Doğaner Fulya, Turgut Coşan Didem, Güneş Hasan Veysi, Değirmenci Irfan, Bal Cengiz
Department of Medical Biology, Medical Faculty, Eskisehir Osmangazi University, 26480, Eskisehir, Turkey,
Mol Biol Rep. 2014 May;41(5):2923-7. doi: 10.1007/s11033-014-3148-z. Epub 2014 Jan 28.
p38 mitogen-activated protein kinases (MAPK) are members of the MAPK family that are activated by inflammatory cytokines and a variety of environmental stresses. It mediates various biological processes. p38 MAPK activity play important roles in tumour progression. Excessive p38 expression is observed in invasive breast cancers. The aim of the present study was to investigate whether the p38 siRNA transfection of breast cancer cells is a putative preventive treatment for human breast cancer. p38 siRNA was used at a concentration of 15, 30, and 100 nM in human breast cancer cell lines (MCF-7) and normal fibroblast cell lines (NIH 3T3). After 48 and 72 h of transfection, the reduction in p38 expression was measured using quantitative real-time PCR. The activation of p38 signalling was measured by ELISA. XTT cell proliferation assay was performed to determine the effect of p38 silencing on MCF-7 and NIH 3T3 cell lines. The results demonstrated that approximately 96% gene silencing occurred by the selected siRNA targeting p38 mRNA. The most effective silencing was observed at 72 h post-transfection using 30 nM p38 siRNA. The results of ELISA showed that the expression of p38 protein was inhibited by p38 siRNA at 30 nM siRNA and 100 nM at 72 h post transfection. XTT results showed that cells stimulated by 30 nM siRNA at 72 h post transfection were the lowest in proliferation. p38 siRNA can interfere with the expression of p38 at protein level in MCF-7 cells, result in inhibition of cell proliferation. p38 siRNA may be a critical regulator to promote the proliferation and protein expression in MCF-7 cells. In this study, we demonstrate that p38 silencing is a preventive maintenance for treating breast cancer.
p38丝裂原活化蛋白激酶(MAPK)是MAPK家族的成员,可被炎性细胞因子和多种环境应激激活。它介导各种生物学过程。p38 MAPK活性在肿瘤进展中起重要作用。在浸润性乳腺癌中观察到p38表达过高。本研究的目的是调查乳腺癌细胞的p38 siRNA转染是否是一种针对人类乳腺癌的潜在预防性治疗方法。在人乳腺癌细胞系(MCF-7)和正常成纤维细胞系(NIH 3T3)中使用浓度为15、30和100 nM的p38 siRNA。转染48小时和72小时后,使用定量实时PCR测量p38表达的降低情况。通过ELISA测量p38信号的激活情况。进行XTT细胞增殖试验以确定p38沉默对MCF-7和NIH 3T3细胞系的影响。结果表明,通过选择靶向p38 mRNA的siRNA发生了约96%的基因沉默。在转染后72小时使用30 nM p38 siRNA观察到最有效的沉默效果。ELISA结果表明,在转染后72小时,30 nM siRNA和100 nM的p38 siRNA抑制了p38蛋白的表达。XTT结果表明,转染后72小时受30 nM siRNA刺激的细胞增殖最低。p38 siRNA可在蛋白质水平干扰MCF-7细胞中p38的表达,导致细胞增殖受到抑制。p38 siRNA可能是促进MCF-7细胞增殖和蛋白质表达的关键调节因子。在本研究中,我们证明p38沉默是治疗乳腺癌的一种预防性手段。