The Circular RNA Hsa＿circ＿0006401 Encodes A New Functional Peptide To Promote The Proliferation And Metastasis Of Colorectal Cancer

Objective The dysregulation of circular RNA(circRNA)expression is closely related to the progression of cancer.Colorectal cancer(CRC)is one of the most common gastrointestinal malignancies with a high mortality rate.In many developing countries,the incidence and mortality of colorectal cancer are still increasing year by year.Here,we aim to study the expression level of hsa＿circ＿0006401 in colorectal cancer(CRC)tissues and cells and its role in the proliferation and metastasis of colorectal cancer,and to explore the potential mechanism of hsa＿circ＿0006401 affecting the biological functions of colorectal cancer.Methods We used real-time quantitative PCR(qRT-PCR)and circRNA in situ hybridization(RNA-ISH)to analyze the expression levels of CircRNA hsa＿circ＿0006401in colorectal cancer tissues and adjacent non-tumor tissues,and collected clinicopathological data of colorectal cancer patients for the correlation analysis.The effect of hsa＿circ＿0006401 on the proliferation of colorectal cancer cells was evaluated by cell count and clone formation experiments;wound healing experiments and Transwell analysis were used to detect the effect of hsa＿circ＿0006401 on the migration of colorectal cancer cells;flow cytometry was used to detect the effect of hsa＿circ＿0006401 on the apoptosis of colorectal cancer cells.In addition,we established a nude mouse subcutaneous tumor xenograft model to explore the effect of hsa＿circ＿0006401 on the proliferation and migration of colorectal cancer in vivo.Hematoxylin-Eosin(H&E)staining is used to identify local tumor infiltration and liver metastasis.In order to verify the function of hsa＿circ＿0006401 encoding small peptides,we constructed a circRNA-ORF overexpression vector plasmid and an overexpression plasmid circRNA-ORFmut with a mutation in the ORF start codon(the start codon ATG was mutated to TTG),and prepared a targeting hsa＿circ＿0006401 Antibodies specific to the splice site.The expression level of hsa＿circ＿0006401 peptide in colorectal cancer tissue and adjacent non-tumor tissues was detected by immunohistochemistry(IHC)method.Western blotting is used to detect changes in related proteins.Through mRNA attenuation analysis,we determined the effect of hsa＿circ＿0006401 peptide on the stability of COL6A3 mRNA.Finally,we identified the protein that directly binds to the hsa＿circ＿0006401 peptide by immunoprecipitation and mass spectrometry,and used Gene Ontology(GO)enrichment analysis to deduce the potential molecular mechanism of hsa＿circ＿0006401.Results In our study,we found that hsa＿circ＿0006401 is closely related to colorectal cancer metastasis,and showed an up-regulated expression in metastatic colorectal cancer tissue samples.When the expression of hsa＿circ＿0006401 is silenced,the ability of proliferation and migration in vitro is inhibited.The down-regulation of hsa＿circ＿0006401 expression reduces the proliferation and liver metastasis of tumor grafts in vivo.There is a 594 nt open reading frame(ORF)spanning the splice site in hsa＿circ＿0006401,which can encode a 198 aa peptide.The 198-aa peptide is encoded by a splice site sequence that does not exist in COL6A3,and hsa＿circ＿0006401 encodes the hsa＿circ＿0006401 peptide to promote the proliferation and migration of CRC.We performed immunohistochemical staining to evaluate the expression of hsa＿circ＿0006401peptide in colorectal cancer tissues and adjacent tissue samples from 12 patients with colorectal cancer.We found that the IHC score of hsa＿circ＿0006401 peptide in colorectal cancer tissue specimens was higher than that of adjacent tissues,and its expression is closely related to lymph node metastasis.Conclusion Our research shows that the circRNA hsa＿circ＿0006401 produced from COL6A3 contains an open reading frame(ORF),which can encode a new type of 198-aa functional peptide.The hsa＿circ＿0006401 peptide can promote the stability of the host gene COL6A3 mRNA,thereby promoting the proliferation and metastasis of colorectal cancer cancer.This study clarified the role and molecular mechanism of hsa＿circ＿0006401 in the occurrence and development of colorectal cancer,and provided a new target for effective treatment and prevention of colorectal cancer.