摘要
目的探讨miRNA-125b-1对乳腺癌SKBR-3细胞放射敏感性的影响,及其对靶基因HER-2蛋白表达的影响。方法合成miRNA-125b-1并通过lipofectamineTM2000转染SKBR-3细胞,分别用逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)和Western blot法检测HER-2mRNA和蛋白质表达水平的变化;流式细胞仪检测细胞周期;四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)法检测细胞增殖抑制率;经不同剂量X线照射后,平板克隆形成实验计算细胞存活率,用单击多靶数学模型进行曲线拟合作图,求曲线参数D0、Dq和N值,比较细胞放射敏感性的变化。RT-PCR和Western blot定量结果的组间比较采用单因素方差分析,细胞周期及细胞增殖实验结果的组间比较采用重复测量方差分析。结果 miRNA-125b-1转染后的SKBR-3细胞中HER-2mRNA(F=447.78,P=0.00)和蛋白质水平(F=10.07,P=0.01)显著下降;转染后的SKBR-3细胞出现了G2M期阻滞;miRNA-125b-1组细胞增殖抑制率明显高于阴性对照组(F=163.28,P=0.00);曲线拟合后显示,转染后的细胞(D0=1.87,Dq=2.68,N=4.21)与对照组(D0=2.51,Dq=4.10,N=5.11)相比D0,Dq和N值均下降(SER=1.34,P<0.05),提示细胞放射敏感性增加。结论 miRNA-125b-1使乳腺癌SKBR-3细胞的放射敏感性增加,可为HER-2阳性的乳腺癌提供新的治疗方法。
Objective To investigate the effect of miRNA-125b-1 on the radiosensitivity of SKBR-3 breast cancer cells, and its influence on HER-2 protein expression. Methods A recombinant miRNA-125b-1 was introduced by lipofeetamineTM2000 mediated gene transfeetion into cultured SKBR-3 cells. The transfeeted cell clones were collected and analyzed by RT-PCR to determine the level of HER-2 mRNA. The protein level of HER-2 was detected by Western blot. The cell cycle phases were determined by flow cytometry, and the cell proliferation inhibitory rate was measured using MTI＂ assay. The surviving fraction of the cells was examined by colony forming assay after the irradiation of different doses of X-ray. A single-hit, multi-target mathematic model was established to estimate the values of Do, Dq and N. The radiosensitivity of cells was compared accordingly. One-way ANOVA was used for comparison of the results by RT-PCR and Western blot between groups, and ANOVA of repeated measurement data was carried out for comparison of cell cycle phases and cell proliferation between groups. Results In the experiment group, both the mRNA and protein expression levels of HER-2 gene in the transfected SKBR-3 cells were reduced significantly（ all P〈0.05）. Most of the transfected SKBR-3 cells were blocked in G2M phase of cell cycle. The cell proliferation inhibitory rate was obviously higher than that in the negative control group （P〈0. 05）. The values of Do, Dq and N were obtained through the cell survival curves. The values of transfected SKBR-3 cells （ DO = 1.87, Dq = 2. 68, N = 4. 21 ） were significantly lower than those in the control group （ Do = 2.51, Dq = 4. 10, N = 5. 11 ; SER = 1.34, P 〈 0.05 ）, which demonstrated that the radiosensitivity of these cells was enhanced. Conclusion miRNA-125b-1 can markedly increase the radiosensitivity of SKBR-3 breast cancer cells,which inspires a new treatment for HER-2 positive breast cancer.
出处
《中华乳腺病杂志（电子版）》
CAS
2011年第6期33-37,共5页
Chinese Journal of Breast Disease(Electronic Edition)
基金
广东省科技计划基金资助项目(2008B030301345)
