D for 48h plus the expression degree of miR-223 was determined by quantitative PCR (left panel). Inside the treated cells the expression amount of IGF-1R was measured by quantitative PCR (proper panel). (B) Wright-Giemsa staining of the cells. Far more post-mitotic cells (metamyelocytes, segmented neutrophils) were seen inside the treated group. Original magnification 1006 (C) NB4 cells were infected with miR-223 construct. High expression of miR-223 (left panel) was confirmed by quantitative PCR. The expression degree of IGF-1R was also examined and down regulated in miR-223 group. (D) Western blot evaluation of IGF-1R expression in NB4 cells infected with miR-223 construct. (E) Development curve of BEL-7704, SMMC-7721, Huh-7, and NB4 cells infected with miR-223 construct. The measurement of cell growth rate was performed by utilizing CCK-8 kit. P,0.01. N = six. (F) IGF-1R expression was measured by quantitative PCR and was down-regulated in each Tiaprofenic acid Inhibitor SMMC-7721 and BEL-7404 cells infected with miR-223 construct. (G) Just after infection of miR-223 into Huh-7 cells, IGF-1R mRNA was also down regulated. All figures are representative from the study and moreover no less than 3 independent experiments yielded related outcomes. p,0.05. doi:10.1371/journal.pone.0027008.gknockdown of IGF-1R by shRNA could induce related inhibitory effects with miR-223 on IGF-1R and Akt/mTOR/p70S6K signal pathway. Inside the present study, miR-223 suppressed IGF-1R and its signaling and acted as a tumor suppressor. However, miR223 is drastically up-regulated in bladder cancers [15], recurrent ovarian cancer [16] and increases cyclin E protein and activity levels, and elevates genomic instability [23]. Thus miR-223 might serve as a doubled-edge sword by targeting opposite functional targets, considering the fact that a single miRNA can target a dozen mRNAs which effect quite a few molecules which might be involved in unique signal pathways. The dominant influence of a miRNA on the regulated function of cells could rely on the relative importance in the targets which are involved in diverse signal pathways. Within this model, miR-223 targeted IGF-1R and its downstream signal pathway, which exerted a significant function in tumor cell development regulation. IGF-1R was the major mRNA among the miR-223 targets in our study. LMO2, STMN1, Mef 2C, FBXW7 and NF1A did not drastically reduce in our program. Indeed many other molecules which includes Rasa1 have been also observed to become targeted by miR-223 in present study at each mRNA and protein levels. The luciferase reporter assay did show that the 39UTR of Rasa1 gene was targeted by miR-223 directly. Rasa1 exerts a tumor suppressor function by removing GTP from RAS-GTP. Its down-regulation ought to activate the Rasa1/RAF/ MEK/ERK signal pathway. On the other hand, ERK1/2 was downregulated not simply at total protein level, but also at phosphorylation level in miR-223 group as compared together with the group, whichindicated that ERK pathway was inhibited although Rasa1 was targeted. Rasa1 encodes p120-RasGAP — a RasGTPase which reverts active GTP-bound into inactive GDP-bound kind [24]. Consequently Rasa1 couldn’t be the functional target of miR-223 because it failed to regulate ERK pathway soon after miR-223 targeting. On the other hand, IGF-1R may be the functional target of miR-223, which was responsible for the inhibition of cell development. Suppression of IGF-1R-mediated pathway might cause inhibition of ERK signaling, which made Rasa1 lose its regulatory role in signaling. Moreover, IGF-1R was also down regulated in NB4 (pr.
