沉默MSH3表达增强舌癌细胞对顺铂的敏感性

Silencing MSH3 expression enhances cisplatin sensitivity of human tongue cancer cells

目的设计靶向DNA错配修复基因MSH3的si RNA干扰片段,观察MSH3有效沉默对舌癌细胞顺铂耐药性的影响。方法针对MSH3基因CDS区序列,设计3条si RNA片段,体外化学合成小干扰RNA(si RNA)片段,通过脂质体介导转染人舌癌细胞CAL27。Real-time PCR及Western检测干扰后MSH3的表达。MTS,凋亡双染法及细胞免疫荧光检测顺铂处理后细胞存活、凋亡及DNA双链断裂的情况。结果 3条si RNA片段转染细胞后,与对照组相比,3号si RNA片段转染后能显著降低MSH3 m RNA及蛋白质表达水平,该片段被选为后续实验。MSH3表达下调后,顺铂的半数抑制浓度(IC50值)分别由21.32降至13.95μmol/L(P<0.05),凋亡指数分别由4.23±1.27升至11.32±1.82(P<0.05),舌癌细胞中γ-H2AX焦簇(Foci)的数量显著增加。结论 MSH3表达下调可以明显增加舌癌细胞对顺铂的敏感性,减少DNA双链断裂修复是其主要机制之一。

Objective To explore the effect of MSH3 knock-down on sensitivity of tongue cancer cells to cisplatin. Methods Three small interfering RNA(si RNA) fragments targeting MSH3 CDS region were synthesized and transfected into CAL27 cells via Lipofectamine. Real-time PCR and Western blotting were used to assess the efficiency of MSH3 silencing. MTS,apoptosis staining and cell immunofluorescence assay were used to examine the cisplatin sensitivity, apoptosis and DNA repair of transfected CAL27 cells. Results One of the 3 si RNAs was found to significantly reduce the expression of MSH3 protein in CAL27 cells(P<0.05). MTS assay showed that MSH3 silencing resulted in an significant reduction of IC50 of cisplatin from 21.32 to 13.95 μmol/L(P<0.05) and increased the apoptotic index of the exposed cells from 4.23 ± 1.27 to 11.32 ± 1.82(P<0.05). Immunofluorescence assay demonstrated that silencing MSH3 markedly reduced the number of γ-H2 AX foci.Conclusion Silencing MSH3 can significantly increase cisplatin sensitivity of tongue cancer cells, the mechanism of which involves mainly attenuation of repair of DNA double-strand damage in the cells.