p38蛋白激酶信号通路对骨桥蛋白介导的三阴性乳腺癌细胞自噬、凋亡活性的影响

Effect of p38 mitogen-activated protein kinase signaling pathway on autophagy and apoptosis activity of triple negative breast cancer cell mediated by osteopontin

目的探讨骨桥蛋白(OPN)和自噬相关蛋白Beclin 1在三阴性乳腺癌组织中的表达及OPN下调对p38蛋白激酶(p38MAPK)、Beclin 1表达及乳腺癌细胞自噬、凋亡活性的影响。方法选取2015年1月至2016年12月南阳市中心医院收治的60例女性三阴性乳腺癌患者的癌组织标本为研究对象,另选取同期手术治疗的40例女性乳腺纤维腺瘤或乳腺增生结节患者的正常乳腺组织作为对照;采用免疫组织化学方法检测三阴性乳腺癌组织和正常乳腺组织中OPN和Beclin 1的表达。将MDA-MB-231细胞株分为特异性OPN小干扰RNA(siRNA)实验组(OPN-siRNA组)、非同源阴性CON-siRNA对照组(CON-siRNA组)和mock空白对照组(mock组);以慢病毒介导基因干扰技术制备OPN低表达MDA-MB-231细胞模型,应用透射电镜及流式细胞术检测OPN下调后细胞自噬体形成和凋亡率,实时荧光定量聚合酶链反应(PCR)和Western blot法检测OPN下调后p38MAPK及Beclin 1的表达。结果 OPN蛋白在乳腺癌组织和正常乳腺组织中的阳性表达率分别为68. 3%(41/60)和12. 5%(5/40),OPN蛋白在乳腺癌组织中的阳性表达率高于正常乳腺组织(χ~2=56. 881,P=0. 000); Beclin 1蛋白在乳腺癌组织和正常乳腺组织中的阳性表达率分别为18. 3%(11/60)和42. 5%(17/40),Beclin 1蛋白在乳腺癌组织中的阳性表达率低于正常乳腺组织(χ~2=54. 692,P=0. 000);三阴性乳腺癌组织中OPN蛋白表达与Beclin 1蛋白表达呈负相关(r=-0. 713,P=0. 003)。病毒转染48 h后,OPN-siRNA组细胞中OPN mRNA和蛋白相对表达量显著低于CON-siRNA组和mock组(F=57. 170、50. 410,P=0. 001)。OPN表达下调后,OPN-siRNA组细胞中自噬体个数、早期凋亡率显著高于CON-siRNA组和mock组(F=17. 590,P=0. 004; F=23. 180,P=0. 003); OPN-siRNA组细胞中Beclin 1 mRNA和蛋白相对表达量明显高于CON-siRNA组和mock组(F=29. 873、32. 174,P=0. 001),p38MAPK mRNA和蛋白相对表达量低于mock组和CON-siRNA组(F=32. 735、37. 110,P=0. 000); mock组与CON-siRNA组细胞中自噬体个数、早期凋亡率、Beclin 1 mRNA和蛋白相对表达量及p38MAPK mRNA和蛋白相对表达量比较差异均无统计学意义(P> 0. 05)。结论抑制OPN表达可促进三阴性乳腺癌细胞自噬和凋亡活性,其作用机制可能与p38MAPK表达下调、Beclin 1表达上调有关。

Objective To study the expression of osteopontin(OPN)and autophagy re-lated protein Beclin 1 in triple negative breast cancer tissues,and to explore the effect of OPN knockdown on the expression of p38 mitogen-activated protein kinase(p38MAPK)、Beclin 1 and autophagy,apoptosis of breast cancer cells.Methods A total of 60 female patients with triple negative breast cancer admitted to the the Central Hospital of Nanyang City from January 2015 to December 2016 were selected as study objects,and forty normal breast tissue specimens of female patients with breast fibroadenoma or breast hyperplasia nodules treated at the same time were selected as the control.Immunohistochemistry were used to detect the expression of OPN and Beclin 1 in the triple negative breast cancer tissues and normal breast tissues.MDA-MB-231 cell lines were divided into specific OPN small interference RNA experimental group(OPN-siRNA group),the same negative CON-siRNA control group(CON-siRNA group)and mock blank control group(mock group).Lentivirus mediated RNA interfer-ence technology were employed to build OPN low expression MDA-MB-231cells,then transmission electron microscope and flow cytometry were used to detect auto-phagy and apoptosis of MDA-MB-231 cells after OPN knockdown,western blot and real-time fluorescence quantitative polymerase chain reaction were used to detect the expression of p38MAPK and Beclin 1.Results The positive expression rate of OPN protein in breast cancer tissue and normal breast tissue was 68.3%(41/60)and 12.5%(5/40),the positive ex-pression rate of OPN protein in breast cancer tissue was higher than that in the normal breast tissue(χ^2=56.881,P=0.000).The positive expression rate of Beclin 1 protein in breast cancer tissues and normal breast tissues was 18.3%(11/60)and 42.5%(17/40)respectively,and the positive expression rate of Beclin 1 protein in breast cancer tissues was lower than that in normal breast tissue(χ^2=54.692,P=0.000).OPN protein expression in tri-negative breast cancer tissues was negatively correlated with Beclin 1 protein expression(r=-0.713,P=0.003).After 48 h viral transfection,the relative expression of OPN mRNA and protein in the OPN-siRNA group was significantly lower than that in the CON-siRNA group and the mock group(F=57.170,50.410,P=0.001).After the down-regulation of OPN expression,the number of autophagosomes and early apoptosis rate in cells of the OPN-siRNA group were significantly higher than those of the CON-siRNA group and the mock group(F=17.590,P=0.004;F=23.180,P=0.003).In the OPN-siRNA group,the relative expression of Beclin 1 mRNA and protein was significantly higher than that of the CON-siRNA group and the mock group(F=29.873,32.174,P=0.001),and the relative expression of p38MAPK mRNA and protein was lower than that of the mock group and the CON-siRNA group(F=32.735,37.110,P=0.000).There was no statistically significance in the number of autophagosomes,early apoptosis rate,relative expression of Beclin 1 mRNA and protein,and relative expression of p38MAPK mRNA and protein in cells between the mock group and the CON-siRNA group(P>0.05).Conclusion Inhibition of OPN expression could promote autophagy and apoptosis of triple negative breast cancer cells,the underlying mechanism of this phenomenon may be related to down-regulation of p38MAPK and up-regulation of Beclin 1.