AKT2基因的过表达抑制H_2O_2诱导的乳腺癌细胞凋亡

Overexpression of AKT2 Suppresses H_2O_2-induced Breast Cancer Cells Apoptosis

研究AKT2基因对H2O2诱导的乳腺癌细胞凋亡的影响.RT-PCR扩增人AKT2基因全长cDNA序列后克隆入pcDNA3.1/myc-His(-)A质粒中,构建AKT2基因的真核表达载体(野生型,WT-AKT2);应用QuikChange定点诱变,制备AKT2激酶活性丧失的负性表达载体(DN-AKT2).WT-AKT2和DN-AKT2分别转染人乳腺癌MCF-7细胞,新霉素筛选稳定转染细胞克隆;设计并合成针对人AKT2基因2个不同部位的siRNA片段,转染入同样的细胞.通过TUNEL和DNA ladder测定,研究转染AKT2基因及AKT2 siRNA前后细胞对H2O2诱导凋亡的抵抗作用.测序鉴定证实,WT-AKT2和DN-AKT2表达载体构建成功.Western印迹结果显示,WT-AKT2和DN-AKT2基因在MCF-7细胞中表达良好,而AKT2 siRNA则可有效地抑制AKT2的表达;TUNEL和DNA ladder测定结果表明,WT-AKT2表达上调的稳定克隆组,可显著提高MCF-7细胞对H2O2诱导的凋亡的抵抗作用(转染DN-AKT2具相反作用).经H2O2作用后,其凋亡细胞数显著低于空载体稳定转染克隆及未转染亲代细胞的,差异具有统计学意义(P<0.05),而后两者之间差异无统计学意义(P>0.05).AKT2抑制乳腺癌细胞对H2O2诱导凋亡的作用可被AKT2 siRNA和PI3K/AKT信号途径抑制剂wortmannin所阻断.内源性的结果进一步证明了AKT2的功能.本研究表明,AKT2能够抑制H2O2诱导的MCF-7人乳腺癌细胞凋亡,可能成为乳腺癌治疗的靶点之一,并为研究乳腺癌细胞抵抗活性氧诱导的细胞凋亡的分子机制奠定了基础.

To study the effect of AKT2 gene on the apoptosis induced by H2O2 in breast cancer cells,the full length AKT2 cDNA was amplified by RT-PCR,and then inserted into pcDNA3.1/myc-His（-）A vector to construct pcDNA3.1/myc-His（-）A-AKT2 wild type（WT-AKT2）.Dominant negative mutant of AKT2（DN-AKT2） were generated by QuikChange site-directed mutagenesis and confirmed by sequencing.The eukaryotic expression vector of WT-AKT2 or DN-AKT2 were transfected into MCF-7 breast cancer cells.Clones stably expressing AKT2 or DN-AKT2 were obtained by neomycin selections.Two siRNAs targeted AKT2 were designed and synthesized,then transfected into the same cell line.Apoptosis with or without H2O2 treatments was determined by TUNEL and DNA Ladder assays.The expression of WT-AKT2 and DN-AKT2,as well as siRNA inhibition in MCF-7 cells was demonstrated by Western blotting.WT-AKT2 over-expression significantly increased the resistance of H2O2-induced apoptosis in MCF-7 cells,but not DN-AKT2.The number of apoptotic cells following H2O2 treatment was significantly lower in WT-AKT2 stably transfected cells than those in empty vector control or nontransfected cells（P0.05）.The inhibition of apoptosis was abolished in AKT2 siRNA,or PI3K/ AKT2 inhibitor wortmannin treated cells.Our study suggested that AKT2 inhibit H2O2-induced apoptosis in cancer cells,and interference of AKT2 might be used as a therapeutic approach for breast cancers.