miRNA-125b-1 对乳腺癌SKBR-3 细胞放射敏感性的影响

doi:10.3877/cma.j.issn.1674-0807.2011.06.007

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lipofectamine^TM 2000 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-2 mRNA 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-2 mRNA (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,D_q=2.68,N=4.21)与对照组(D₀=2.51,D_q=4.10,N=5.11)相比D₀,D_q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。

关键词: 乳腺肿瘤, 放射疗法, miRNA-125b-1, 微小RNA, 人表皮生长因子受体2

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 lipofectamine^TM 2000 mediated gene transfection into cultured SKBR-3 cells. The transfected 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 MTT 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 D₀, D_q 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 D₀, D_q and N were obtained through the cell survival curves. The values of transfected SKBR-3 cells (D₀=1.87,D_q=2.68,N=4.21) were significantly lower than those in the control group(D₀ =2.51,D_q=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.

Key words: breast neoplasms, radiotherapy, miRNA-125b-1, microRNA, HER-2

图1 miRNA-125b-1 转染SKBR-3 细胞后绿色荧光蛋白的表达(×100) a:转染浓度为40 nmol/L,脂质体为0.6 μl; b:转染浓度为40 nmol/L,脂质体为0.8 μl;c:转染浓度为40 nmol/L,脂质体为1.0 μl;d:转染浓度为80 nmol/L,脂质体为0.6 μl;e:转染浓度为80 nmol/L,脂质体为0.8 μl;f:转染浓度为80 nmol/L,脂质体为1.0 μl。

表1 转染miRNA-125b-1 对SKBR-3 细胞HER-2 基因mRNA 的影响

组别	HER-2 mRNA表达量	F值	P值
脂质体对照组	0.82±0.00^a	447.78	0.00
阴性对照组	0.81±0.00^b
miRNA-125b-1组	0.66±0.01

表1 转染miRNA-125b-1 对SKBR-3 细胞HER-2 基因mRNA 的影响

组别	HER-2 mRNA表达量	F值	P值
脂质体对照组	0.82±0.00^a	447.78	0.00
阴性对照组	0.81±0.00^b
miRNA-125b-1组	0.66±0.01

图2 各组SKBR-3 细胞HER-2 mRNA 的表达情况 M:DNA 标记条带;1:脂质体对照组;2:阴性对照组;3:miRNA-125b-1 组

表2 转染miRNA-125b-1 对SKBR-3 细胞HER-2 基因蛋白的影响

组别	HER-2 mRNA表达量	F值	P值
脂质体对照组	0.79±0.05^a	10.07	0.01
阴性对照组	0.59±0.13^b
miRNA-125b-1组	0.36±0.15

表2 转染miRNA-125b-1 对SKBR-3 细胞HER-2 基因蛋白的影响

组别	HER-2 mRNA表达量	F值	P值
脂质体对照组	0.79±0.05^a	10.07	0.01
阴性对照组	0.59±0.13^b
miRNA-125b-1组	0.36±0.15

图3 miRNA-125b-1 对乳腺癌SKBR-3 细胞HER-2 蛋白表达的影响 1:脂质体对照组;2:阴性对照组;3:miRNA-125b-1 组

表3 流式细胞仪对各组SKBR-3 细胞周期的检测值 (%)

组别	G₀-G₁期	G₂M期	S期
脂质体对照组	67.22±7.30	8.07±1.73	24.71±6.41
阴性对照组	64.56±6.88	10.42±1.47	25.02±5.66
miRNA-125b-1组	50.56±0.93	23.20±1.07	26.13±0.93

表3 流式细胞仪对各组SKBR-3 细胞周期的检测值 (%)

组别	G₀-G₁期	G₂M期	S期
脂质体对照组	67.22±7.30	8.07±1.73	24.71±6.41
阴性对照组	64.56±6.88	10.42±1.47	25.02±5.66
miRNA-125b-1组	50.56±0.93	23.20±1.07	26.13±0.93

表4 MTT 法检测miRNA-125b-1 对SKBR-3 细胞增殖的抑制作用

组别	抑制率(%)			F值	P值
组别	24 h	48 h	72 h	F值	P值
阴性对照组	14±3	14±3	13±2	163.28	0.00
miRNA-125b-1转染组	40±5	44±6	46±13

表4 MTT 法检测miRNA-125b-1 对SKBR-3 细胞增殖的抑制作用

组别	抑制率(%)			F值	P值
组别	24 h	48 h	72 h	F值	P值
阴性对照组	14±3	14±3	13±2	163.28	0.00
miRNA-125b-1转染组	40±5	44±6	46±13

图4 阴性对照组和miRNA-125b-1 组在不同时间段的细胞增殖抑制情况

图5 SKBR-3 细胞的存活曲线

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Effect of miRNA-125b-1 on radiosensitivity of SKBR-3 breast cancer cells

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Effect of miRNA-125b-1 on radiosensitivity of SKBR-3 breast cancer cells