| MicroRNA-223(miR-223) was first identified and subsequently characterized in the haematopoietic system, where it highly expresses in myeloid compartment, monocytes, and myeloid leukemic cells. The role of miR-223has not been elucidated, but is believed to be involved in the differentiation and maturation of granulocyte precursors. One of the mechanisms of retinoic acid (RA)-induced differentiation is to up-regulate the expression of miR-223. Therefore, miR-223may play an important role in the induction of leukemia cells.A target gene of miR-223identified as a target of miR-223mechanism during their differentiation of granulocytes wais Mef2C (Myocyte Enhancer Factor (Mef2C) which can promote myeloid progenitor proliferation. Other researchers suggested that Nuclear Factor (NF-1A) iwas another target gene of miR-223. and NF-IA can also inhibite the expression of miR-223which can overcome through competitive binding of NF-1A on the miR-223promoter region against C/EBP α. C/EBP α can induce the expression of miR-223by binding to its promoter region as well, and at the same time it can relieve the blocking of leukemia specific fusion protein PML/RAR α which stops the process of granulocytic differentiation. There is also a document indicating that miR-223is suppressed in hepatocellular carcinoma cells and identified STMN1as theis the target gene of miR-223, which is quite different from those in leukemic cells. The protein level of STMN1is negatively correlated with the expression of miR-223. However, the detailed mechanism of miR-223action still remains unclear. Does miR-223work as an initial molecule during the hematopoietic cell differentiation? Or miR-223just is a phenotype molecule after differentiation? Is there any similar role of miR-223in non-hematopoietic cells compared with haematopoietic cells? Is there the a similar expression profile of miR-223inside and outside thein non-haematopoietic system tumor? Is a single target important enough to influlence all cells? Does a regulation, crsotalk or influence exists among targets of the same microRNA? In this experiment, we seek for the target and its function of miR-223. The thesis is divided into two parts. Part I Establishment of miR-223over-expression system and observation of its biological functionsLentivirus vector can integrate the carried gene to host genome and constitutively express its carried miRNA or siRNA. We conducted miR-223recombinant construct by lentivirus vector PLL3pLL3.7for over-expression of miR-223. We srorted transduced human cervical carcinoma cells (line HeLa cells) after infectiontransducted with either pLL3.7-miR-223or control vector EV, which served as stablely transduction infected cells after sorting by a fluorescence activated cell sorter. The expression of miR-223was quantified through microRNA specific stem-loop RT primer mediated RT-PCR and Real-time PCR,. It was found that miR-223-over-expressed in HeLa cells infected with (HeLa-pLL3.7-miR-223,) but the vehicle in comparisoncontrol group infected with the control vector(HeLa-EV) expressed very low level of miR-223. the The capacity of proliferation and migration assay in vitro showed striking suppression in HeLa-miR-223as compared to with HeLa-EV, and the significant inhibition of proliferation and. The tumor weight after inoculation of miR-223-over-expressed cells in nude mice was observed in nude mice in vivo much less than those of control and we concluded that miR-223can suppress the growth rate of HeLa cells either in vivo or in vitro. MiR-223had its function in the hematological system, as well as in non-hematological system.Part Ⅱ The mechanism for miR-223inhibition of HeLa cell growthTo address the mechanism of miR-223inhibiting HeLa cell growth, we screened the potential target genes of miR-223by informatics and constructed a series of3’UTR of suggested target genes to the Dual-Luciferase Report Gene vector psi-CHECK-2and observed the response of luciferases. After analysis of the response to miR-223and the level of mRNA and protein of the tested genes, we identified IGF-1R, Fox01, Fox03and Rasal are new targets of miR-223in HeLa cells, because the response of its their3’UTR-luciferase recombinant to miR-223was greatly inhibited as compared to with HeLa-EV and the inhibition can be reversed by miR-223inhibitor. The levels of their mRNA and protein were negatively correlated with the expression of miR-223. The down-stream molecules such as PI3K/AKT/mTOR/p70S6K in IGF-1R-mediated pathway were also suppressed as well. It showed that the Receptor Tyrosine Kinase AKT were was down-regulated at phosphorylation level, but not at total protein level. Two molecules including GSK3β and p27that were downstream and directly inhibited by of AKT were up-regulated at both phosphorylation level and protein levels, respecitively. In contrary, the molecules including Bcl-2and p70S6K that were promoted directly by AKT were significantly inhibited. One was Bcl-2is Bcl-2, an important apoptosis inhibitor, and the other was p70S6K is, an essential protein kinase in mTOR pathway, as well as the Hypoxia-Inducible Factor-1a (HIF-1α), which is a direct target of p70S6K. tTherefore, we concluded that miR-223block HeLa cell growth, at least partly by direct targeting IGF-1R and its downstream PI3K/AKT/mTOR/p70S6K pathway. Interference of IGF-1R in HeLa cells could mimic the inhibition mediated by miR-223. In contrary, rescuing IGF-1R expression in HeLa-miR-223can reverse the inhibition of miR-223. After rescuing IGF-1R expression, a similar proliferation rate was also observed in the cells as the control.Fox01, Fox03, and Rasa are tumor suppressors, which could be new targets of miR-223. We noticed that HeLa cells grew fast, but became very slow after miR-223infection. These results indicated that the signaling of these targets was regulated and inhibited by IGF-1R signaling. The target molecules of the same miRNA may influence one another with complex regulations. Surely, there might be dozens of other targets of miR-223existed, but the molecular pathway performed by IGF-1R is important for miR-223action in HeLa cells.All above, we identified new targets of miR-223and found that miR-223suppressed HeLa cell growth mainly by targeting IGF-1R and its signal pathway. Rasa and Foxo signaling co-operated IGF-1R pathway. We think that multiple targets work together to fully exert the function of a microRNA. |
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