The Function And Molecular Mechanisms Of CircITGA7 In The Growth And Metastasis Of Human Colorectal Cancer

Backgroud and ObjectiveCircular RNAs(circRNAs)are significantly dysregulated in various cancer types.However,the roles and mechanisms of circRNAs in cancer remain largely unknown.In this study,we investigated the expression,functions and mechanisms of a novel circRNA(circITGA7)in colorectal cancer(CRC),and preliminarily explored its expressional regulation mechanisms.MethodsCircITGA7 was validated by PCR amplification,Sanger sequencing,RNase R digestion and actinomycin D treatment.Real-time quantitative PCR(qRT-PCR)was used to detect the expression of circITGA7 in CRC tissues and CRC cell lines.The relationship between the expression levels of circITGA7 and the clinicopathological parameters was analyzed.CircITGA7 was overexpressed or knocked down in CRC cell lines to investigate the function of circITGA7 on CRC cell proliferation and metastasis in vitro and in vivo by MTT,colony formation,flow cytometry,wound healing,transwell migration and invation assays,and subcutaneous xenograft,intrasplenic injection and orthotopic metastasis models.The signalling pathways that may be involved in circITGA7 functions were screened using RNA-seq and western blot.Fluorescence in situ hybridization(FISH)and RNA subcellular localization analysis were used to determine the subcellular location of circITGA7 in CRC cells.Bioinformatic analysis,dual-luciferase reporter assay,western blot and immunohistochemistry(IHC)were used to investigate whether circITGA7 could bind to miRNAs and then modulate the signalling pathways.In addition,RNA pulldown and mass spectrometric analysis were performed to investigate the interactions of circITGA7 and proteins.Finally,the mechanisms of downregulated circITGA7 expression were preliminarily explored using methylation detection of circITGA7 promoter,transcription factor analysis and chromatin immunoprecipitation(ChIP)assay.ResultsCircITGA7 was significantly downregulated in CRC tissues and CRC cell lines compared to adjacent noncancerous tissues and the normal colonic epithelial cell line FHC.Futhermore,the expression levels of circITGA7 were significantly correlated with tumour size,lymph metastasis,distant metastasis and TNM stage of CRC patients.Functional assays showed that circITGA7 inhibited CRC cell growth and metastasis in vitro and in vivo.RNA-seq and western blot determined that circITGA7 is a negative regulator of Ras signalling pathway.It decreased Ras protein level and the phosphorylation levels of Erk and Akt.FISH and RNA subcellular localization analysis showed circITGA7 predominantly resided in the cytoplasm of CRC cells.Further experiments revealed the mechanism that circITGA7 could bind to hsa-miR-370-3p to increase its target gene NF1 expression and then suppress the Ras signalling pathway.RNA pulldown and mass spectrometric analysis revealed that circITGA7 may be combined with multiple tumour-related proteins.In addition,we found that the methylation status of circITGA7 promoter,the expression of the transcription factor RREB1,and the upstream and downstream intron sequences of circITGA7 may be related with the expression of circITGA7.ConclusionCircITGA7 inhibits CRC cell growth and metastasis by suppressing Ras signalling pathway through binding to hsa-miR-370-3p and upregulating the expression of its target gene NF1.