他莫昔芬对三阴乳腺癌细胞饥饿耐受的影响及其机制研究

Effect of tamoxifen on starvation tolerance in triple negative breast cancer and its mechanism

目的探讨他莫昔芬(TAM)对三阴乳腺癌(TNBC)MDA-MB-468细胞耐受饥饿的影响,研究G蛋白偶联雌激素受体-自噬相关蛋白Beclin-1(GPER-Beclin-1)通路在其中的作用。方法用血清饥饿方法建立模型细胞,并分为对照组和实验组。另取常规培养的MDA-MB-468细胞作为空白组。对照组和实验组分别以1‰二甲基亚砜和5μmol·L^(-1)TAM处理48 h,空白组常规培养48 h。用siRNA转染技术沉默模型细胞中GPER表达,并分为GPER-NC组(空载转染)和GPER-si组(转染GPER)。GPER-NC组和GPER-si组均以5μmol·L^(-1)TAM处理8 h。用结晶紫染色法检测细胞贴壁存活率,用蛋白印迹法检测LC3B和Beclin-1蛋白表达,并评估自噬能力。结果血清饥饿48 h,实验组、对照组和空白组细胞的贴壁存活率分别为(128.77±6.66)%,(101.00±3.26)%和(177.25±4.45)%,LC3Ⅱ/LC3Ⅰ相对蛋白表达量分别为(142.15±5.25)%,(100.33±4.55)%和(65.34±8.78)%,Beclin-1相对蛋白表达量分别为(156.22±7.88)%,(101.25±3.80)%和(97.97±8.90)%,实验组和对照组的上述指标比较,差异均有统计学意义(均P<0.05)。血清饥饿48h,GPER-NC组和GPER-si组细胞的贴壁存活率分别为(103.55±6.68)%和(49.74±5.15)%,LC3Ⅱ/LC3Ⅰ相对蛋白表达量分别为(98.36±6.68)%和(64.54±5.25)%,Beclin-1相对蛋白表达量分别为(105.39±6.68)%和(31.03±8.76)%,差异均有统计学意义(均P<0.05)。结论 TAM能增强TNBC MDA-MB-468饥饿耐受能力,该效应可能与GPER-Beclin-1通路激活自噬有关。

Objective To investigate the effect of tamoxifen（ TAM） on triple-negative breast cancer（ TNBC） cells,and To investigate the role of the G protein-coupled estrogen receptor-autophagy-related protein Beclin-1（ GPER-Beclin-1） pathway. Methods Model cells were established by Serum starvation method,and then were divided into control group and test group. Another conventional cultured cells were taken as blank group. The control group and the experimental group were treated with 1 ‰ dimethyl sulfoxide and 5 μmol·L^（-1） TAM for 48 h respectively. The blank group was cultured for 48 h. Cell viability was measured by crystal violet staining. The expression of GPER in the model cells was silenced by siRNA transfection,and divided into GPER-NC group （ no-load transfection） and GPER-si group（ GPER transfection）. GPER-NC group and GPER-si group were treated with 5 μmol·L^（-1） TAM for 8 h. Western blotting was used to detect protein indexes（ LC3 B and Beclin-1）.Results After 48 h of serum starvation,the main indexes in test,control and blank groups were compared： the adherent survival rates were（ 128. 77 ± 6. 66） %,（ 101. 00 ± 3. 26） % and（ 177. 25 ± 4. 45） %,autophagy-related protein LC3Ⅱ/LC3Ⅰ were（ 142. 15 ± 5. 25）,（ 100. 33 ± 4. 55） and（ 65. 34 ± 8. 78） %,relative protein expression of Beclin-1 were（ 156. 22 ± 7. 88） %,（ 101. 25 ± 3. 80） % and（ 97. 97 ± 8. 90） %,respectively. The differences were statistically significant between test group and control group（ all P〈0. 05）. After 48 h of serum starvation,the main indexes in GPER-NC and GPER-si groups were compared： the adherent survival rates were（ 103. 55 ± 6. 68） % and（ 49. 74 ± 5. 15） %,relative protein expression of LC3Ⅱ/LC3Ⅰ were（ 98. 36 ± 6. 68） % and（ 64. 54 ± 5. 25） %,relative protein expression of Beclin-1 were（ 105. 39 ± 6. 68） % and（ 31. 03 ± 8. 76） %. There were significant differences between GPER-si group and GPER-NC group（ all P〈0. 05）. Conclusion TAM enhanced TNBC MDA-MB-468 starvation tolerance,which may be related to the activation of autophagy by GPER-Beclin-1 pathway.