| MicroRNAs (miRNAs) are endogenous non-coding RNA that exist widely in eukaryotes, the length of which are usually 19-25 nucleotides. Previous studies show that miRNA could bind to the seed squences of the target messenger RNAs (mRNAs) 3’ untranslated regions (3’UTRs), blocking the translation of mRNA or degradating the target mRNA. Thus miRNAs play an important role in the regulation of cell functions, including poliferation, cell apotosis, cell migration, lipid synthesis and stem cell differentiation etc. Meanwhile, miR-218 was first to be reported high expressed in nervus centralis. miR-218 could regulate the invasion, migration, proliferation and sternness of glioblastoma (GBM) through different target genes. Thus it brings a new target of clinical treatment for the inhibition of GBM. In recent reports, there are also some assays about miR-218 during the cardiac differentiation and development. Regulatory circuit between miR-218 and Tbx5 affects the heart development of zebrafish. Downregulation of miR-218 could rescue the defects generated by Tbx5 over-expression during heart development. Over-expression of miR-218 in zebrafish embryos results in irregular walls in ventricles and failing to form atria and hearts looping. While the role of miR-218 during embryonic stem cell cardiomyogenesis still remains undiscoverd, it is urgent to start the research in a representative mammalian embryonic system.Since murine embryonic stem (ES) cells were first found in the inner cell mass of blastocysts, they are widely used in varieties of researches because of their infinite multiplication capacity in suitable vitro circumstances and the ability of differentiating into any kinds of somatic cells in vivo or in vitro now. It is reported that miR-1 and miR-133 are involved in regulating the formation of embryonic cardiac development and cardiac differentiation. The Let-7 family also take part in the regulation of cardiovascular disease and myocardial differentiation of ES cells. miR-218 was reported regulating the self-renewal of stem cell. Rictor was identified as a novel target of miR-218 in oral squamous cell carcinoma (OSCC), through which miR-218 inhibit the activation of the mTOR-Akt signaling pathway, results in the regulatory of oral carcinogenesis. Rictor is the core subunits of mTORC2, which could affect the stability and integrity of mTORC2. Rictor can affect the differentiation fate of embryonic stem cell to the developing embryonic, promote the formation of cardiovascular and restrain the myocardial atrophy. However, there is no reports about any direct targeting genes of miR-218 in ES cells that regulate the cardiomyocyte differentiation of ES cells in vitro up to now.Cell migration which runs through the process of ES cells myocardial differentiation, plays an important role in the differentiation of ES cells. In the model of ES cells cardiac differentiation in vitro, the formation of embryoid bodies (embryonid bodies, EBs) imitates the formation of gastrula with three germ layers, during which involved a series of morphogenetic movements including cell proliferation, migration, accumulation and rearrangement. miR-124a downregulated the modules rearrangement of the migratory cytoskeleton by reduce the expression of SLUG and IQGAP1, thus inhibit the gastrulation of human embryonic stem cells. miR-218 regulates cancer cell migration through Slit2/Robol pathway and affects the expression of Cell division cycle 42 (Cdc42) and Racl. But there are still no reports about the relationship between miR-218 and cell migration during the mouse ES cells cardiac differentiation.Therefore, this research mainly explore the regulatory role of miR-218 during the process that ES cells oriented differentiate to cardiomyocytes in vitro. Further research was taken by bioinformatics. Bioinformatics play a key role in the understanding of the complex interactions between miRNAs and genome. Approaches such as Targetscan, miRanda and Pictar are used to analysis the complementary sequences and structure. Further function analysis like the signaling pathway and functional prediction could be predicted through approaches such as miRPath, Tarbase and MicroT-CDS. And the gene expression of the predicted results is further detected by PCR. In order to elucidate the relationship between the regulation of miR-218 expression and the differentiation of ES cells into myocardial cells, it is important to reveal the potential target gene of miR-218 that may be involved in the regulation of cardiac differentiation process. Furthermore, this research could provide experimental basis for further study on the mechanism of ES cell differentiation.Objective:To investigate the expression of miR-218 and its role in the regulation of mouse ES cells cardiomyogenesis and the potential target genes of miR-218. To elucidate the relationship between miR-218 and the differentiation of mouse ES cells. To analysis the post transcriptional regulatory molecules of miR-218 in the differentiation of ES cells.Methods:This study were based on the classsical ES cell cardiomyogenesis model: first hanging drops with definite number of cells as embryoid bodies for 3 days, then culture EBs in bacteriological dishes