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Objective To investigate the relationship between miR-411 and breast cancer,and the expression of miR-411 and its effects and mechanism research on proliferation and metastasis in breast cancer.Methods Real-time polymerase chain reaction (RT-PCR) was used to detect the expression of miR411 in breast cancer cells and tissues.Methyl thiazolyl tetrazolium (MTT),clone formation assay and Transwell assay were used to detect the effect of miR-411 expression on the proliferation,migration and invasion in breast cancer cells.The effect of miR-411 on growth factor receptor-bound protein 2 (GRB2) expression in breast cancer cells was detected by RT-PCR and Western blot.The direct effect of miR-411 target on GRB2 was detected by dual luciferase reporter assay.MTT,clone formation assay and Transwell assay detect the effect of GRB2 expression on the proliferation and invasion in breast cancer cells.Detection the effect high expression of miR-411 on GRB2 downstream signaling pathway related molecules expression in breast cancer cell with Western blot.Results The expression of miR-411 in breast cancer tissues was significantly lower than that in adjacent non-cancerous tissues (P ＜ 0.05).The expression of miR-411 in breast cancer cells SK-BR-3,BT-549 and MDA-MB-231 was significantly lower (P ＜ 0.05).Compared to the negative control group,the transfection of miR-411 mimic inhibited the proliferation,migration and invasion of MDA-MB-231 breast cancer cells (P ＜ 0.05).Targetscan showed that miR-411 could bind to GRB2 3'UTR at position 741-747.Compared with the negative control group,GRB2 3'UTR wild-type plasmid and miR-411 co-transfection reduced the fluorescence activity (P ＜ 0.05).Transfection of siGRB2 significantly reduced the expression of GRB2 protein in MDA-MB-231 breast cancer cells (P ＜ 0.05).Compared to the negative control group,the inhibition of GRB2 expression reduced the proliferation and the number of colony formation of MDA-MB-231 breast cancer cells (P ＜ 0.05).Transwell assay showed that transfection of siGRB2 significantly reduced the number of invasive cells in MDA-MB-231 breast cancer cells (P ＜ 0.05).Conclusions miR-411 is related closely to the occurrence and development of breast cancer,miR-411-GRB2-Ras axis is expected to become a new target for biological treatment of breast cancer.
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OBJECTIVE: To investigate the effect(s) of exogenous gonadotropin on the cytoplasmic and nuclear maturation of cumulus-partially enclosed immature human oocytes in vitro derived from ovarian stimulation cycles. DESIGN: Experimental human study. SETTING: University-based laboratory. PATIENT(S): Women, aged 26-35 years, with infertility secondary to male factors, underwent ovarian stimulation and intracytoplasmic sperm injection cycles using a long protocol of pituitary down-regulation. INTERVENTION(S): Cumulus-partially enclosed immature human oocytes that were retrieved from the stimulated cycles were collected at the time of intracytoplasmic sperm injection. The cumulus-partially enclosed immature human oocytes were allocated into two groups: [1] oocytes at the germinal vesicle (GV) stage; and [2] oocytes at the metaphase I (MI) stage. Each group was cultured in vitro with and without gonadotropin supplements. Some metaphase II (MII) oocytes derived from the two groups were parthenogenetically activated and exposed to subsequent embryonic development for 168 hours in vitro. Other MII oocytes were tested for meiotic apparatus analysis, including spindle morphology and chromosomal alignment, by immunofluorescence staining and scanning confocal microscopy. MAIN OUTCOME MEASURE(S): Oocyte maturation and activation rates, percentages of embryonic development, and spindle normalization were analyzed by χ(2) analysis, whereas oocyte maturation time was analyzed by one-way analysis of variance. RESULT(S): For GV oocytes the maturation and activation rates were significantly higher during in vitro maturation with supplementation with FSH/LH (68% vs. 60% and 82% vs. 62%, respectively). However, maturation time (22.78 ± 0.87 vs. 23.70 ± 0.94 hours), embryonic development (cleavage: 84% vs. 83%; four-cell: 72% vs. 66%; eight-cell: 48% vs. 43%; blastocyst: 5% vs. 7%), and meiotic apparatus normalization rates (55% vs. 61.1%) were similar. For MI oocytes there were no significant differences in the maturation rates (85% vs. 84%), maturation time (14.81 ± 0.65 vs.15.73 ± 0.58 hours), activation rates (77% vs. 80%), embryonic development (cleavage rates: 80% vs. 83%; four-cell: 68% vs. 72%; eight-cell: 56% vs. 51%; blastocyst: 7% vs. 6%), and meiotic apparatus normalization rates (52.4% vs. 54.5%). CONLUSION(S): Gonadotropin supplements to the maturation medium play an important role in cumulus-partially enclosed oocytes at the GV stage; however, MI stage-derived oocytes from stimulated cycles fail to acquire improved maturity after in vitro maturation. Furthermore, gonadotropin at the current concentration did not increase spindle or chromosomal abnormalities in MII oocytes maturated from either GV- or MI-stage oocytes in vitro.
