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Objective: To investigate the expression and clinical significance of microRNA-34a (miR-34a) in human breast cancer tissues, and to analyze the correlation of methylation status of miR -34a gene promoter with the expression of mature miR-34a and the survival of patients with breast cancer. Methods: The expression level of mature miR-34a in breast cancer tissues (n = 93) and benign breast lesions (n = 5, as the control) was detected by TaqMan probe-based real-time fluorescent quantitative PCR (TaqMan® MicroRNA Assay Kit). The bisulfite conversion and purification of genomic DNA from each formalin-fixed and paraffin-embedded breast cancer samples were performed by EpiTect Fast FFPE Bisulfite Kit. The methylation status of miR-34a gene promoter in breast cancer samples was assessed by methylation-specific PCR. The relationship between miR-34a expression and miR -34a gene methylation was statistically analyzed. The prognostic value of miR -34a gene methylation was analyzed by Kaplan-Meier curve. Results: The expression level of miR-34a in breast cancer was significantly lower than that in benign breast lesions (P = 0.006). The expression of miR-34a was negative correlated with tumor size (r = -0.312, P = 0.021), lymph node metastasis (r = -0.378, P = 0.004) and tumor grade (r = -0.341, P = 0.011). However, the expression of miR-34a was not associated with estrogen receptor (ER), progesterone receptor (PR) or human epidermal growth factor receptor-2 (HER-2) (all P>0.05). The level of mature miR-34a in unmethylated group was significantly higher than that in methylated/unmethylated group or methylated group (both P<0.001). Compared with unmethylated group and methylated/unmethylated group, the recurrence-free survival (P<0.001, P = 0.019) and overall survival (P = 0.002, P<0.001) of patients with breast cancer were significantly reduced in miR -34a gene methylated group. Conclusion: In some patients with breast cancer, the methylation of miR -34a gene may lead to the down-regulation of mature miR-34a expression. Furthermore, the methylation status of miR -34a gene is associated with the risk of tumor recurrence and poor prognosis in patients with breast cancer.
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Objective To elucidate the mechanism of radiation resistant effect of LyGDI on NSCLC A549 cells.Methods A549 and H460 cells were irradiated with X-rays of 0,2,4 and 6 Gy.The clone-forming assay was used to detect cell survival and radiosensitivity.The expressions of LyGDI and COX-2 (Cyclooxygenase-2),a key radiosensitivity-related protein,were detected using Western blot.The miR-34 families were analyzed with RT-PCR.50 nmol/L mature miR-34c was transfected into A549 cells.Results The expression levels of LyGDI and COX-2 were much higher in radioresistive A549 cells than that in H460 cells.While the expression of miR-34a was quite low and miR-34b/c was hardly found in both NSCLC cells.Transfection of miR-34c into A549 cells strongly enhanced X-ray induced apoptosis by inhibiting the activations of LyGDI,COX-2,Bcl-2 and p21.Conclusions Up-regulation of LyGDI could induce COX-2 expression.The low expression of miR-34 family might be responsible for the radiation resistance of NSCLC cells.
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Objective To understand the molecular aberration at whole genomic level,CGH (comparative genomic hybridization) was used to investigate genetic abnormality in lung cancer.Methods Comparative genomic hybridization was performed in 17 cases to detect the global genomic aberration in cancer tissue cells.Results All of 17 cases detected by CGH showed chromosomal aberrations.The average numbers of chromosomal gains and losses in each case were 7.0 and 4.8 in NSCLC and 8.4 and 9.6 in SCLC,respectively.The frequency of gains and losses on chromosome had no significant differences between NSCLC and SCLC.The frequencies of gains on chromosomal arms 3q24 -28 and 11q13(58.3％ and 58.3％ ) in NSCLC were significantly higher than that in SCLC(0％ and 0％ ) ( P ＜0.05 and ＜0.05,respectively).Conclusions The cytogenetic aberration generally existed in lung cancer cells.Several regions ( more than one) of chromosomal aberration were involved in the carcinogenesis of NSCLC and SCLC.The regions and frequencies of chromosomal aberration in NSCLC were somewhat different from that in SCLC,which might result in the different biological behavior of the two types of lung cancer.The chromosomal aberration might be served as a marker to differentiate the two types of lung cancer.
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Objective To elucidate the function and regulatory mechanism of LyGDI involved delayed cell death in the human K562 cells and HL-60 cells induced by 60Co γ-rays. Methods Erythrosine B cells staining was used to count the apoptosis rate. PI staining and flow cytometry were applied to check the cell cycle. The expression of LYGDI and Rac1 was resolved by Western blot by using monoclonal antibody of LyGDI and Racl. The distribution of Racl protein in cells was observed with immunofluorescence by using the confocal microscope. Results The K562 cells showed G2/M phase arrest and the percent age was 71.3%. The apoptosis rate was very low at early post-irradiation stage in the K562 cells. The apoptosis rate was 14% in the K562 cells at 24 h post-irradiation with 8 Gy of γ-rays, and delayed cell apoptosis was present. LyGDI was cleaved in the K562 cells irradiated by 4 Gy 60 Co γ-rays after 24 hours post-irradiation. The expression of Racl protein was not altered at all, but the distribution was changed in the irradiated cells while the Racl protein moved to cell membrane and a little in cell nucleus. The Racl was activated with the losing the binding affinity with the LyGDI. Conclusion LyGDI could promote the delayed cell apoptosis, which is through the activation of the Rac1.