Molecular Mechanism Of Small Molecule Aicar Maintaining Embryonic Stem Cell Pluripotency

Embryonic stem cells (ES cells) are generated from the inner cell mass ofblastocyst-stage embryos. These cells can propagate indefinitely, maintain self-renewal, anddifferentiate into almost any cell type of the body. As a result, they are considered to be thebest model system for research into early embryonic development. Therefore, studies on thepluripotency maintenance and differentiation of ES cells can provide new insight intoembryonic development, regenerative medicine, and organ transplantation.In ES cells, the balance between pluripotency and differentiation should be tightlycontrolled. It is well known that the core transcription network and signal pathways playcrucial roles in ES cells. However, this homeostasis is also influenced by the regulation ofepigenetic modifications. As an important mechanism of epigenetic regulation, microRNAs(miRNAs) play crucial roles in the regulation of ES cell pluripotency maintenance anddifferentiation.It was reported that，strong external stimuli, such as a transient low-pH stressor andhypoxia stress, were conducive to the formation of induced pluripotent stem cells (iPS cells).5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) is a membrane permeableactivator of AMP-activated protein kinase (AMPK). As an AMP analog, AICAR can activatekey protein of energy metabolism AMPK and repress anabolism, but do not influence thelevel of ATP, ADP and AMP. AICAR is thought to affect ES cell function and let cells in thestate of energy stress, but its role in ES cell fate decision is unclear. In this study, weinvestigated J1ES cells cultured with or without AICAR and aimed to uncover the functionof AICAR in ES cell stemness maintenance. The main contents and results of this research areas follows:1. AICAR combined with LIF (leukaemia inhibitory factor) can maintain J1ES cellcolony morphology and elevate expression levels of core pluripotent ES cell markers. Wecultured J1ES cells with1,000U/mL LIF and either1mM AICAR or an equal volume ofDMSO. In the presence of AICAR, J1ES cells showed high alkaline phosphatase (AP) activity, comparing with those cultured without AICAR. Immunofluorescence staining andreal-time PCR (quantitative real-time polymerase chain reaction) results indicated AICARtreatment can elevate the expression level of all four ES cell markers Nanog, Oct4, Sox2andKlf4. In addition, Although AICAR cannot maintain ES cell identity without LIF, it canantagonize the action of RA-induced differentiation.2. Whole-genome gene expression profiling indicated that AICAR maintains ES cells in apluripotent state. We performed microarray analysis to investigate AICAR downstreamtargets and further understood its effect on ES cells using the total RNA of cells cultured inthe presence or absence of AICAR. Our microarray data demonstrated that AICAR cansignificantly up-regulate pluripotency-associated genes and down-regulatedifferentiation-associated transcription factors. Using those differentially expressed genesidentified, we performed GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes andGenomes) pathway analysis with the DAVID (Database for Annotation, Visualization andIntegrated Discovery) online system. AICAR was not only shown to influence the AMPKpathway, but also act on other signaling pathways such as BMP, MAPK and TGF-β, tomaintain the stemness of J1ES cells.3. The function of AICAR to J1ES cells may associate with BMP signal pathway.Real-time PCR results suggested that the expression of BMP pathway associated genes Bmp2,Bmp4and their downstream regulators Ids, Coch and Dusp9were up-regulated following thetreatment of AICAR. Meanwhile, AICAR can antagonize the down-regulation function of RAon the above mentioned genes. Dorsomorphin (Dorso) is the inhibitor of BMP pathway. Itsrepression on the function of AICAR further demonstrated AICAR can maintain ES cellpluripotency through BMP pathway.4. AICAR induced epigenetic modification changes in J1ES cells. AICAR modulated EScell epigenetic modification by altering the expression of epigenetic-associated proteins,including Dnmt3a, Dnmt3b, Smarca2, Mbd3, and Arid1a. Our study indicated, the globalDNA5-mC (5-methylcytosine) level was decreased, while5hmC (5-hydroxymethyl cytosine)level was unchanged. As to histone modification, AICAR treatment down-regulate H3K9Ac(histone H3lysine9acetylation) level and up-regulated H3K27me3(histone H3lysine27tri-methylation) level. In addition, AICAR also influence the transcription of lincRNAs (longintervening non-coding RNA).5. sRNA (Small RNA) high-throughput sequencing results suggested AICAR cansignificantly modulate the expression of multiple miRNAs, including those have crucialfunctions in ES cell development. We identified the miRNAs expression patterns of theAICAR-treated J1ES cells and those without treatment. The selected differentially expressed miRNAs were further validated and confirmed by real-time PCR. For the differentlyexpressed miRNAs identified, further study was conducted regarding the pluripotency anddifferentiation associated miRNAs with their targets. Particularly, mostdifferentiation-associated miRNAs were down-regulated and pluripotency-associatedmiRNAs were up-regulated.6. Down-regulation of miR-134was partly responsible for increased expression ofpluripotency markers. Mir-134is a differentiation-associated miRNA and its expression wasup-regulated during RA-induced ES cell differentiation. Compared with J1ES cells withouttreatment, expression of miR-134was significantly decreased following AICAR treatment,while its targets Nanog and Sox2was increased. Overexpression of miR-134can inducedown-regulation of pluripotency markers Nanog, Oct4, Sox4and Klf4, and the addition ofAICAR can antagonize the decreased expression of Nanog and Sox2.7. Prediction and validation of miRNAs that may target Myc. Myc was significantlydown-regulated after AICAR treatment. To explore the reasons of Myc down-regulation, wepredicted miRNAs that may target the3‘-UTR (untranslated region) of Myc using theTargetScan and miRanda database, and selected those up-regulated miRNAs to make furtherresearch. Our results identified miR-34a,34b and34c can repress the expression of Myc in J1mouse ES cells. Furthermore, miR-135b and miR-340may also regulate Myc expression bydirectly target the3‘-UTR of Myc mRNA.Overall, our work suggests that AICAR is capable of maintaining ES cell self-renewaland pluripotency, which could be useful in future medical treatment and provide insight intothe clinical application of ES cells.