| AbstractCardiovascular diseases, especially myocardial ischemic diseases which cause large amount of functional myocardial cell injury and deaths, including heart failure, have become the primary threat to human health. For all types of end-stage cardiovascular diseases, current conventional treatments can only slow the progression of the diseases, but cannot completely cure the patients. The development of regenerative medicine and modern biomedical engineering has enabled us to regenerate parts of tissues / organs that would be helpful in repairing, regenerating or replacing damaged tissues and organs in vivo. Stem cells can be induced into cardiomyocytes, and the use of tissue engineering technology enables the in vitro construction of myocardial tissue based on stem cells. Progression during this field has brought new hopes for cardiovascular diseases treatment and it has become a research hotspot now. Stem cells have some specific characteristics, such as the ability to self-renewal and to differentiate into certain types of somatic cell. It has been reported that some kinds of stem cells can regenerate functional cardiac cells in vitro or in vivo and can repair damaged cardiac tissue, thus restore cardiac function. It has become a new strategy and future treatment of cardiovascular diseases. Induced pluripotent stem cells, i PSCs, was first reported in 2006, the researchers inserted four genes of transcriptional factors into mouse skin fibroblasts, and successfully obtained a new kind of stem cell similar to embryonic stem cell. The i PSC can be obtained from the somatic cells of patients and has become the current hot pet, for circumventing the ethics controversy and potential immune rejection. However, there are still many obstacles concerning the efficiency, differentiation mechanisms and target cell maturation of i PSCs induced cell differentiation that have hindered its usage in cardiac tissue regeneration project.Micro RNAs(mi RNAs) is a class of endogenous small non-coding RNAs, widely present in the eukaryotic cells. They have highly conserved nucleotide sequences and have a wide variety of biological functions. Mi RNA inhibits protein translation by degradation of m RNA expression, and regulates cell growth, differentiation, migration, apoptosis, and tissue development. Numerous studies have reported that about 40 kinds of micro RNA are involved in the regulation of cardiac physiological and pathological activities. Among them, micro RNA-1(mi RNA-1) and micro RNA-133(mi RNA-133) has been confirmed that are involved in the development of embryonic cardiac. The high expression levels of mi RNA-1 and mi RNA-133 in muscle tissue also have been confirmed. To this end we speculate: mi RNA-1 and mi RNA-133 may be involved in the regulation of i PSC differentiation into cardiomyocytes, and revealing the underlying mechanism helps promoting effective cardiomyocytes differentiation from i PSCs. ObjectiveThe objective of this research was to study the effects of mi RNA-1 and mi RNA-133 on inducing cardiomyocytes differentiation of mi PSC, and to further explore mi RNA-1 and mi RNA-133-related mechanisms to promote mi PSC differentiation into cardiomyocytes. Methods1) Using RT-PCR to detect expression levels of mi RNA-1 and mi RNA-133 in undifferentiated mi PSC, neonatal rat cardiomyocytes and differentiating mi PSC, respectively. For the differentiating mi PSC group, samples at different time points of 0 d, 3 d, 5 d, 10 d were collected and compared.2) Using the transient transfection method to transfect mi PSC with mi RNA-1, mi RNA-133 and both, respectively. The transfection efficiencies were verified by detecting expression levels of mi RNA-1 and mi RNA-133 48 hours after transfection.3) After transfection, the cells were submitted to spontaneous differentiation for another 14 days(15 days total). The number of beating cell cluster o on the 15 th day was counted, and m RNA expression of the cardiac-specific transcription factor NKX2.5 and two cardiac-specific proteins c Tn T and α-MHC were detected by RT-PCR and q RT-PCR. Statistical analysis were performed using one-way ANOVA comparison.4) To verify the existence of mi PSC derived cardiomyocyte, immunocytotochemical staining of specific markers of myocardial structural protein c Tn T was performed.5) Inhibitors of ERK1 / 2 / MAPK, JNK, FAK, AMPK pathways were administrated respectively in the mi PSC differentiation culture medium, and cardiac markers were investigated by q RT-PCR after 15 days. By comparing the specific effect on cardiac marker expression, we pinpointed a possible signaling pathways involved in mi RNA-1 and mi RNA-133 related cardiac differentiation.6) Detect protein phosphorylation levels of ERK expression by Western blot, verify its relationship with c Tn T. Results1) Oct4 and Nanog were expressed in mi PSC cultured in gelatin-coated tissue culture plates using a monolayer culture method. The expressions of mi RNA-1 and mi RNA-133 were not detected in undifferentiated mi PSC, but were highly expressed in mouse cardiomyocytes. Spontaneous beating cells were spotted after 10 days of differentiation. From the fifth day onwards, the NKX2.5 and c Tn T gene expressions were detected and were gradually increased. The mi RNA-1 and mi RNA- 133 expressions were also co-related with time increasing. These results confirmed that monolayer cultured mi PSC can spontaneously differentiate into cardiomyocytes; and mi RNA-1 and mi RNA-133 expressions were elevated with the progress of mi PSC differentiation. Thus it suggested that mi RNA-1 /-133 may be involved in i PSC directed differentiation into cardiomyocytes.2) The transfection of mi PSC with mi RNA-1 and mi RNA-133 vectors were highly success, because the results of RNA expression in the 3rd day revealed both mi RNAs were significantly expressed in target cells.3) The overexpression of either mi RNA-1 or mi RNA-133 has no significant effect on cell differentiation into cardiomyocytes of mi PSC, but the co-transfection could significantly enhance myocardial differentiation, suggesting that a synergy between mi RNA-1 and mi RNA-133 in promoting cardiac differentiation. RT-PCR detection of cardiac-specific markers Nkx2.5, α-MHC, c Tn T and MLC2 a / 2v demonstrated significantly higher expression levels of mi PSC differentiate into cardiomyocytes when overexpressing both mi RNA-1 and mi RNA-133 at the same time.4) After the differentiation for 15 days, immunofluorescence staining of cardiac-specific protein c Tn T convinced mi PSC spontaneous differentiation into beating cardiomyocytes in vitro. The single transfection of mi RNA-1 or mi RNA-133 had no effect on myocardial differentiation, but co-transfection could significantly enhance the generation of differentiated cardiomyocytes, and the cardiac sarcomeres were more obvious.5) ERK1 / 2 / MAPK, JNK, FAK, AMPK pathway inhibition screening found that only ERK1 / 2 / MAPK pathway inhibitor could significantly prohibited the effect of mi RNA-1 and mi RNA-133 in inducing cardiomyocyte differentiation of mi PSC. It was thus proposed that the ERK1 / 2 / MAPK pathway is involved in mi RNA-1 / mi RNA-133-related cardiomyocyte inducing effect.6) Western blot detected the expression levels of ERK1 / 2 and phospho-ERK(p-ERK1 / 2) during mi PSC differentiation after mi RNA-1 /-133 co-overexpression, and found that, mi RNA-1 and mi RNA-133 joint overexpression could increase the ERK1 / 2 and phospho-ERK(p-ERK1 / 2) expression levels. While adding p-ERK1 / 2 inhibitor PD98059 could significantly decrease p-ERK-1 / 2 ratio, and inhibit the expression of c Tn T. ConclusionThe mi RNA-1 and mi RNA-133 participate in cardiomyocytes differentiation of mi PSC, and co-overexpression of mi RNA-1/-133 at the beginning of mi PSC differentiation can promote cardiomyocyte differentiation through up-regulation of p-ERK1 / 2 of ERK1 / 2 /MAPK signal pathway. |
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