沉默SND1重组慢病毒制备及其对乳腺癌细胞增殖和侵袭的影响

Preparation of lentivirus silencing SND1 and its influence on breast cancer cell proliferation and invasion

目的:探讨沉默葡萄球菌核酸酶结构域包含蛋白1(Staphylococcal nuclease domain containing 1,SND1)表达对乳腺癌细胞系MCF-7增殖和侵袭迁移的影响。方法:制备可介导SND1靶向沉默的重组慢病毒,感染MCF-7细胞建立沉默SND1的稳定亚细胞系;用qRT-PCR和Western blot检测SND1沉默效果;用噻唑蓝(MTS)染色法、细胞划痕和transwell实验,检测沉默SND1后MCF-7细胞增殖及侵袭迁移能力的变化。结果:成功构建了表达靶向SND1shRNA及对照shRNA的重组慢病毒质粒,经包装获得重组慢病毒(LV-SH1、LV-SH2、LV-SH3和LV-NC);用上述慢病毒感染MCF-7细胞,筛选出SND1沉默效果最佳的稳定亚细胞系MCF-SH3;增殖与侵袭迁移检测结果显示,MCF-SH3的增殖活性及侵袭迁移能力显著低于对照组细胞,差异有统计学意义(P<0.01)。结论:SND1可以促进乳腺癌细胞增殖及侵袭迁移,沉默SND1表达可抑制其增殖及侵袭迁移,提示SND1表达异常与乳腺癌密切相关,可能通过影响细胞增殖及侵袭迁移在乳腺癌的发生发展中起重要作用。

Objective： This work aimed to construct stable MCF-7 cell sublines from which staphylococcal nuclease domain con-taining 1 （SND1） expression was interfered to analyze the effect of SND1 silencing on the proliferation and metastasis of MCF-7 cells. Methods： The lentivirus that could mediate SND1 silencing was prepared. MCF-7 cells were infected with the lentiviruses to construct stable sub-cell lines. Quantitative real-time polymerase chain reaction and Western blot analysis were employed to determine SND1 ex-pression level. MTS, wound healing, and transwell assays were applied to analyze the effect of SND1 silencing on the proliferation, mi-gration, and invasion of MCF-7 cells. Results： A lentivirus expression vector that contains sequences encoding shRNAs targeting SND1 and an shRNA negative control were successfully established. The lentiviruses （LV-SH1, LV-SH2, LV-SH3, and 和 LV-NC） were then collected and packaged. Stabilized MCF-7 sublines were prepared through infection with lentiviruses. The most efficient MCF-7 stable cell subline, MCF-SH3, was selected for SND1 silencing. Compared with the control cell, the proliferation, migration, and inva-sion potential of MCF-SH3 were significantly decreased. Conclusion： SND1 could promote the proliferation, migration, and invasion of breast cancer cells. Thus, silencing SND1 expression will inhibit such proliferation, migration, and invasion. These results indicated that the unusual expression of SND1 is associated with breast cancer and may participate in cancer progression by affecting prolifera-tion, migration, and invasion.