Premature ovarian failure (POF) is a heterogeneous disease. Though dozens of candidate genes have been identified for the genetic etiology of POF, it is largely unexplained in majority of patients. Recently, Wt1+/R394W mice was found to present POF-like phenotype, which indicates that WT1 might be a plausible candidate gene for non-syndromic POF. The coding region of WT1 gene was screened in 384 patients with POF and 6 novel variations were identified, including two missense mutations (p. Pro126Ser in exon1 and p. Arg370His in exon7) and four intronic variants (c.647-27C > T, c.647-13G > C, c.647-13G > A in intron1 and c.950 + 14T > C in intron 4). In vitro experiments showed that both mutant p. Pro126Ser and p. Arg370His repressed the expression of Amh and Cdh1, and induced the expression of Fshr and Cyp19 in mRNA level (P < 0.05). The expression changes of AMH, FSHR, CYP19 and CDH1 were confirmed by western blot. These genes (AMH, FSHR, CYP19 and CDH1) are required for granular cells (GCs) proliferation, differentiation and oocyte-GCs interaction. The novel mutant p. P126S and p. R370H in the WT1 gene potentially impaired GCs differentiation and oocyte-GCs interaction, which might result in loss of follicles prematurely. Therefore, WT1 is a plausible causal gene for POF.

MicroRNAs (miRNAs) have been improved to regulate oocyte development in a cell- or stage-specific manner. In this study, we aimed to clarify microRNA-224's (miR-224) role in cumulus cells (CCs), to find out whether a change level of miR-224 in CCs could influence the maturation of oocyte. We found that overexpression of miR-224 of CCs led to the impairment of cell expansion, along with a decrease in the gene expression associated with cell expansion and maturation of oocyte. The increased expression of miR-224 in CC interrupted oocyte cell cycle at the GV stage. The GDF9, BMP15 and ZP3 of the oocytes were also down-regulated. The following in vitro fertilization had yielded a lower number of oocytes from cumulus-oocyte complexes (COCs) overexpressing miR-224 when reaching the blastocyst stage. The suppressive effect of miR-224 in the maturation of COC is validated by the miR-224 knockdown model, where the expansion of cumulus cell was increased and oocyte was developed to MII stage. In addition, the expression of aromatase in CCs was down-regulated by miR-224, resulting in a decreased level of estradiol (E2). A further investigation found that miR-224 down-regulated the expression of protein and mRNA of Ptx3 by targeting its 3'UTR. Our study revealed that miR-224 regulates the gene expression and function of CCs, which influences the maturation of oocyte, at least in part, via targeting Ptx3.

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BackgroundCarcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a member of CEACAM family and has been reported to be upregulated in various types of human cancer and involved in tumor progression and metastasis. However, the biological roles and clinical significances of CEACAM6 in osteosarcoma still remain to be elucidated.Materials and methodsReal-timePCR, immunohistochemistry and Western blot analysis were used to determine CEACAM6 expression in osteosarcoma cell lines and clinical specimens. Then the clinical relevance of CEACAM6 was analyzed in osteosarcoma. The function of CEACAM6 in osteosarcoma was examined by wound-healing and cell invasion assays, and expression levels of epithelial–mesenchymal transition markers.ResultsIn the present study, we found that CEACAM6 was markedly upregulated in metastatic osteosarcoma tissues when compared with the nonmetastatic osteosarcoma tissues. Upregulation of CEACAM6 was significantly associated with lung metastasis status (P=0.006) in patients with osteosarcoma. Survival analyses suggested that osteosarcoma patients with high CEACAM6 expression had a significantly shorter overall survival time and lung metastasis-free survival time than those with low CEACAM6 expression. Knockdown of CEACAM6 inhibits osteosarcoma cell migration and invasion. Moreover, silencing CEACAM6 suppressed osteosarcoma cells epithelial–mesenchymal transition.ConclusionTaken together, this study suggests that CEACAM6 might be a promising biomarker and a potential therapeutic target for the treatment of metastatic osteosarcoma.