MicroRNA-7Inhibits Human Colorectal Cancer Cell Lines Invasion And Proliferation By Targeting FAK Expression

Background:Colorectal cancer is not only one of the most common malignancy in the world,but also one of the important causes of death caused by cancer-related deaths. In ourcountry, the incidence of colorectal cancer increasing year by year, surgery is still theprimary means of treatment. The general effect of cancer treatment are stilldissatisfied, especially metastatic colorectal cancer often has poor therapeutic effectsand prognosis, the life quality of patients severely affected, along with targetedmolecular therapy in-depth research, more and more molecular target for thetreatment appear, so that the treatment of colorectal cancer have a new tool.Compared with the traditional cancer research molecular such as gene, mRNA,protein, microRNA as a novel research focus has more quality to become tumormarkers and therapeutic targets.MicroRNA is a class of18-25nucleotides non-protein-coding single-strandedsmall molecules of RNA, by complementary base pairing way to identify the targetmRNA, and to inhibit the target mRNA translation depending on the degree ofcomplementarity, or degrade mRNA like siRNA, thereby negatively regulating theexpression of target gene. MicroRNAs function continuous deepening, MicroRNAshave been found closely associated with tumors and affect tumor growth andmetastasis, therefore, it may as a target for cancer diagnosis, treatment, prognosis. Itsimportant role can not be separated by changes in gene target.FAK (Focal adhesion kinase) is a cytoplasmic non-receptor protein tyrosinekinase, and it is an important molecule of integrin-mediated signal transduction. FAKinvolved in multiple signal elements on the membrane transduction induced by thecell physiology, including cytoskeletal reorganization, adhesion, migration,proliferation and other processes. Research shows that FAK is a target gene of miR-7,miR-7binding to FAK mRNA3’non-coding region targeted inhibition of FAKprotein translation, and it inhibits a variety of tumor cell growth, invasion andmetastasis. Our previous studies have shown that FAK expression in colorectal cancer is higher than the adjacent tissues. In our study, we discussed miR-7and FAKexpression in colorectal cancer, further elaborate the true mechanism of CRCmetastasis, to find a new target.ObjectivesThe aim of this research is to definite the change of FAK protein andproliferation or invasion on human colon cancer cells after up-regulate or downmiR-7, clearly whether miR-7is targeting FAK protein regulation of proliferation andinvasion of colon cancer cells, to find new targets for colorectal cancer treatment.MethodsSubculture of human colon cancer cells Caco-2and HCT-8, by lipofectionmiR-7mimic/inhibitor and the corresponding negative controls to increase ordecrease the expression of miR-7, the transfection efficiency was detected byfluorescent staining and Real-time PCR. Invasion and related proteins expression ofcolon cancer cells such as FAK, MMP-2and MMP-9protein detected after miR-7changes by Western Blot. Cell proliferation rates were determined using MTT.Migration ability was measured using wound-healing assay. SPSS18.0statisticalsoftware was used to evaluate the date. Each assay was performed at least three times.Results1. MiR-7mimic and miR-7inhibitor were transfected by Lipofectamine intohuman colon cancer cells and regulate the expression of miR-7successfully. MiR-7mimic transfected into cells increased expression of miR-7detected by qRT-PCR,miR-7inhibitor transfected into cells decreased expression of miR-7, by transfectionof miR-7mimic negative-5’-cy3into cells and then fluorescence assay showedtransfection efficiency of almost100%.2. The ectopic miR-7levels significantly decreased FAK protein expression asdetermined. FAK protein expression detected after transfected miR-7mimic, itshowed a clear downward trend contrast with the group of transfected with miR-7mimic negative and the non-transfected.(P≤0.05).3. The significantly increased FAK protein expression as determined afterdecreased miR-7levels. FAK protein expression detected after transfected miR-7inhibitor, it showed a clear upward trend contrast with the group of transfected with miR-7inhibitor negative and the non-transfected.(P≤0.05).4. Proliferation of colorectal cancer cells changed accordingly after miR-7expression changed. We discovered that over-expression of miR-7significantlyinhibited the proliferation, whereas down-regulation of miR-7expression promotedthe proliferation of HCT-8and Caco-2cells at24,48h after transfection, respectively(P≤0.05).5. Changes in colorectal cancer cells migration after miR-7expression changed.The ectopic miR-7levels of miR-7significantly inhibited cells migration, howeverdown-regulation of miR-7expression promoted the migration (P≤0.05).6. Cell invasion-related protein expression such as MMP-2and MMP-9changedaccordingly after miR-7expression changed. The over-expression of miR-7obviouslysuppressed cells MMP-2and MMP-9expression, the significantly increased MMP-2and MMP-9protein expression as determined after decreased miR-7levels (P≤0.05).Conclusion1. Lipofectamine transfected miR-7mimic and miR-7inhibitor into colorectalcancer cells and regulates the expression of miR-7successfully.2. Alteration of miR-7expression changed FAK protein expression level.Over-expression of miR-7significantly inhibited the FAK expression, whereasdown-regulation of miR-7expression promoted the FAK expression, miR-7canregulate the biological activity of colorectal cancer cells by targeting FAK.3. miR-7can inhibit the proliferation and migration of colorectal cancer cells.The ectopic miR-7expression levels suppressed cells proliferation and migration.4. After changing miR-7expression in colorectal cancer cells, MMP-2andMMP-9protein expression was negative. miR-7can inhibit cell invasion relatedproteins.