Effect of siRNA transfection targeting P75 gene on proliferation and apoptosis in human breast cancer cells

陈素莲; 马佳; 张枫; 余新超; 李翠云; 杨清玲; 陈昌杰

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Effect of siRNA transfection targeting P75 gene on proliferation and apoptosis in human breast cancer cells

更新时间：2026-01-09

- Effect of siRNA transfection targeting P75 gene on proliferation and apoptosis in human breast cancer cells
- Journal of Army Medical University Vol. 35, Issue 11, Pages: 1124-1128(2013)
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  CLC： R394.2;R730.23;R737.9
- Published：2013
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陈素莲,马佳,张枫,余新超,李翠云,杨清玲,陈昌杰.靶向P75的siRNA对人乳腺癌细胞增殖和凋亡的影响[J].陆军军医大学学报(原第三军医大学学报),2013,35(11):1124-1128.

　Chen Sulian,Ma Jia,Zhang Feng,Yu Xinchao,Li Cuiyun,Yang Qingling,Chen Changjie.Effect of siRNA transfection targeting P75 gene on proliferation and apoptosis in human breast cancer cells[J].J Amry Med Univ (J Third Mil Med Univ),2013,35(11):1124-1128.
陈素莲,马佳,张枫,余新超,李翠云,杨清玲,陈昌杰.靶向P75的siRNA对人乳腺癌细胞增殖和凋亡的影响[J].陆军军医大学学报(原第三军医大学学报),2013,35(11):1124-1128. DOI：

　Chen Sulian,Ma Jia,Zhang Feng,Yu Xinchao,Li Cuiyun,Yang Qingling,Chen Changjie.Effect of siRNA transfection targeting P75 gene on proliferation and apoptosis in human breast cancer cells[J].J Amry Med Univ (J Third Mil Med Univ),2013,35(11):1124-1128. DOI：

摘要

目的构建P75 特异小干扰RNA(siRNA) 表达载体，并观察其对神经生长因子(NGF) 促乳腺癌细胞生长及凋亡的影响。方法通过GenBank提供的P75 mRNA序列，设计3对能转录短发夹状RNA的DNA序列，构建pSilencer™4.1-CMV neo质粒重组载体，转染乳腺癌SKBR3细胞；通过实时荧光定量PCR和Western blot筛选RNA干扰效果最强的干扰片段；以此干扰片段转染至乳腺癌SKBR3细胞，利用G418 筛选稳定表达P75-siRNA 的SKBR3 细胞株，命名为P75-siRNA；MTT检测P75干扰后NGF对细胞增殖作用的影响；流式细胞术检测细胞凋亡；建立荷乳腺癌小鼠模型，观察P75干扰后对小鼠肿瘤生长和凋亡相关蛋白表达的影响。结果序列测定表明成功构建P75-siRNA表达载体，实时荧光定量PCR和Western blot检测结果显示P75 mRNA和蛋白表达下调，尤以P75 2-siRNA干扰片段效果显著；MTT检测显示，NGF能刺激细胞增殖，与NGF组相比，P75-siRNA干扰后细胞增殖率均明显增高（

0.05）；与空白对照组相比，NGF+P75-siRNA组和NGF组的细胞凋亡率明显降低（

0.05)，NGF+P75-siRNA组更为明显；荷乳腺瘤小鼠中，P75-siRNA 组小鼠的肿瘤生长明显超过未转染组和空载体转染组（

0.05)，且P75-siRNA组Caspase-3表达下降，Bcl-2表达增加，Bax表达下降（

0.05)。结论 P75特异干扰表达载体能有效降低P75的表达，增强NGF促乳腺癌SKBR3细胞的增殖作用和抗凋亡作用，从而使肿瘤生长更为明显。

Abstract

Objective To construct an expression vector of P75 small interfering RNA (siRNA)

and to determine its effects on the cell proliferation and apoptosis in breast cancer cells SKBR3 induced by nerve growth factor (NGF). Methods According to the mRNA complete sequence of P75 gene in GenB

ank

3 pairs of DNA sequence transcribing short hairpin RNAs of P75 were selected and designed

and then constructed with the vector of pSilencer™4.1-CMV neo plasmid. After the vectors were transfected into SKBR3 cells

the strongest interference effect of them was chosen by fluorescent quantitation PCR and Western blotting. Then the obtained vector was transfected into SKBR3 cells after confirmed by sequencing. The stable cell line expressing P75 siRNA was selected by G418 assay. MTT assay was performed to assess the cell proliferation induced by NGF after P75 interference and the cell apoptosis was evaluated by flow cytometry (FCM). The mouse model of transplanted breast carcinoma was constructed by SKBR3 cells. The effect of P75 siRNA on the growth of the tumor and the expression of apoptosis relevant proteins were observed on the tumor-bearing mice. Results The expressing vector of P75 siRNA was successfully constructed. The mRNA and protein levels of P75 were decreased after P75 interference

especially with the vector of P75 2-siRNA. MTT assay showed that NGF significantly stimulated the proliferation of SKBR3 cells and the proliferation was more significant when combined with P75-siRNA in comparison with blank control（

0.05）. The apoptotic rate was obviously lower in the cells treated by NGF+P75-siRNA and NGF along compared with the cells transfected with blank vector（

0.05）. Those cells after NGF+P75-siRNA treatment had the lowest apoptotic rate （

0.05）. In the mouse model of transplanted breast cancer

tumor growth in P75-interfering group was more obvious than un-transfection group and blank carrier transfection group（

0.05）as well as a decrease in Caspase-3 and Bax expression and an increase in Bcl-2 expression. Conclusion The vector of P75 siRNA decreases the expression of P75 in SKBR3 cells effectively

and enhances the cell proliferation and anti-apoptosis of NGF. So it leads to tumor grow more obviously.

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