The Role And Molecular Mechanism Of MiR-320a In Progression Of Human Colorectal Cancer

OBJECTIVE:(1) To evaluate the expression of miR-320a in colorectal cancer tissuesamples, then investigate the biological function and molecular mechanism ofmiR-320a in colorectal cancer cells.(2) To investigate the molecularmechanism of how the expression of miR-320a was regulated in colorectalcancer cells.METHODS:(1) Expression levels of miR-320a in60colorectal cancer tissue samples and5colorectal cancer cell lines were detected with real-time qualified RT-PCR.(2)The migration and proliferation ability of the5colorectal cancer cell lineswere examined by trans-well assay and CCK-8assay, respectively.(3) Theeffect of miR-320a on colorectal cancer cell metastasis, proliferation and cellcycle distribution was investigated by trans-well assay, CCK-8assay and flow cytometry, with stable expression clones established with lentiviraltransfection compared with wild type cell lines. In vivo tumour growth assaywas also performed to examine the influence of miR-320a inhibition tocolorectal cancer cell proliferation and invasion.(4) The variation ofE-cadherin and Vimentin caused by miR-320a overexpression or inhibitionwas shown by Western blot with colorectal cancer cell lines andimmunohistochemistry with tumors formed in vivo.(5) Ras-related C3botulinum toxin substrate1(Rac1) was one of protein targets predicted withbioinformatics analysis, with two putative miR-320a binding sites formiR-320a at site611and1140in Rac1mRNA3′-UTR region. Western blotand semi-quantitive PCR were used to show whether Rac1protein andmRNA level were affected by miR-320a overexpression or inhibition.(6)The3′-UTR of Rac1mRNA containing both miR-320a binding sites wasamplified by PCR and then inserted in pMIR-REPORT luciferase vector,Mutation of miR-320a binding sites at661and1140were, respectively,generated to produce mutant constructs, pMIR/Rac1-mut611andpMIR/Rac1-mut1140. Luciferase activity was measured using the DualLuciferase Reporter Assay according producer’s instruction after the vectorswere transfected in SW620cells with miR-320a mimics and pRL-TK vector.(7) Rac1overexpression vector and siRNA/Rac1were used to investigate whether the Rac1is responsible for the miR-320a-induced inhibition of cellaggressiveness.(8) Influence of E2A on miR-320a expression was verifiedby gain or loss function assay based on TESS tool result, then ChIP assaywas used on detect whether E2A can binding directly on miR-320a DNAsequence.(9) Investigate the role of E2A protein in regulating themigration/invasion ability of colorectal cancer cell by overexpression andknockdown technology.(10) Verify the role of miR-320a in E2A regulatingthe progression of colorectal cancer.RESULTS：(1) miR-320a inversely associates with aggressiveness and metastasis in CRC.(2) miR-320a suppresses CRC cell migration/invasion and proliferation invitro and in vivo.(3) miR-320a inhibits the epithelial–mesenchymal transitionof colorectal cancer cells.(4) miR-320a directly targets Rac13′-UTR toreduce Rac1expression.(5) Overexpression of Rac1restores themiR-320a-induced inhibition of cell migration/invasion and proliferation,while Knockdown of Rac1antagonizes the anti-miR-320a-induced cellmigration/invasion and proliferation.(6) E2A induced miR-320a expressionby directed binding to miR-320a DNA sequence.(7) E2A protein reducedEMT and inhibited metastasis/proliferation ability in colorectal cancer cells, and miR-320a plays an important role in proliferation part.CONCLUSIONS:miR-320a suppresses colorectal cancer cell metastasis and proliferation bydirectly targeting Rac1, the expression of which was affected by E2A.