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目的:研究携带特异性抑制叉头框蛋白M1(Forkhead box protein M1,FOXM1)表达的腺病毒(Ad5.sh FOXM1)对人涎腺腺样囊性癌(Adenoidcystic carcinoma,ACC)细胞周期的调控、细胞凋亡的影响及其作用机理。方法:根据人FOXM1 m RNA的序列,设计合成针对FOXM1基因的三对sh RNA,转染ACC-2细胞系,利用实时荧光定量PCR、Western blot筛选获得最佳干扰片段,并构建入腺病毒载体,利用流式细胞术、MTT、Western blot等检测其对ACC-2细胞周期、细胞凋亡的调控及分子作用。结果:1成功筛选获得了抑制FOXM1表达的sh RNA,并构建了特异性抑制ACC-2细胞中FOXM1表达的腺病毒Ad5-sh FOXM1;2Ad5-sh FOXM1能明显抑制ACC细胞的增殖,并引起ACC-2细胞S期的阻滞(P<0.05);2Ad5-sh FOXM1通过下调c-Myc蛋白的表达抑制了细胞的增殖,通过下调P21Cip1,P27Kip1蛋白的表达抑制了ACC-2细胞周期(P<0.05)。结论:下调FOXM1的表达能够明显抑制ACC-2细胞周期和ACC-2细胞的增殖。
OBJECTIVE: To study the regulation of the cell cycle of human salivary adenoid cystic carcinoma (ACC) carrying adenovirus (Ad5.sh FOXM1) which specifically inhibits the expression of Forkhead box protein M1 (FOXM1) Effect of apoptosis and its mechanism of action. Methods: According to the sequence of human FOXM1 m RNA, three pairs of sh RNA targeting FOXM1 gene were designed and synthesized and transfected into ACC-2 cell line. The optimal interference fragments were obtained by real-time fluorescence quantitative PCR and Western blot screening, and constructed into adenoviral vector The cell cycle and apoptosis of ACC-2 cells were detected by flow cytometry, MTT and Western blot. Results: 1shRNA was successfully screened to inhibit FOXM1 expression, and adenovirus Ad5-sh FOXM1 which specifically inhibited FOXM1expression in ACC-2cells was constructed. 2Ad5-sh FOXM1can significantly inhibit the proliferation of ACC cells and cause ACC (P <0.05). 2Ad5-sh FOXM1 inhibited the proliferation of ACC-2 cells by down-regulating the expression of c-Myc protein and down-regulating the expression of P21Cip1 and P27Kip1 (P < 0.05). Conclusion: Down-regulation of FOXM1 expression can significantly inhibit ACC-2 cell cycle and ACC-2 cell proliferation.