for 2 days, finally plating EBs for further differention. The expression of miR-218 was detected by quantitative real-time polymerase chain reaction (qRT-PCR). miR-218 mimic/inhibitor was used to investigate whether the dosage of miR-218 affects the cardiac differention. Western-blot was used to detect the expression of proteins. Flow cytometry, immunostaining experiments were taken to reveal the effects of miR-218 on the number and the structure sarcomere of cardiac cell. The intracellular Ca2+ transients were tested by Fluo4-AM staining. Wound migration assays and transwell migration assays were taken to detect the effect of miR-218 on ES migration in vitro, and Western-blot tested the expression of cell adhesion and migration interrelated proteins. The target gene of miR-218 in ES cells was analysised by bioinformatics. RT-PCR and qRT-PCR detected the expression level of target gene after the transfection of miR-218 mimic into ES cells.Results:1. miR-218 mimics inhibited the formation of myocardial with 12±2% and 32±7% beating EBs at day 5+2 and day 5+3, much more lower than the beating percent 29±4% and 56±5% in the control group (P<0.01). The amplitude of the intracellular Ca2+ transients was fallen in the cardiomyocytes derived from ES cells. And the specific protein marker of Mesoderm brachyury, cardiac specific transcription factor Nkx2.5, GATA4, TBX5, myocardial structure protein a-Actinin, specific marker of ventricle-like cells MLC-2v, atrial-like cells ANP and pacemaker-like cells HCN4,desmosomal protein Desmoplakin, cell junction proteins Cx43 and N-cadherin decreased significantly. Flow Cytometry found that myocardial cells numbers reduced remarkably. Sarcomere structure turned into disorder. miR-218 inhibitor promoted ES cells cardiomyogenesis and upregulated the expression of Nkx2.5, GATA4, TBX5, a-Actinin, MLC-2v, ANP, HCN4, L-type Ca2+ CPαlD, CaMKII5, Desmoplakin, Cx43 and N-cadherin. EBs which were transfected by miR-218 inhibitor presented higher beating percent in contrast with the control group, with 68±4% and 84±8% at day 5+2 and day 5+3 (P<0.01). The amplitude of the intracellular Ca2+ transients was elevated in the cardiomyocytes derived from ES cells. miR-218 inhibitor also increased the number of myocardial cells and showed a ladder-like sarcomere structure. However, the change of the miR-218 in ES cells did not affect the expression of cardiac specific transcription factor MEF2C.2. Bioinformatics analysis showed that there were 164 target genes through which miR-218 participated in the regulation of 20 pathways. Combine the prediction results with literature reports, eight genes from four signaling pathways (Rictor, Pik3r1, Pi4k2a, Adcy1, Shank2, Pdgfr-a, Grml and Gnai2 from Phosphatidylinositol signaling system, Glutamatergic synapse, Gap junction and mTOR signal pathway) which may be involved in myocardial differentiation were chosen for further research. PCR results showed that miR-218 mimic could significantly downregulate the expression of Rictor, Pik3r1, Shank2, Pdgfr-a, Grml.3. On the base of the prediction and verification of miR-218 target genes Pdgfr-a, Grml and Rictor and the reported of the regulation of these genes on the caidiomyogenesis of ES cells or cell migration of tumor cells. We detected the regulation of miR-218 on the cell migration of ES cells. miR-218 mimic promoted cellular migration away from EBs at day 5+3, the distance between the edge of the EBs and the nucleus of the most distant cell was 431.1 μm, much more longer than the distance of the control EBs 350.1 μm. While miR-218 inhibitor restrained the migration, the distance was 235.9 μm, lower than the distance of the control EBs 318.7 μm. In the wound healing model, miR-218 mimic significantly increased the ES cell migration distance compared with the negative control group at 12 h and 24 h. The transwell experimental results showed that miR-218 mimic can promote murine ES cell migration in vitro. miR-218 mimic can also inhibit cell migration related proteins Racl and Cdc42 expression through the target gene of Grml, Pdgfr-a or Rictor.Conclusions:1. During the differentiation of mouse ES cells into cardiomyocytes, the expression of miR-218 is closely related to the cardiac differentiation. miR-218 mimic inhibited cardiomyocyte differentiation, while miR-218 inhibitor significantly promoted the differentiation of ES cells into cardiomyocytes.2. Bioinformatics results showed that miR-218 might regulate 164 genes that are involved in 20 intracellular signaling pathways. miR-218 mimic coud downregulate the mRNA expression of Rictor, Pik3rl, Shank2, Pdgfr-a, Grml. These five genes can be important targets for the following study of the regulation of miR-218 on the cardiomyocyte differentiation of ES cells.3. The expression of miR-218 in ES cells was related to the directional spreading ability of EB cells during the myocardial differentiation. The increased expression of miR-218 could promote the migration of ES cells in vitro, while the decreased expression of miR-218 could inhibit the migration of ES cells in vitro. miR-218 could also regulate the cell migration related proteins Cdc42 and Racl. |
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