The Effect Of MiR-200b On Proliferation, Migration, Invasion Of Breast Cancer Cell And The Study Of Molecular Mechanism

Objective: microRNA is a kind of endogenous non-coding small molecule RNA which involved in activities of cell proliferation, differentiation and apoptosis. miR-200 b is one member of miR-200 family who is over expressed in high degree of malignant breast cancer cell and low expressed in low degree of malignant breast cancer cell. Fucosyltransferase 4(FUT4) is an enzyme protein catalyze L-fucose transfer of fucose residues from GDP-Fuc to receptors to form α-1,3 linkage. The expression level of FUT4 is high in breast cancer which could promote the occurrence of EMT and is positively correlated with cell proliferation and metastasis. This study aimed to explore the relationship between miR-200 b and FUT4 and investigated whether it influenced the proliferation, migration and invasion of breast cancer cell(MCF-7) through PI3K/Akt signaling pathway by targeting FUT4. To explore whether miR-200 b could be a new target for diagnosis and treatment of breast cancer.Methods:1. The expression of miR-200 b, FUT4 in breast cancer(1)Using real-time PCR and immunochemistry to detect the expression of miR-200 b and FUT4 in breast cancer tissues.(2)Transfecting mi R-200 b mimics into MCF-7 cell of breast cancer,using real-time PCR,Western blot, immunofluorescence to assay the expression changes of miR-200 b and FUT4.2. Regulation of FUT4 by miR-200b(1)Using software such as TargetScan, PicTar to predict the binding sites between miR-200 b and FUT4 and determine whether FUT4 was one candidate target gene of miR-200 b.(2)Transfecting mi R-200 b mimics into MCF-7 cell to increase the expression level of miR-200 b, then using real-time PCR to determine the expression change of miR-200 b, FUT4 and western blot to determine the change of FUT4 at protein level.( 3) Constructing psi-CHECK-2 vector with 3’-untranslated region(3’-UTR) of FUT4(psi-CHECK-2-FUT4-3’UTR), co-transfected with mi R-200 b mimics into MCF-7 cell.FUT4 luciferase activity was detected by dual luciferase reporter gene assay.3. Impact of miR-200 b on cell proliferation, migration, invasion of MCF-7(1)Transfecting miR-200 b mimics into MCF-7 cell, using CCK-8 assay,colony formation assay to detect the influence of miR-200 b on cell proliferation.(2)The miR-200 b mimics was transfected into MCF-7 cell, the function of cell migration was detected by Cell scratch experiment.(3)Transfecting miR-200 b mimics into MCF-7 cell, the impact of miR-200 b on cell invasion was determined by transwell assay.4. The influence of miR-200 b on PI3K/Akt signaling pathway through regulating the expression of FUT4After transfecting miR-200 b mimics into MCF-7 cell, detected the related factors changes of PI3K/Akt signaling pathway and the expression of FUT4 by western blot.Results:1. Increased expression of miR-200 b decreased the expression of FUT4 in MCF-7 cellAfter transfecting mi R-200 b mimics with Lipofectamine 2000, real-time PCR was used to confirm the expression of mi R-200 b which increased while FUT4 decreased; Western blot and imunofluorescence were used to confirm the expression of FUT4 decreased in protein level.2. FUT4 was a target gene of miR-200bThe software predicted that there were binding sites between miR-200 b and FUT4 which may become a candidate target gene of miR-200 b. We co-transfected miR-200 b mimics and psi-CHECK-2-FUT4-3’UTR which containing FUT4 3’-untranslated region into MCF-7 cell.Compared with miR-NC group, we found miR-200 b could inhibit FUT4 luciferase activity of cells who transfected with reporter gene significantly. So it indicated that FUT4 was a targetgene of miR-200 b.3. The effect of miR-200 b on cell proliferation, migration, invasion of MCF-7(1)CCK-8 cell proliferation assay and Colony formation assay showed that the increased expression of miR-200 b could inhibit cell proliferation when compared with the control group.(2)Cell scratch experiment showed that the migration capacity of MCF-7 from miR-200 b mimics transfection group reduced obviously.(3)Vitro cell invasion assay(Transwell) results showed that upregulation of miR-200 b could inhibit cell invasion ability significantly when compared with the control group.4. The impact of miR-200 b on PI3K/Akt signaling pathway by regulating the expression of FUT4Increased expression of miR-200 b led to decreased expression of FUT4, p-PI3 K, p-PDK,p-Akt(ser473) of PI3K/Akt signaling pathway which was consistent with the previous results that miR-200 b could inhibit cell proliferation, migration and invasion.Conclusions:1. Dual luciferase gene reporter assay confirmed that FUT4 was a target gene of miR-200 b.2. Upregulation of miR-200 b could reduce the ability of proliferation, migration, invasion of MCF-7 cell. It indicated that miR-200 b could affect cell proliferation, migration and invasion.3. miR-200 b could regulate the expression of FUT4 and may affected proliferation,migration, invasion of MCF-7 through PI3K/Akt signaling pathway.4. miR-200 b may become a new target for diagnosis and treatment of breast cancer because it could influence cell proliferation, migration, invasion of MCF-7 by regulating the expression of FUT4.