Zhang Y, Zhou SY, Yan HZ, XU DD, Chen HX, Wang XY, Wang X, Liu YT, Zhang L, Wang S, Zhou PJ, Fu WY, Ruan BB, et al. right. Data are represented as mean SD. Anti-miR-203 increases the expression of SOCS3 and decreases pStat3 expression in breast cancer cells Our previous study have identified IACS-8968 S-enantiomer that SOCS3 is a direct target of miR-203 [10]. SOCS3 is known to be down-regulated in various types of cancers including breast cancer [19, 20]. We observed that SOCS3 expression was increased in the MCF-7-antimiR-203 and ZR-75-1-antimiR-203 cells as compared to control cells (Figure ?(Figure2,2, panel A). SOCS3 is regulated by Stat3 via the IL6/Stat3/NFkB mediated signaling pathway and SOCS3 negatively regulates the expression of Stat3 [21]. Therefore, we examined the expression of pStat3 in breast cancer cells. We observed that the level of pStat3 was reduced in both MCF-7-antimiR-203 and ZR-75-1-antimiR-203 cells as compared to control cells (Figure ?(Figure2,2, panel B). However, total Stat3 expression remained AIbZIP unchanged. These data suggested that miR-203 inhibits SOCS3 expression and concurrently increased the expression of pStat3 in these breast cancer cells. Open in a separate window Figure 2 Inhibition of miR-203 increases the expression of SOCS3 and enhances phospho-STAT3 expression in breast cancer cellsMCF-7 control, MCF-7-antimiR-203, ZR-75-1 control and ZR-75-1-antimiR-203 cells lysates were subjected to Western blot analysis using SOCS3 IACS-8968 S-enantiomer (A), pStat3 and total Stat3 (B) antibodies. The blots were reprobed with an antibody to actin for comparison of protein loading in each lane. Densitometric analyses of all these proteins were done by using Image J software and shown on the right. Data are represented as mean SD. Anti-miR-203 decreases the expression of pERK and c-Myc in breast cancer cells Activated ERK is associated with differentiation and proliferation of cells in various types of cancers including breast cancer [22]. We examined the expression of pERK by Western blot analysis. We observed that the pERK was decreased in both MCF-7-antimiR-203 and ZR-75-1-antimiR-203 cells as compared to control cells (Figure ?(Figure3,3, panel A). Interestingly, total ERK expression remained unchanged in MCF-7 cells whereas decreased in ZR-75-1 cells and reason is unknown at present. c-Myc is an essential transcription factor that has been extensively studied due to its vital functions in the regulation of cancer cell growth [23]. It also plays a critical role in tumor initiation, progression, and survival of cancer [24]. Therefore, we examined the expression of c-Myc in breast cancer cells by Western blot analysis. We observed that c-Myc expression was decreased in both MCF-7-antimiR-203 and ZR-75-1-antimiR-203 cells as compared to control cells (Figure ?(Figure3,3, panel B). Open in a separate window Figure 3 ERK-c-myc signaling pathways are inhibited by anti-miR-203 in breast cancer cellsMCF-7 control, MCF-7-antimiR-203, ZR-75-1 control and ZR-75-1-antimiR-203 cells lysates were analyzed for pERK, ERK (A) and c-Myc (B) expression by Western blot analysis. The blots were reprobed with an antibody to actin for comparison of protein loading in each lane. Densitometry analyses of all these proteins were done by IACS-8968 S-enantiomer using Image J software and shown IACS-8968 S-enantiomer on the right. Data are represented as mean SD. Inhibition of miR-203 decreases tumor growth in the nude mice Our data revealed that miR-203 expression is associated with proliferation of breast cancer cells. We next investigated whether inhibition of miR-203 could also suppress tumor progression data suggested that cyclin D1 expression was inhibited in miR-203 knockdown breast cancer cells as compared with control cells (Figure ?(Figure1).1). We further examined the expression level of cyclin D1 and PCNA by Western blot analysis. Our results demonstrated that the expression of cyclin D1 and PCNA was significantly lower in tumors from miR-203 knockdown MCF-7 cells as compared to control cells (Figure ?(Figure4,4, panel C). Thus, these results indicated that miR-203 inhibition plays a role, in part, for reduction of IACS-8968 S-enantiomer MCF-7 tumor growth. Open in a separate window Figure 4 Inhibition.