In mammals, the sperm deliver mRNA of unknown function into the oocytes during fertilization. The role of sperm microRNAs (miRNAs) in preimplantation development is unknown. miRNA profiling identified six miRNAs expressed in the sperm and the zygotes but not in the oocytes or preimplantation embryos. Sperm contained both the precursor and the mature form of one of these miRNAs, miR-34c. The absence of an increased level of miR-34c in zygotes derived from α-amanitin–treated oocytes and in parthenogenetic oocytes supported a sperm origin of zygotic miR-34c. Injection of miR-34c inhibitor into zygotes inhibited DNA synthesis and significantly suppressed first cleavage division. A 3′ UTR luciferase assay and Western blotting demonstrated that miR-34c regulates B-cell leukemia/lymphoma 2 (Bcl-2) expression in the zygotes. Coinjection of anti–Bcl-2 antibody in zygotes partially reversed but injection of Bcl-2 protein mimicked the effect of miR-34c inhibition. Oocyte activation is essential for the miR-34c action in zygotes, as demonstrated by a decrease in 3′UTR luciferase reporter activity and Bcl-2 expression after injection of precursor miR-34c into parthenogenetic oocytes. Our findings provide evidence that sperm-borne miR-34c is important for the first cell division via modulation of Bcl-2 expression.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of other genes by transcriptional inhibition or translational repression. miR-34a is a known tumor suppressor gene and inhibits abnormal cell growth. However, its role in other tumorigenic processes is not fully known. This study aimed to investigate the action of miR-34a on cell invasion. We found that miR-34a is expressed at various levels in cervical cancer (HeLa, SiHa, C4I, C33a and CaSki) and trophoblast (BeWo and JAR) cell lines. Transient forced expression of miR-34a did not affect the proliferation of these cell lines. Computational miRNA target prediction suggested that Notch1 and Jagged1 were targets of miR-34a. By using functional assays, miR-34a was demonstrated to bind to the 3' untranslated regions of Notch1 and Jagged1. Forced expression of miR-34a altered the expression of Notch1 and Jagged1 protein as well as Notch signaling as shown by the response of Hairy Enhancer of Split-1 protein to these treatments using western blot analysis. Forced expression of miR-34a suppressed the invasiveness of HeLa and JAR cells. By using gamma-secretase inhibitor (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) that interfered Notch signaling and RNA interference that knockdown Notch1 expression, we confirmed that downregulation of Notch1 reduced the invasiveness of the cells. Transfection of intracellular domain of Notch nullifies the effect of miR-34a on the invasiveness of the cells. Besides, we identified that miR-34a affected cell invasion by regulating expression of urokinase plasminogen activator through Notch. Our results provide evidence that miR-34a inhibits invasiveness through regulation of the Notch pathway and its downstream matrix degrading enzyme.

Cancer metastasis is determined by the formation of the metastatic niche and the ability of cancer cells to adapt to microenvironmental stresses. Anoikis resistance is a fundamental feature of metastatic cancer cell survival during metastatic cancer progression. However, the mechanisms underlying anoikis resistance in ovarian cancer are still unclear. Expressions of miRNA-141 and its downstream targets were evaluated by qPCR, Western blotting, Immunohistochemical (IHC) and in situ hybridization (ISH) assays. The luciferase assays were used to prove KLF12 as the downstream target of miR-141. The cDNA microarray and apoptotic protein arrays were used to identify the targets of miR-141 and KLF12. The competition of KLF12 and Sp1 on survivin promoter was examined by ChIP assay. IHC analysis on ovarian cancer tissue array was used to evaluate the expressions of KLF12 and miR-141 and to show the clinical relevance. The functional studies were performed by in vitro and in vivo tumorigenic assays. Enforced expression of miR-141 promotes, while knockdown of miR-141 expression inhibits, cell proliferation, anchorage-independent capacity, anoikis resistance, tumor growth and peritoneal metastases of ovarian cancer cells. Bioinformatics and functional analysis identified that Kruppel-related zinc finger protein AP-2rep (KLF12) is directly targeted by miR-141. Consistent with this finding, knockdown of KLF12 phenocopied the effects of miR-141 overexpression in ovarian cancer cells. In contrast, restoration of KLF12 in miR-141-expressing cells significantly attenuated anoikis resistance in ovarian cancer cells via interfering with Sp1-mediated survivin transcription, which inhibits the intrinsic apoptotic pathway and is crucial for ovarian cancer cell survival, anoikis resistance and peritoneal metastases. Immunohistochemical (IHC) and in situ hybridization (ISH) assays confirmed that miRNA-141 expression is inversely correlated with KLF12 expression and significantly associated with advanced ovarian cancers accompanied with distal metastases, underscoring the clinical relevance of our findings. Our data identify a novel signaling axis of miR-141/KLF12/Sp1/survivin in enhancing anoikis resistance and likely serves as a potential therapeutic target for metastatic ovarian cancer.

Perfluorooctanesulfonate (PFOS) was produced and used by various industries and in consumer products. Because of its persistence, it is ubiquitous in