Rna Binding Protein Qki-5 On The Post-transcriptional Regulation Of Foxo1 In Retinoic Acid Inhibits The Function Of Breast Cancer Cell Proliferation And New Gene Apr3 Involved In The Function Of Retinoic Acid-induced Cell Cycle Arrest

QKI is an RNA binding protein essential for normal myelination, and are abundantly expressed in the central nervous system. QKI viable mice exhibit extensive body tremors detectable by postnatal day 10 resulting from a severe deficit in myelination. Although the most extensive study about QKI are foused on nervous system, profound and abundant distribution of QKI-5, was observed in various other tissues of adult, such as heart, lung or testis.Analyzing the functions of QKI isoform in nervous system disclosed that QKI-6, 7 are mainly responsible for transport and stability of target RNAs. Whereas, QKI-5 binds target transcripts to retain them in the nucleus., Specific role of QKI-5 has been largly unknownIn addition to its fundamental role in myelination, QKI also participated in vascular development, apoptosis, cell adhesion, cell growth, morphogenesis and organogenesis. In light of the validated target motif and RNA binding specificity of QKI with its targets, we select FoxO1 as a candidate gene from 1430 new putative mRNA targets predicted by other researcher.1. As we known, FoxO proteins have an important role in the regulation of metabolism, resistance to oxidative stress, cell cycle progression, apoptosis, longevity, senescence and vascular development. Interestingly, there is a common embryonic lethal phenotype shared by QKI or FoxO1 knockout mice due to vascular development disturbance, hinting the functional overlap between them. The underlying mechanism are both related with dysregulation of retinoic acid (RA) signaling pathways.2. One the basis of bioinformatics analysis on FoxO1 3'UTR., it contains 3 predicted QRE and may be a QKI target transcript, its mRNA sequence is evolutionarily conserved among organisms, and secondary structure is highly complicated. These features highly suggested that 3'UTR of FoxO1 is functionally important in vivo.In order to assess whether QKI-5 could regulate FoxO1 expression at post-transcriptional level, we made a reporter constructs containing 3"UTR of FoxO1 at the downstream of luc reporter gene. Upon QKI-5 overexpression, the luciferase activity were gradually decreased, implying that QKI-5 could negatively regulate FoxO1 expression at post-transcriptional level. Furthermore we performed RNA co-immprecipitation assay, and verified that QKI-5 could associate specifically with 3'UTR of FoxO1 in vivo. Is this type of regulation functionally important in vivo?In model of RA-induced cell cycle arrest of mammary cancer cells, the expression of QKI-5 and FoxO1 were both up-regulated. we analyzed the effect of QKI-5 on cell cycle regulation following RA induction by specifically silencing the expression of QKI-5. As expected, the FoxO1 and related cell cycle regulators were moderately altered, implying that the effects of RA is partially mediated by QKI-5. Besides the stability of FoxO1 mRNA was greatly enhanced upon RA induction in QKI-5-silenced cells compared with that of controls, confirming the effects of RA on the stability of FoxO1 mRNA was mediated by QKI-5.It was known that ATRA is able to induce the differentiation of breast epithelium normally. In MCF-7 breast cancer line, ATRA induced cell cycle arrest, Under the condition of lacking QKI-5 , or at very low level, mimicked by qki-5 RNAi treatment, we found the incidence of senescence was dramatically enhanced determined by SA-gal detection assay. Our results have disclosed an underlying importance of QKI-5 in the posttranscriptional regulation of FoxO1 during ATRA induced cell cycle and differentiation process, without QKI-5, the cell tends to be more susceptible to become senescent under the ATRA treatment. All-trans-retinoic acid (ATRA) is a classic differentiation and apoptosos agent. ATRA treatment induce terminal differentiation and growth arrest of several established human myeloid cell lines in vitro and has also proven to be effective in the clinical treatment of acute promyelocytic leukemia (APL) by inducing differentiation and apoptosis of the immature blasts. Besides, ATRA is capable of directing the differentiation of several solid tumor cell lines.Although the biologic effects of ATRA are well characterized, the molecular mechanisms involved are largely unknown. Our previous experiment identified a series of differentially expressed genes in HL-60 cells upon treatment with ATRA by use of PCR-based subtractive hybridization method. Apr3 was one of the novel genes among them.The structural analysis by use of InterPro and PROSITE predicted that Apr3 contains several putative functional domains, including a signal sequence at N-terminus, following with one EGF-domain, one transmembrane region and the extremely short intracellular region at C-terminus. Our specific interest is to characterize if Apr3 was a membrane protein and participate in RA signal pathway in vivo.Since Apr3 was a differentially expressed genes in HL-60 cells upon ATRA treatment, In order to define its expression pattern in other cell line, RT-PCR analyses was employed to observe that Apr3 has a wide distribution among several cell lines, and be obviously up-regulated upon ATRA treatment. Indirect immunofluorescence assay disclosed that Apr3 was localized on the cell membrane, but its truncated mutant, lacking the transmembrane region and intracellular domain, was shown to be dotted and be likely a secreted one. Previous studies have indicated that retinoic acids could inhibit cell proliferation by inducing G1 arrest in many different cell types. In order to study the effects of Apr3 on cell cycle progression, FACS showed a higher ratio of cells arrest at G1 cell cycle phase after transient Apr3 transfection, while its mutant form overexpression showed the opposite effects with higher number of cells at S phase, denoting the high rate of cell proliferation. Cyclin D1 is one of the most important marker in G1 phase. we first performed cyclin D1 promoter reporter assay. Apr3 overexpression strongly inhibited the cyclin D1 promoter activities, in contrast, its mutant showed an opposite enhancing effects on cyclin D1. Both RT-PCR and Western Blot analyses confirmed the above results.Our data provide the first evidence to show that Apr3 is a membrane protein and plays a critical role in inducing cell cycle arrest at G1 phase by inhibiting Cyclin D1 expression. Its mutant form Apr3? showed opposite effects, suggesting that Apr3 is functionally important to regulate the cell proliferative status in vivo.