丙戊酸通过下调EGFR的表达抑制三阴性乳腺癌细胞生长增殖并诱导凋亡

The role and effects of EGFR down-regulated by valproic acid in growth inhibition and apoptosis induction effects in triple-negative breast cancer cells

目的探讨丙戊酸(VPA)对三阴性乳腺癌可能的治疗作用,并初步探索其可能的分子机理。方法MTS法结合克隆形成实验分析不同浓度VPA(0、0.25、0.5、1、2、4 mmol/L)处理对三阴性乳腺癌MDA-MB-231细胞存活的影响,结晶紫染色法检测不同浓度VPA(0、0.125、0.25、0.5 mmol/L)作用后前述细胞克隆形成情况;Western blot法检测凋亡指示蛋白多聚腺苷二磷酸核糖聚合酶(PARP),并结合凋亡特异性ELISA法分析VPA(0.5 mmol/L)处理24、48 h诱导的MDA-MB-231细胞凋亡效应,qRT-PCR法检测其表皮生长因子受体(EGFR)mRNA的表达水平;Western blot法分析VPA(0.25、0.5、1 mmol/L)处理24 h对MDA-MB-231细胞中EGFR的表达及其下游Akt信号通路的影响;miRNAs特异qRT-PCR法分析VPA(0.5 mmol/L)处理24、48 h对MDA-MB-231细胞中miR-133a和miR-133b表达的影响;采用单因素方差分析及Dunnet-t检验进行统计学分析。结果与对照组VPA(0 mmol/L)比较,不同浓度(0.25、0.5、1、2、4 mmol/L)VPA干预组MDA-MB-231细胞的存活率(100﹪±0.46﹪比86.97﹪±0.21﹪、77.66﹪±0.17﹪、66.81﹪±0.59﹪、46.68﹪±0.45﹪、15.89﹪±0.25﹪)及细胞平均克隆数均[(769.33±22.03)个比(599.33±39.00)个、(492.00±52.00)个、(263.33±14.74)个]降低,且呈浓度依赖性(P均<0.05)。0.5 mmol/L VPA处理24 h对体外培养MDA-MB-231细胞凋亡率差异无统计学意义(0.13﹪±0.00﹪比0.14﹪±0.00﹪,P>0.05),但处理48 h后MDA-MB-231凋亡率(0.13﹪±0.00﹪比0.80﹪±0.00﹪)升高,差异有统计学意义(P<0.05)。RT-qPCR结果表明,0.5 mmol/L VPA处理24、48 h对MDA-MB-231细胞中EGFR mRNA的表达水平无明显影响(差异具有统计学意义但无生物学意义,P<0.001)。Western blot结果则显示VPA(0.25、0.5、1 mmol/L)处理24 h可明显下调MDA-MB-231细胞中EGFR的表达,相应的下游Akt信号通路被抑制。此外,miRNA特异RT-qPCR分析结果表明,与对照组(0 h)比较,0.5 mmol/L VPA处理24、48 h上调靶向EGFR mRNA的miR-133b的表达(1±0比6.21±0.89、4.58±0.62)(P<0.001)。结论VPA在体外显示较好的抑制三阴性乳腺癌MDA-MB-231细胞生长增殖并诱导其凋亡效应;初步的机制探讨提示,这可能与VPA处理上调靶向EGFR mRNA的miR-133b的表达特异下调MDA-MB-231细胞中EGFR的表达,进而抑制下游Akt信号通路有关;VPA作为三阴性乳腺癌患者治疗可能的候选药物值得进一步深入研究。

Objective To explore the potential anti-tumor effects and mechanisms of valproic acid(VPA)against triple-negative breast cancer(TNBC).Methods MTS assay was carried out to evaluate the effects of VPA(0,0.25,0.5,1,2,4 mmol/L)on survival of TNBC cell MDA-MB-231.The effects of VPA(0,0.125,0.25,0.5 mmol/L)on clone formation of MDA-MB-231 were detected by crystal violet staining.Induction of apoptosis of MDA-MB-231 treated with VPA(0.5 mmol/L)for either 24 h,48 h was analyzed by detecting of cleavage of poly ADP-ribose polymerase(PARP)by Western blot as well as apoptotic-ELISA,and real-time quantitative reverse transcriptase PCR(qRT-PCR)was used to analyze the mRNA expression level of EGFR in MDA-MB-231.The effects of VPA(0.25,0.5,1 mmol/L)on expression of EGFR and its downstream Akt signaling pathway in MDA-MB-231 was also detected by Western blot analysis.MiRNAs specific qRT-PCR was carried out to analyze the effect of 0.5 mmol/L of VPA for either 24 h,48 h on expression of both miR-133a and miR-133b in MDA-MB-231.One-way ANOVA and dunnet-t test were used for statistical analysis.Results Compared with control(0 mmol/L of VPA),the survival rate(100﹪±0.46﹪vs 86.97﹪±0.21﹪,77.66﹪±0.17﹪,66.81﹪±0.59﹪,46.68﹪±0.45﹪,15.89﹪±0.25﹪,P<0.0001)and the mean clone numbers(769.33±22.03 vs 599.33±39.00,492.00±52.00,263.33±14.74,P<0.05)of MDA-MB-231 were significantly decreased after treated by VPA(0.25,0.5,1,2,4 mmol/L),which in a dose-dependent manner.There was no effect in apoptosis rate of MDA-MB-231 cells 24 h after treated with 0.5 mmol/L of VPA(apoptosis rate being 0.13﹪±0.00﹪vs 0.14﹪±0.00﹪,P>0.05),while a longer term treatment with 0.5 mmol/L of VPA(48 h)did significantly induce apoptosis as compared with control(apoptosis rate being 0.13﹪±0.00﹪vs 0.80﹪±0.00﹪,P<0.05).Although treatment with 0.5 mmol/L of VPA for 24 h,48 h didn't alter the mRNA expression of EGFR in MDA-MB-231,our Western blot analysis indicated that the protein expression of EGFR was significantly down-regulated 24 h after treated with VPA along with the downstream Akt signaling pathway,while there was no significantly difference in expression level of EGFR mRNA even 48 h treated by VPA.In addition,compared with control group,the expression of miR-133b targeting EGFR mRNA was significantly up-regulated in MDA-MB-231 treated with 0.5 mmol/L of VPA either for 24 h,48 h as indicated by qRT-PCR analysis(1±0 vs 6.21±0.89,4.58±0.62,P<0.001).Conclusions VPA exhibits favorable effects on inhibition of growth and induction of apoptosis of TNBC cell MDA-MB-231 in vitro.Our preliminary mechanism study suggested that treatment with VPA could down-regulate the expression of EGFR via up-regulation of the expressioin of miR-133b which targeting EGFR mRNA,inhibiting the downstream Akt signaling pathway and induces growth inhibition and apoptosis of MDA-MB-231.VPA as a potential candidate of therapeutic drug for patient with TNBC is worthy of further investigation.