MiR-301a promotes embryonic stem cell differentiation to cardiomyocytes.

BACKGROUND: Cardiovascular disease is the leading cause of death worldwide. Tissue repair after pathological injury in the heart remains a major challenge due to the limited regenerative ability of cardiomyocytes in adults. Stem cell-derived cardiomyocytes provide a promising source for the cell transplantation-based treatment of injured hearts. AIM: To explore the function and mechanisms of miR-301a in regulating cardiomyocyte differentiation of mouse embryonic stem (mES) cells, and provide experimental evidence for applying miR-301a to the cardiomyocyte differentiation induction from stem cells. METHODS: mES cells with or without overexpression of miR-301a were applied for all functional assays. The hanging drop technique was applied to form embryoid bodies from mES cells. Cardiac markers including GATA-4, TBX5, MEF2C, and α-actinin were used to determine cardiomyocyte differentiation from mES cells. RESULTS: High expression of miR-301a was detected in the heart from late embryonic to neonatal mice. Overexpression of miR-301a in mES cells significantly induced the expression of cardiac transcription factors, thereby promoting cardiomyocyte differentiation and beating cardiomyocyte clone formation. PTEN is a target gene of miR-301a in cardiomyocytes. PTEN-regulated PI3K-AKT-mTOR-Stat3 signaling showed involvement in regulating miR-301a-promoted cardiomyocyte differentiation from mES cells. CONCLUSION: MiR-301a is capable of promoting embryonic stem cell differentiation to cardiomyocytes.

[Cloning of ESTs of spermatogenesis-related genes from early stage spermatogenic cells of mice using a modified DDRT-PCR method].

To avoid the shortcomings of radioactive pollution and high rate of false positives in DDRT-PCR method, the technique have been modified by replacement of radioactive reagents with fluorescent reagents followed by confirming the results using reverse RNA dot blot. The modified DDRT-PCR method was used in this study to clone spermatogenesis-related genes from early stage spermatogenic cells of mice. Primitive spermatogonia were isolated from 6-day-old mice and type B spermatogonia from 9-day-old mice. The purity of isolated cells was over 90%. Total RNA was extracted from the cells, fluorescent differential display technique was performed to screen the differentially expressed genes. Differences in expression of the screened fragments were identified by reverse RNA dot blot. 16 ESTs were selected for sequencing. The analysis results in GenBank/Blast database revealed that 7 of them were novel ESTs, and they were then registered in GenBank. All of them expressed stronger in B spermatogonia than in primitive spermatogonia. 3 of them were chosen to further identify their expression patterns by semi-quantitative RT-PCR. Compared with traditional differential display technique, the modified method used in this study can avoid radioactive pollution and eliminate false positive results. The present study suggests that the gene activation or up-regulation in B spermatogonia may be related to some specific process in the following steps of spermatogenesis.

NYD-SP16, a novel gene associated with spermatogenesis of human testis.

By hybridizing human adult testis cDNA microarrays with human adult and embryo testis cDNA probes, a novel human testis gene NYD-SP16 was identified. NYD-SP16 expression was 6.44-fold higher in adult testis than in fetal testis. NYD-SP16 contains 1595 base pairs (bp) and a 762-bp open reading frame encoding a 254-amino acid protein with 73% amino acid sequence identity with the mouse testis homologous protein. The NYD-SP16 gene was localized to human chromosome 5q14. The deduced structure of the NYD-SP16 protein contains one transmembrane domain, which was confirmed by GFP/NYD-SP16 fusion protein expression in the cytomembrane of the transfected human choriocarcinoma JAR cells, suggesting that it is a transmembrane protein. Multiple tissue distribution indicated that NYD-SP16 mRNA is highly expressed in the testes and pancreas, with little or no expression elsewhere. Further analysis of abnormal expression in infertile male patients revealed complete absence of NYD-SP16 in the testes of patients with Sertoli-cell-only syndrome and variable expression in patients with spermatogenic arrest. Homologous gene expression in mouse testis was confirmed in spermatogenic cells by in situ hybridization. The results of cDNA microarray, in situ hybridization, and semiquantitative polymerase chain reaction in mouse testis of different stages indicated that NYD-SP16 expression is developmentally regulated. These results suggest that the putative NYD-SP16 protein may play an important role in testicular development/spermatogenesis and may be an important factor in male infertility.

[Characterization of expression of the centrosomal protein, Cenexin, in rat spermatogenesis].

The centrosomal protein, Cenexin, is a molecular marker of mature centriole. To elucidate the variability and function of mature centriole in spermatogenesis, the high titer polyclonal antibody against rat cenexin was obtained by immunizing mice with recombinant cenexin which was made up in this study. The expression of cenexin in rat spermatogenesis was carried out by semi-quantitative RT-PCR, immunofluorescence and Western Blot. The results demonstrated that the level of Cenexin mRNA was higher in spermatogonia and spermatocytes, then decreased in following stages, while Cenexin protein was located on one centriole from spermatogonia to spermatids, showing mature centriole existed in these stages. Cenexin protein was localized to the basal body of the flagellum in elongated spermatids and the stained signal disappeared in the most of epidydimal sperms. These results suggested that the expression pattern of cenexin in rat spermatogenesis might be related to the initiation of the flagella formation.