| ?Objective?Colorectal cancer is a commonly diagnosed cancer in the digestive system and metastasis is the leading cause of related deaths,however the mechanism of conlorectal cancer metastasis is still unclear.This study aims: 1.To compare the migration,adhesion and anoikis resistance activities of SW480 and SW620 cells,which are respectively estabilished from the primary colorectal cancer tissues and paired lymph nodes tissues of the same patient,and detect expression level of epithelial-mesenchymal-transition(EMT)makers and the protein moleculars associated with adhesion and anoikis,in order to make sure whether the two cells could be used as an ideal model for research of colorectal cancer metastasis.2.To identify differentially expressed microRNA(miRNA)between the two cells and clinical samples,and the relevance of its expression level with colorectal cancer metastasis.Analysis the impact of this miRNA on proliferation,migration,adhesion,anoikis resistance activities and the protein moleculars associated with EMT,adhesion and anoikis in the two cells.To comfirm the function of this miRNA on metastases in vivo.3.To clarify the mechanism of colorectal cancer metastasis regulated by the miRNA through modulating its target genes.?Methods?1.Transwell migration,cell adhesion and flow cytometry assay were used to analyze the activities of migration,adhesion and anoikis resistance in SW480 and SW620 cells.The protein levels of E-cadherin,vimentin,?-integrin and Bcl-2 in the two cells were detected by Western blot assay.2.miRNA microarray and RT-qPCR were used to screen and verify the miRNA differently expressed between SW480 and SW620 cells.RT-qPCR was also applied to evaluate the expression of this miRNA in 26 pairs of colon cancer samples.In addition,the effects of this miRNA on the capabilities of proliferation,migration,adhesion and anoikis resistance and the expression of E-cadherin,vimentin,?-integrin and Bcl-2 were assessed.Furthermore,the nude mice model was adopted to research the effect of miRNA on metastases.3.The candidate target genes regulated by miRNA were screened and verified by bioinformatics,double fluorescence report assay,RT-qPCR and Western blot assay.Moreover,RT-qPCR and Immunohistochemistry were used to detect the mRNA and protein levels of target genes in colon cancer tissues.Finally,Transwell migration,cell adhesion,flow cytometry and Western blot assay were applied to determine the target genes effects on migration,adhesion,anoikis resistance and the expression of ?-integrin and Bcl-2 in colorectal cancer cells.?Redults? 1.Compared with SW620 cells,SW480 cells exhibited stronger migration,but weaker cell adhesion and anoikis resistance abilities,and de-regulated expression of E-cadherin,?-integrin and Bcl-2.2.miR-10 a was upregulated in primary colorectal cancer cells and tissues compared to those of lymph nodes,and its expression in primary colorectal cancer tissues compared to the metastatic cancer tissues was inversely correlated with distant metastasis and invasion depth(P<0.05).Furthermore,miR-10 a knockdown notably inhibited EMT and migration in SW480 cells,but uptaked the adhesion and anoikis resistance abilities by enhancing ?-integrin and Bcl-2.Besides,the opposite results can be acquired by miR-10 a overexpression.The xenograft model demonstrated miR-10 a significantly impaired the metastatic capability of SW620 cells in vivo(P<0.05).3.Based on bioinformatics analysis,double fluorescence report,RT-qPCR and Western blot assay,MMP14 and ACTG1 were identified as direct target genes negatively regulated by miR-10a(P<0.05).MMP14 and ACTG1 expression were respectively inversely correlated with miR-10 a in the same samples(P<0.05).Their hyperexpression significantly facilitates migration and adhesion of SW480 cells(P<0.05)and their down-regulation significantly suppressed adhesion activity of SW480 cells(P<0.05)that might be related to ?-integrin expression.On the other hand,knockdown of MMP14 and ACTG1 markedly enhanced while overexpression of them could suppress sensitive to anoikis of SW620 cells that were associated with ?-integrin and Bcl-2(P<0.05).?Conclutions?1.SW480 and SW620 cells can be used as an ideal model to study metastasis of colorectal cancer.2.miR-10 a promotes colon cancer cells migration and invasion in vitro but inhibites them metastasis in vivo by promoting EMT and anoikis during metastasis process.3.MMP14 and ACTG1 are direct target genes which are negatively regulated by miR-10 a and contribute to migration,adhesion and anoikis resistance of colon cancer cells by positively regulating ?-integrin and Bcl-2.This study clarifies a mechanism of colorectal cancer metastasis and provides a new sight for therapeutic of colorectal cancer. |
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