[Impact of the expression of DNA methyltransferase 1 on the differentiation of spermatogonial stem cells in mice].

OBJECTIVE: To investigate the influence of the expression of DNA methyltransferase 1 (DNMT1) on the differentiation of spermatogonial stem cells (SSC) in mice. METHODS: SSCs were isolated from the testis tissue of 1-week-old BALB/c male mice by two-step enzyme digestion. DNMT1-siRNA and negative control siRNA (NC-siRNA) were transfected into the third-generation SSCs after isolation and purification, and the untransfected cells were used as the control. At 24 hours after transfection, the mRNA and protein expressions of DNMT1 were detected by real-time quantitative PCR (RT-qPCR) and Western blot, respectively, and the methylation level of DNMT1 was determined. The SSCs were induced to differentiate into spermatocytes using the stem cell growth factor, and the expressions of the germ cell proliferation-related protein (Nanos2), promyelocytic leukemia zinc finger protein (PLZF) and retinoic acid-stimulated protein 8 (Stra8) were measured by RT-qPCR and Western blot after 48 hours of differentiation. RESULTS: At 24 hours after transfection, the relative mRNA and protein expressions of DNMT1 and the DNA methylation level were significantly decreased in the DNMT1-siRNA group compared with those in the control and DNMT1-NC groups (P < 0.05), but showed no statistically significant difference between the latter two (P > 0.05). The relative mRNA and protein expressions of Nanos2 and PLZF were also decreased while those of Stra8 increased in the DNMT1-siRNA group in comparison with those in the control and DNMT1-NC groups after 48 hours of differentiation (P < 0.05), but none exhibited any statistically significant difference between the control and DNMT1-NC groups (P > 0.05). CONCLUSION: Knockdown of DNMT1 promotes the differentiation of SSCs into spermatocytes in mice, which may be related to the reduction of the genome methylation level, inhibition of the expressions of Nanos2 and PLZF, and promotion of the expression of Stra8.

Transcriptional activation of human CDCA8 gene regulated by transcription factor NF-Y in embryonic stem cells and cancer cells.

The cell division cycle associated 8 (CDCA8) gene plays an important role in mitosis. Overexpression of CDCA8 was reported in some human cancers and is required for cancer growth and progression. We found CDCA8 expression was also high in human ES cells (hESCs) but dropped significantly upon hESC differentiation. However, the regulation of CDCA8 expression has not yet been studied. Here, we characterized the CDCA8 promoter and identified its cis-elements and transcription factors. Three transcription start sites were identified. Reporter gene assays revealed that the CDCA8 promoter was activated in hESCs and cancer cell lines. The promoter drove the reporter expression specifically to pluripotent cells during early mouse embryo development and to tumor tissues in tumor-bearing mice. These results indicate that CDCA8 is transcriptionally activated in hESCs and cancer cells. Mechanistically, two key activation elements, bound by transcription factor NF-Y and CREB1, respectively, were identified in the CDCA8 basic promoter by mutation analyses and electrophoretic motility shift assays. NF-Y binding is positively correlated with promoter activities in different cell types. Interestingly, the NF-YA subunit, binding to the promoter, is primarily a short isoform in hESCs and a long isoform in cancer cells, indicating a different activation mechanism of the CDCA8 transcription between hESCs and cancer cells. Finally, enhanced CDCA8 promoter activities by NF-Y overexpression and reduced CDCA8 transcription by NF-Y knockdown further verified that NF-Y is a positive regulator of CDCA8 transcription. Our study unearths the molecular mechanisms underlying the activation of CDCA8 expression in hESCs and cancer cells, which provides a better understanding of its biological functions.

FOXJ2 controls meiosis during spermatogenesis in male mice.

Spermatogenesis is a highly complex cell differentiation process necessary for production of haploid spermatozoa. Central to this unique process is spermatocyte meiosis. FOXJ2 (Forkhead box J2), a FOX transcription factor, is specifically expressed in meiotic spermatocytes in adult mouse testes, so we used a germ cell specific conditional knockout model (Foxj2(flox/flox) , Mvh-Cre) to explore its role in spermatogenesis. Loss of FOXJ2 in the male germ line led to meiotic arrest and complete infertility. Although, DNA double-strand breaks (DSBs) were initiated, Foxj2-deficient spermatocytes failed to form chromosomal synapses and perform DSB repair. Furthermore, Foxj2-deficient spermatocytes contained significantly less mRNA encoding DSB repair-associated factors (Rad18, Rad51, Brca1, Brca2, and Tex15) and meiotic arrest-related proteins (Fzr1, Hsp70-2, Spata22, Eif4g3, and Zpac); in contrast, no change was observed in the expression of spermatogonia markers (Gfra1, Zbtb16, and c-Kit) and germ cell markers (Dazl, Mvh, and Tra98). Taken together, FOXJ2 appears to promote meiotic progression in male mice by a mechanism that needs further investigation. Mol. Reprod. Dev. 83: 684-691, 2016 © 2016 Wiley Periodicals, Inc.

Overexpression of RASAL1 Indicates Poor Prognosis and Promotes Invasion of Ovarian Cancer.

RAS protein activator like-1 (RASAL1) exists in numerous human tissues and has been commonly demonstrated to act as a tumor suppressor in several cancers. This study aimed to identify the functional characteristics of RASAL1 in ovarian adenocarcinoma and a potential mechanism of action. We analyzed RASAL1 gene expression in ovarian adenocarcinoma samples and normal samples gained from the GEO and Oncomine databases respectively. Then the relationship between RASAL1 expression and overall survival (OS) was assessed using the Kaplan-Meier method. Furthermore, the biological effect of RASAL1 in ovarian adenocarcinoma cell lines was assessed by Quantitative real time-PCR (qRT-PCR), Cell Counting Kit-8 (CCK-8), western blot, wound healing and transwell assay. The statistical analysis showed patients with higher RASAL1 expression correlated with worse OS. The in vitro assays suggested knockdown of RASAL1 could inhibit cell proliferation, cell invasion and migration of ovarian adenocarcinoma. Moreover, the key proteins in the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathway were also decreased in ovarian adenocarcinoma cells with RASAL1 silencing. These findings provide promising evidence that RASAL1 may be not only a powerful biomarker but also an effective therapeutic target of ovarian adenocarcinoma.