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目的观察外源性雌激素受体β1(ERβ1)基因转染人乳腺癌MDA-MB-231细胞后对人端粒酶逆转录酶(hTERT)基因表达和细胞增殖能力的影响。方法应用脂质体法将ERβ1真核表达质粒转染至乳腺癌MDA-MB-231细胞中(pcDNA3.1-ERβ1转染组),另分别设空载体组和未转染组作为对照,分别用实时荧光定量PCR法和Western blot法检测各组细胞中ERβ1h、TERT mRNA和蛋白表达的变化;流式细胞仪观察细胞凋亡率的变化;细胞生长曲线观察转染后细胞增殖能力的改变。结果 pcDNA3.1-ERβ1转染组MDA-MB-231细胞中ERβ1 mRNA表达量为0.449±0.077,明显高于未转染组的0.153±0.035(P=0.001)或空载体组的0.160±0.020(P=0.000);相应的ERβ1蛋白表达量为0.847±0.065,亦明显高于未转染组的0.356±0.050(P=0.001)或空载体组的0.390±0.030(P=0.000)。pcDNA3.1-ERβ1转染组MDA-MB-231细胞中hTERT mRNA表达量为0.127±0.020,明显低于未转染组的0.283±0.025(P=0.001)或空载体组的0.283±0.049(P=0.002);相应的hTERT蛋白表达量为0.147±0.023,也明显低于未转染组的0.783±0.025(P=0.001)或空载体组的0.802±0.019(P=0.002)。细胞生长曲线提示,从第3天开始pcDNA3.1-ERβ1转染组细胞的增殖能力较未转染组明显减弱(P<0.05);流式细胞仪结果显示,pcDNA3.1-ERβ1转染组的细胞凋亡率为(6.15±0.94)%,明显高于未转染组的(1.41±0.42)%(P=0.001)。结论 ERβ1可能通过下调hTERT基因表达抑制乳腺癌细胞的增殖。
Objective To investigate the effect of exogenous estrogen receptor β1 (ERβ1) gene on human telomerase reverse transcriptase (hTERT) gene expression and cell proliferation in human breast cancer MDA-MB-231 cells. Methods ERβ1 eukaryotic expression plasmid was transfected into breast cancer cell line MDA-MB-231 by lipofectamine 2000 (pcDNA3.1-ERβ1 transfection group) and empty vector group and untransfected group The changes of ERβ1h, TERT mRNA and protein expression in each group were detected by real-time fluorescence quantitative PCR and Western blot. The changes of apoptosis rate were observed by flow cytometry. The proliferation of cells was observed by cell growth curve. Results The level of ERβ1 mRNA in MDA-MB-231 cells transfected with pcDNA3.1-ERβ1 was 0.449 ± 0.077, which was significantly higher than that of untransfected cells (0.153 ± 0.035, P = 0.001) or 0.160 ± 0.020 P = 0.000). The corresponding expression level of ERβ1 protein was 0.847 ± 0.065, which was significantly higher than that of non-transfected group (0.356 ± 0.050, P = 0.001) or 0.390 ± 0.030 (P = 0.000) The expression of hTERT mRNA in MDA-MB-231 cells transfected with pcDNA3.1-ERβ1 was 0.127 ± 0.020, which was significantly lower than that in untransfected cells (0.283 ± 0.025, P = 0.001) or 0.283 ± 0.049 = 0.002). The corresponding expression of hTERT protein was 0.147 ± 0.023, which was also significantly lower than that of the untransfected group (0.783 ± 0.025, P = 0.001) or the empty vector group (0.82 ± 0.019, P = 0.002). The cell growth curve indicated that the proliferation of pcDNA3.1-ERβ1 transfected cells was significantly weaker than that of untransfected cells from day 3 (P <0.05). The results of flow cytometry showed that pcDNA3.1-ERβ1 transfection group (6.15 ± 0.94)%, which was significantly higher than that of the untransfected group (1.41 ± 0.42)% (P = 0.001). Conclusion ERβ1 may inhibit the proliferation of breast cancer cells by down-regulating hTERT gene expression.