Mechanism Of Circular RNA CircEMB＿003 In The Proliferation And Migration Of Colorectal Cancer

BackgroundCircular RNA(Circ RNA)is a single-stranded closed circular non-coding RNA without 3’ end,5’ end and poly A tail.Circular RNAs are more stable than linear RNAs,because they are not easy to be broken down by exonuclease due to their closed circular structures.It has been reported that circular RNAs are abnormally expressed in a variety of tumors,and they are associated with other diseases such as diabetes and cardiovascular diseases.Circular RNAs have been the biomarkers and targets for the treatment of many diseases because they are abnormally expressed in a variety of diseases and they can regulate the occurrence and development of the diseases.However,the functions and mechanisms of circ RNAs in tumors are still largely unknown.This study tests the expression of a circular RNA CircEMB＿003(also known as Circ＿0001481)in human colorectal cancer and elucidates its roles in the proliferation and migration of colorectal cancer.ObjectivesTo test the expression of CircEMB＿003 in colorectal cancer,to clarify the effect of CircEMB＿003 on the biological behavior of colorectal cancer cells,and to provide a new strategy for clinical gene therapy of colorectal cancer.Methods1.CircEMB＿003 was identified by PCR amplification,Sanger sequencing,and RNase R digestion.2.Real-time q PCR detection of CircEMB＿003 expression in colorectal cancer tissues and colorectal cancer cell lines.The receiver operating characteristic(ROC)curve was used to analyze the diagnostic value of CircEMB＿003.3.Lentiviral packaging system was used to construct the colorectal cancer cell line overexpressing CircEMB＿003 stably.4.CCK8 and plate colony formation assays were used to detect the effect of CircEMB＿003 on the proliferation of colorectal cancer cells HCT116 and LOVO.5.Transwell migration assay and wound healing assay were used to detect the effect of CircEMB＿003 on the migration of colorectal cancer cells HCT116 and LOVO.6.The effect of CircEMB＿003 on the growth of colorectal cancer was detected in vivo by subcutaneous tumorigenesis experiment in nude mice.Immunohistochemistry was used to detect the effect of CircEMB＿003 on the expression of Ki67 which is a marker for proliferation.Results1.Experiments such as PCR,agarose gel electrophoresis and Sanger sequencing confirmed that CircEMB＿003 is a closed circular RNA formed by back shearing,which is stable and resistant to exonuclease degradation.2.The results of q RT-PCR showed that,the expression of CircEMB＿003in colorectal cancer tissues and cell lines was significantly lower than that in adjacent tissues and normal intestinal epithelial cell NCM460.The difference is statistically significant(P<0.05).The ROC curve results showed that CircEMB＿003 has diagnostic value.3.The results of CCK8 cell proliferation assay and plate colony formation assay showed that overexpression of CircEMB＿003 could inhibit the proliferation of colorectal cancer cells HCT116 and LOVO.The difference is statistically significant(P<0.05).4.Transwell migration assay and wound healing assay showed that CircEMB＿003 inhibited the migration of colorectal cancer cells HCT116 and LOVO.The difference was statistically significant(P<0.05).5.In vivo tumour xenograft experiments showed that overexpression of CircEMB＿003 could inhibit the growth of colorectal cancer.The difference is statistically significant(P<0.05).The results of Immunohistochemistry also showed that the Ki67 positive index decreased after overexpression of CircEMB＿003,suggesting that CircEMB＿003 inhibited the proliferation of colorectal cancer in vivo.ConclusionsCircEMB＿003 is a closed circular RNA formed by back splicing,and it is down-regulated in colorectal cancer;overexpression of CircEMB＿003 can inhibit the proliferation of colorectal cancer cells in vitro and in vivo and inhibit the migration of colorectal cancer cell in vitro.