抑制生长因子受体连接蛋白-2表达对乳癌细胞生长的影响

Effect of inhibiting expression of growth factor receptor-bound protein-2 on proliferation of breast cancer cells

目的进一步验证生长因子受体连接蛋白-2(Grb2)的表达在乳癌发展中的作用。方法应用脂质体转染技术将Grb2小干扰RNA(siRNA)转染至乳癌细胞SKBr3中,台盼蓝计数法测定存活细胞数,TUNEL技术和AnnexinⅤ/PI染色分析转染后细胞的凋亡,免疫细胞化学技术分析转染后细胞Grb2的表达。Western蛋白质印迹法测定Grb2、细胞外信号调节激酶(p42/44ERK)、磷酸化p42/44ERK(P-p42/44ERK)、原癌基因蛋白质c-akt(Akt)、磷酸化Akt(P-Akt)和STAT5转录因子表达的改变。流式细胞术检测细胞活化的半胱氨酸天冬氨酸蛋白酶(caspase)3的表达。结果台盼蓝计数法结果显示,转染Grb2siRNA可有效抑制SKBr3的生长。TUNEL实验显示,Grb2siRNA转染SKBr3细胞后,随着时间延长,凋亡细胞明显增加。AnnexinⅤ/PI测定结果亦提示,Grb2的抑制可明显诱导SKBr3细胞凋亡。转染48h后,SKBr3的活化caspase3表达水平由0.99%升至17.43%。免疫组化染色表明,Grb2siRNA转染细胞后,SKBr3细胞Grb2表达明显下降,由转染24h后的下降至转染72h后的+～-。Western蛋白质印迹分析证实,Grb2的抑制可导致SKBr3细胞P-p42/44ERK,P-Akt以及STAT5表达明显下降。P-p42ERK与p42ERK的相对吸光度值之比由未转染的(60±17)%下降至转染24h后的(38±13)%,及转染48h后的(21±8)%;P-p44ERK与p44ERK的相对吸光度值之比,由未转染时(104±16)%,分别下降至(49±13)%及(30±10)%;P-Akt与Akt的相对吸光度值之比由未转染的(40±6)%下降至(32±10)%和(15±4)%。与未转染组相比,转染24及48h后STAT5相对吸光度值分别下降为(64±6)%和(52±14)%。结论抑制Grb2的表达可抑制乳癌细胞生长并诱导细胞凋亡。

AIM To confirm the effect of growth factor receptor-bound protein-2 （ Grb2 ） inhibition on the advance of breast cancer. METHODS Grb2 small interference RNA （siRNA） was transfected to breast cancer cells （SKBr3） by Lipofectamine transfection system. Trypan blue exclusion assay was used to observe the inhibition of cell proliferation. The apoptosis of breast cancer cells induced by Grb2 siRNA was analyzed using TUNEL assay and Annexin V/PI staining. The expression of Grb2 in SKBr3 cells transfected with Grb2 siRNA was examined with immunocytochemistry. The expression of signal molecules including Grb2, extracellular signal-regulated kinase （ p42/44 ERK）, phosphorylated ERK （ P-p42/ 44 ERK ）, proto-oncogene proteins c-akt （Akt）, phosphorylated Akt（P-Akt） and STAT 5 transcripition factor was evaluated by Western blot assay. Caspase-3 was examined by flow cytometry（FCM）. RESULTS Trypan blue exclusion assay showed that Grb2 siRNA could inhibit the growth of SKBr3 cells significantly. TUNEL test demonstrated that the percentage of apoptotic cells increased greatly on SKBr3 cells transfected with Grb2 siRNA in a time-dependent manner. Annexin V/PI analysis also suggested that the inhibition of Grb2 lead to the apoptosis of SKBr3 cells obviously. After transfection for 48 h, the level of active caspase 3 of SKBr3 cells was up to 17.43% from 0.99%. Immunocytochemistry demonstrated that the expression of Grb2 decreased greatly in SKBr3 cells transfected with Grb2 siRNA, and the expression degree decreased from ＋＋＋ （24 h after transfection） to ＋ to - （72 h after transfection）. Western Blot assay showed that the inhibition of Grb2 could decrease the level of phosphorylated ERK（including P-p42 and P-p44）, P-Akt and STAT 5 transcription factor of SKBr3 cells significantly. The ratio of relative band density of P-p42 ERK vs p42 ERK was respec-tively（60 ±17 ） % for without transfection, （ 38 ± 13 ）% at 24 h after transfection, and （21± 8 ） % at 48 h after transfection ; the ratio of P-p44 ERK vs p44 ERK was respectively （104 ±16 ）% for without transfection, （49±13） % at 24 h after transfection, and （30 ±10） % at 48 h after transfection; the ratio of P- Akt vs Akt was respectively （40 ± 6 ） % for without transfection, （32 ±10）% at 24 h after transfection, and （15±4）% at 48 h after transfection; the relative band density of STAT 5 transcription factor decreased to （ 64± 6 ） % at 24 h after transfection and （ 52 ± 14 ） % at 48 h after transfection, compared to that with- out transfection. CONCLUSION Inhibition of Grb2 expression has inhibitory effect on breast cancer cells and induces apoptosis.