芍药苷调控MLH1和MSH2蛋白增强黑色素瘤细胞对放疗的敏感性

目的:探讨芍药苷调控MLH1和MSH2蛋白增强黑色素瘤细胞放疗敏感性的机制。方法:体外培养构建放疗抵抗性的黑色素瘤M14细胞株(M14-RR); Western blot实验检测M14和M148-RR细胞中MLH1和MSH2的表达水平,MTT细胞实验检测不同浓度的芍药苷对黑色素瘤细胞活性的影响; Western blot检测芍药苷对黑色素瘤细胞中MLH1和MSH2蛋白水平的影响;免疫荧光检测黑色素瘤细胞γ-H2AX聚集点的情况及对放疗的敏感性; CCK-8实验检测M14-RR细胞株接受放疗后凋亡率和细胞周期的变化。结果:成功构建M14-RR放疗抵抗细胞株后,Western blot检测M14-RR细胞中MLH1和MSH1的表达水平高于M14细胞,MTT细胞实验检测250μg/ml浓度的芍药苷药物对M14细胞最佳抑制率为(63. 61±3. 65)%; Western blot检测250μg/ml浓度的芍药苷组黑色素瘤细胞中MLH1和MSH2的表达水平比对照组细胞的增高[(85. 31±5. 16) vs (38. 26±3. 16),P<0. 05;(74. 15±6. 08) vs (29. 62±3. 57),P<0. 05];免疫荧光检测芍药苷作用黑色素瘤细胞后γ-H2AX聚集点的表达明显增多,DNA损伤增多;芍药苷作用黑色素瘤细胞接受放疗后细胞凋亡率增加,提高黑色素瘤细胞在G1期和S期的分布率,干扰细胞增殖的进程。结论:芍药苷通过调控MLH1和MSH1蛋白增强黑色素瘤细胞的DNA损伤及修复能力,增强其对放疗的敏感性。

Effect of paeoniflorin on MLH1 and MSH2 protein enhances sensitivity of melanoma cells to radiotherapy

Objective:To investigate the mechanism of paeoniflorin regulating MLH1 and MSH2 protein to enhance the sensitivity of melanoma cells to radiotherapy. Methods: The melanoma M14 cell line(M14-RR) was constructed in vitro.Western blot was used to detect the expression of MLH1 and MSH2 in M14 and M148-RR cells.MTT assay was used to detect the effect of different concentrations of paeoniflorin on the activity of melanoma cells.Western blot was used to detect the effect of paeoniflorin on MLH1 and MSH2 protein in melanoma cells.Immunofluorescence detection of melanoma cells γ-H2AX aggregation point of the situation and the sensitivity of radiation,CCK-8 was used to detect the apoptosis rate and cell cycle of M14-RR cell line. Results: The expression of MLH1 and MSH1 in M14-RR cells was higher than that in M14 cells after Western blot.The best inhibitory rate of paeoniflorin was(63.61±3.65)% in M14 cells by MTT assay.Western blot was used to detect the expression of MLH1 and MSH2 in melanoma cells of paeoniflorin group at 250 μg/ml,which was higher than that in control group[(85.31±5.16) vs (38.26±3.16), P <0.05;(74.15±6.08) vs (29.62±3.57), P <0.05].The expression of γ-H2AX in the melanoma cells was significantly increased by immunofluore-scence,and the DNA damage was increased.Effect of paeoniflorin on the expression of melanoma cells in the G1 and S stages of melanoma cells increased the percentage of apoptotic cells. Conclusion: Paeoniflorin enhances its sensitivity to radiotherapy by regulating MLH1 and MSH1 protein to enhance the DNA damage and repair ability of melanoma cells.