Results:Among the candidate miRNAs with complementary sequences to hsa_circ₀009361 screened by bioinformatics methods,the expression of miR-582 in CRC tissues was negatively correlated with hsa_circ₀009361,and the expression of miR-582 was also negatively regulated by hsa_circ₀009361 in CRC cells.RNA FISH showed that hsa_circ₀009361 and miR-582 colocalized in the cytoplasm of HCT-116.RIP and dual luciferase reporter experiments showed that hsa_circ₀009361 could directly bind to miR-582.APC2 was identified as a target gene regulated by miR-582 by bioinformatics methods,RIP and dual luciferase reporter assays.The expression of APC2 and the activity of Wnt/p-catenin pathway were regulated by hsa_circ₀009361 and miR-582.Rescue experiments confirmed that miR-582 and APC2 could reverse the changes in proliferation,migration and invasion ability of CRC cells induced by hsa_circ₀009361.Conclusion:Hsa_circ₀009361 is a sponge of miR-582,and the Wnt/?-catenin pathway inhibitor APC2 is a target gene regulated by miR-582.Hsa_circ₀009361 can act as a ceRNA to indirectly up-regulate the expression of APC2 and inhibit the activity of the Wnt/?-catenin pathway by competitively binding to miR-582.The abnormally down-regulation of hsa_circ₀009361 in CRC attenuates its tumor suppressive effect and promotes the progression of CRC.