The Effect Of Hsa＿circ＿0009361 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 β-catenin is the rate-limiting event of most sporadic CRC.β-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（circ RNA）is a special RNA with a circular closed structure,which can indirectly regulate the expression of target genes by competitively binding to corresponding micro RNAs（mi RNAs）,and plays an important role in post-transcriptional regulation,also known as the competitive endogenous RNA（ce RNA）mechanism.Some circ RNAs are found to be abnormally expressed in CRC and are closely related to tumor size,clinical stage and prognosis.However,as a ce RNA,the competitive regulation pattern between circ RNA and mi RNA 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 circ RNAs between CRC and paracancerous tissues by circ RNA chip,and detected the expression level of hsa＿circ＿0009361 in CRC tissues and cells.Part II clarifies the biological function of hsa＿circ＿0009361 in CRC by in vitro and in vivo experiments.Part III clarified the mechanism of the ce RNA network “hsa＿circ＿0009361/mi R-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 circ RNAs in CRC tissues and paracancerous tissues,and screen for the most differentially expressed circ RNA.Methods:The expression profiles of circ RNAs in 10 pairs of CRC tissues and adjacent non-tumor tissues was analyzed by circ RNA microarray.q RT-PCR was used to verify the circ RNAs expression in 20 cases of fresh CRC tissues.The most differentially expressed circ RNA was selected and its expression level was detected in CRC cell lines by q RT-PCR.Results:A total of 48 circ RNAs with significantly different expression between cancer and paracancerous tissues were screened from 15,095 circ RNAs（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 circ RNAs（hsa＿circ＿0000423,hsa＿circ＿0000512,hsa＿circ＿0000511,hsa＿circ＿0000467 and hsa＿circ＿0000231）and the top five down-regulated circ RNAs（hsa＿circ＿0009361,hsa＿circ＿0012185,hsa＿circ＿0001455,hsa＿circ＿0000775 and hsa＿circ＿0001669）were verified by q RT-PCR.We found that hsa＿circ＿0009361 was the most down-regulared circ RNA in CRC tissues（Fold Change=31.25,P<0.001）.Compared with human normal colonic epithelial cells NCM460,hsa＿circ＿0009361 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 circ RNAs than down-regulated circ RNAs in CRC tissues.Hsa＿circ＿0009361 is the most down-regulated circ RNA in CRC tissues,and its expression level in most CRC cell lines is significantly lower than that in normal colonic epithelial cells.Part II the biological function of hsa＿circ＿0009361 in CRCObjective: To clarify the biological function of hsa＿circ＿0009361 in CRC cells and animal models.Method: The hsa＿circ＿0009361 overexpression vector and the si RNA covering the reverse splicing region of hsa＿circ＿0009361 were transfected into HCT-116 cells and Lovo cells,respectively,and HCT-116 cells with hsa＿circ＿0009361 overexpression and Lovo cells with hsa＿circ＿0009361 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＿circ＿0009361 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 β-catenin in mice tumor tissues were detected by immunohistochemistry（IHC）.Results: Overexpression of hsa＿circ＿0009361 in HCT-116 cells inhibited cell proliferation,promoted apoptosis,and significantly reduced cell migration and invasion.Silencing hsa＿circ＿0009361 in Lovo cells promoted cell migration and invasion,but had no significant effect on cell proliferation and apoptosis.In addition,overexpression of hsa＿circ＿0009361 significantly increased the expression of E-cadherin and decreased the vimentin level in HCT-116 cells,while silencing hsa＿circ＿0009361 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＿circ＿0009361 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＿circ＿0009361 overexpressed were increased compared with the controls,while the expression level of β-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＿circ＿0009361 also significantly inhibits the growth and metastasis of CRC,possibly related to Wnt/β-catenin pathway activity.Part III the regulation mechanism of the ce RNA network “hsa＿circ＿0009361/mi R-582/APC2” involved in CRC progressionObjective: To identify the mi RNA adsorbed by hsa＿circ＿0009361 and the downstream target gene,and elucidate the regulation mechanism of hsa＿circ＿0009361 in the progression of CRC.Method: The candidate mi RNAs with potential binding to hsa＿circ＿0009361 were screened by bioinformatics methods,and their expression levels in CRC tissues were detected by q RT-PCR.Overexpressing or silencing hsa＿circ＿0009361 in CRC cells,and the changes in target mi RNA expression were observed.Fluorescence in situ hybridization（FISH）was used to observe the colocalization of hsa＿circ＿0009361 with the target mi RNA in HCT-116 cells.RNA immunoprecipitation（RIP）and dual luciferase reporter assays were used to verify the binding of hsa＿circ＿0009361 to the target mi RNA.The target genes that may be regulated by the target mi RNA were screened by bioinformatics methods,and RIP and dual luciferase reporter assays were used to verify binding of the target mi RNA to the target gene.Regulating the expression of hsa＿circ＿0009361 and the target mi RNA in HCT-116 cells,the expression of the target gene was detected by Western blotting,and the activity of Wnt/β-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 mi RNAs with complementary sequences to hsa＿circ＿0009361 screened by bioinformatics methods,the expression of mi R-582 in CRC tissues was negatively correlated with hsa＿circ＿0009361,and the expression of mi R-582 was also negatively regulated by hsa＿circ＿0009361 in CRC cells.RNA FISH showed that hsa＿circ＿0009361 and mi R-582 colocalized in the cytoplasm of HCT-116.RIP and dual luciferase reporter experiments showed that hsa＿circ＿0009361 could directly bind to mi R-582.APC2 was identified as a target gene regulated by mi R-582 by bioinformatics methods,RIP and dual luciferase reporter assays.The expression of APC2 and the activity of Wnt/β-catenin pathway were regulated by hsa＿circ＿0009361 and mi R-582.Rescue experiments confirmed that mi R-582 and APC2 could reverse the changes in proliferation,migration and invasion ability of CRC cells induced by hsa＿circ＿0009361.Conclusion: Hsa_circ₀009361 is a sponge of mi R-582,and the Wnt/β-catenin pathway inhibitor APC2 is a target gene regulated by mi R-582.Hsa_circ₀009361 can act as a ce RNA to indirectly up-regulate the expression of APC2 and inhibit the activity of the Wnt/β-catenin pathway by competitively binding to mi R-582.The abnormally down-regulation of hsa＿circ＿0009361 in CRC attenuates its tumor suppressive effect and promotes the progression of CRC.