PAK6影响裸鼠乳腺癌早期放疗疗效的实验研究

Effects of PAK6on the Efficacy of Early Radiotherapy for Breast Cancer in Nude Mice

目的探究分析p21活化激酶6(p21-activated kinases 6,PAK6)基因对裸鼠乳腺癌早期放疗疗效的影响及作用机制。方法采用实时荧光逆转录定量聚合酶链反应(real-time reverse transcription quantitative polymerase chain reaction, RT-qPCR)实验和Western blot实验检测人正常乳腺上皮细胞MCF-10A及人乳腺癌细胞MDA-MB-468、MCF-7中PAK6 mRNA水平及蛋白表达。采用慢病毒载体介导RNA技术(RNAi),构建PAK6基因稳定沉默的人乳腺癌细胞系MDA-MB-468/shRNA-PAK6、MCF-7/shRNA-PAK6并检测转染效率,分别给予0、2、4 Gy单次剂量放射治疗,采用细胞计数试剂盒(cell counting kit 8,CCK-8)实验、克隆形成实验和流式细胞凋亡实验观察沉默PAK6表达对乳腺癌细胞放射敏感性的影响。构建裸鼠皮下乳腺癌移植瘤模型,分别给予0、2、4 Gy单次剂量放射治疗,观察移植瘤生长情况,计算抑瘤率。采用RT-qPCR实验和Western blot实验检测移植瘤组织中PAK6蛋白、β-连环蛋白(β-catenin)、c-myc、半胱氨酸蛋白酶3(Caspase-3)蛋白表达情况。结果与人正常乳腺上皮细胞MCF-10A比较,人乳腺癌细胞MDA-MB-468和MCF-7中PAK6 mRNA及蛋白均呈明显高表达(P<0.05)。在乳腺癌细胞MDA-MB-468和MCF-7中,沉默PAK6表达可抑制细胞活力和细胞克隆形成率(P<0.05),增加细胞凋亡率(P<0.05)。裸鼠成瘤实验结果显示,与2 Gy shRNA-NC组比较,2 Gy shRNA-PAK6组大鼠移植瘤体积明显降低(P<0.05),肿瘤倍增时间和增敏系数明显增大(P<0.05),抑瘤率增加(P<0.05),移植瘤组织中PAK6、β-catenin、c-myc蛋白表达水平明显较低(P<0.05),Caspase-3蛋白表达水平则明显较高(P<0.05);与4Gy shRNA-NC组比较,4Gy shRNA-PAK6组各项指标趋势同前。结论沉默乳腺癌细胞中PAK6基因表达可抑制肿瘤细胞的增殖和克隆,改善肿瘤细胞体内外放疗敏感性,其作用可能与抑制Wnt/β-catenin信号通路作用有关。

Objective To explore the effects and mechanism of p21-activated kinase 6(PAK6) gene on the efficacy of the early radiotherapy for breast cancer in nude mice.Methods The levels of PAK6 mRNA and protein expression in human normal breast epithelial cells MCF-10 A and human breast cancer cells MDA-MB-468 and MCF-7 were detected by real-time reverse transcription quantitative polymerase chain reaction(RT-qPCR) and Western blot. The stable and silent human breast cancer cell lines MDA-MB-468/shRNA-PAK6, MCF-7/shRNA-PAK6 were constructed by lentivirus mediated RNA Technology(RNAi), and its transfection efficiency was detected. Then single dose of 0,2,4 Gy radiotherapy were given respectively, and the effect of silencing PAK6 expression on radiotherapy sensitivity of breast cancer cells were observed by cell counting kit 8(CCK-8) test, clone formation test and flow cytometry test. A subcutaneous breast cancer transplanted tumor model was constructed in nude mice, and single dose of 0,2,4 Gy radiotherapy were given to observe the growth of the transplanted tumor, and the tumor inhibition rate was calculated. PAK6 protein, β-catenin, c-myc and Caspase-3 protein expression in transplanted tumor tissues were detected by RT-qPCR and Western blot.Results Compared with human normal breast epithelial cells MCF-10 A, the expression levels of PAK6 mRNA and protein in human breast cancer cells MDA-MB-468 and MCF-7 were significantly higher(P<0.05). In breast cancer cells MDA-MB-468 and MCF-7, silencing PAK6 expression could inhibit cell viability and cell clone formation rate(P<0.05), and increase cell apoptosis rate(P<0.05). The results of nude mice tumor formation experiments showed that compared with the 2 Gy shRNA-NC group, the transplanted tumor volume of the 2 Gy shRNA-PAK6 group was significantly reduced(P<0.05), and the tumor doubling time and sensitization coefficient were significantly increased(P<0.05),tumor inhibition rate increased(P<0.05), PAK6, β-catenin and c-myc protein expression levels in transplanted tumor tissues were significantly lower(P<0.05), and Caspase3 protein expression levels were significantly higher(P<0.05). Compared with the 4 Gy shRNA-NC group, the trend of the indicators in the 4 Gy shRNA-PAK6 group was the same as before.Conclusion PAK6 gene expression in siliencing breast cancer cells can inhibit the proliferation and cloning of tumor cells, and improve the sensitivity of tumor cells to radiotherapy in vivo and in vitro, which may be related to inhibiting of Wnt/β-catenin signaling pathway.