Ion of miR-183-96182 cluster on gastric cancer cell phenotype, we transfected a miRCURY LNATM miRNA Inhibitor Negative Manage or maybe a mix of miRCURY LNATM inhibitors for miR-183, miR-96 and miR-182 into AGS cells. The upregulation of FoxO1, on the list of targets of miR-18396-182 cluster, indicated the efficiency of miR inhibitors (Figure 7A). Suppression of miR-183-96-182 cluster decreased proliferation and migration of AGS cells (Figure 7B and C). To investigate the effects of GSK3b knockdown on gastric cancer phenotype, we transfected handle siRNA or GSK3b-specific siRNA into AGS cells. Compared with manage siRNA, GSK3b siRNA particularly downregulated GSK3b protein (Figure 7D). Knockdown of GSK3b enhanced AGS cell proliferation (Figure 7E), but had no substantial effect on AGS cell migration (Figure 7F).2996 Nucleic Acids Research, 2014, Vol. 42, No.eight 7 6 five four 3 two 1ARela ve pri-miR-183 level eight 7 six five four three 2 1 0 EVRela ve miRNA levelBEV -CateninmiR-96 -CateninmiR-miR-C 1.Rela ve pri-miR-183 level 1 0.eight 0.6 0.4 0.two 0 Handle siRNA -Catenin siRNAD 1.Rela ve miRNA level 1 0.8 0.six 0.four 0.2 0 miR-96 miR-182 miR-Control siRNA -Catenin siRNAFigure 6. b-Catenin enhances expression of main and mature miR-96, miR-182 and miR-183. An EV, a vector encoding b-Catenin, manage siRNA or b-Catenin siRNA, was transfected into AGS cells, respectively. Total RNA was extracted and utilized for RT-PCR to measure the expression levels of major and mature miRs. All experiments had been repeated 3 occasions with related results (*P 0.05 by Student’s t-test). (A) Overexpression of b-Catenin increases the pri-miR-183 level. (B) Overexpression of b-Catenin increases the expression of miR-96, miR-182 and miR-183. (C) Knockdown of b-Catenin decreases the pri-miR-183 level. (D) Knockdown of b-Catenin decreases the expression of miR-96, miR-182 and miR-183.ABRela ve AGS prolifera on1.2 1 0.eight 0.six 0.4 0.2 0 LNA handle cluster inhibitorsCRela ve AGS migra on 1.2,6-Di(1-pyrazolyl)pyridine site 2 1 0.DSPE-MPEG2000 web eight 0.PMID:23376608 six 0.4 0.two 0 LNA manage cluster inhibitorsFOXO1 GAPDHRela ve AGS prolifera on2 1.5 1 0.five 0 manage siRNA GSK3siRNARela ve AGS migra onDEF2 1.five 1 0.5 0 control siRNA GSK3siRNAGSK3 GAPDHFigure 7. Suppression of miR-183-96-182 cluster or knockdown of GSK3b alters gastric cancer cell phenotype. (A) Suppression of miR-183-96-182 cluster increases FoxO1 protein level. (B) Suppression of miR-183-96-182 cluster decreases AGS cell proliferation. (C) Suppression of miR-183-96182 cluster decreases AGS cell migration. (D) GSK3b siRNA especially downregulates GSK3b protein. (E) Knockdown of GSK3b increases AGS cell proliferation. (F) Knockdown of GSK3b does not affect AGS cell migration significantly. All experiments had been repeated three instances with related benefits (*P 0.05 by Student’s t-test).Nucleic Acids Analysis, 2014, Vol. 42, No. 5DISCUSSION The Wnt signaling plays a pivotal part in tumorigenesis in different cancers including gastric cancer (37,38). Given that the CK1 and CK2 protein kinase families play essential roles in Wnt signaling pathway (39,40), we wondered whether or not KO GSK3b deregulated the expression of these kinases. We located, however, that knocking out GSK3b did not modify the expression of CK1 and CK2, ruling out deregulated activity of these kinases in GSK3b KO cells. As a key element of this pathway, GSK3b has emerged as a potential therapeutic target for cancer therapy (41). Because GSK3b is really a multifunctional protein kinase, inhibition of GSK3b may have critical side effects. To minimize these side e.
