Nef对乳腺癌细胞survivin与RASSF2基因的调控及细胞凋亡

Nef regulates survivin and RASSF2 genes in breast cancer cells and apoptosis

目的探讨甲基莲心碱(Nef)对乳腺癌细胞survivin、RASSF2基因的调控及细胞凋亡情况。方法将在ATCC细胞库购买的乳腺癌细胞株zr-75-30细胞分为癌细胞组(CC组):单纯zr-75-30细胞不做其他处理,正常培养;低浓度组(LC组):将zr-75-30细胞与浓度为5 mg/kg甲基莲心碱混合培养;中浓度组(MC组):将zr-75-30细胞与浓度为10 mg/kg甲基莲心碱混合培养;高浓度组(HC组):将zr-75-30细胞与浓度为20 mg/kg甲基莲心碱混合培养。通过流式细胞术、Hoechst 33258荧光染色法、Western blot法、免疫组化法、qRT-PCR法检测zr-75-30细胞凋亡情况、survivin的蛋白表达、RASSF2的表达情况、survivin、RASSF2 mRNA的表达含量来探讨Nef对乳腺癌细胞survivin、RASSF2基因的调控及细胞凋亡的情况。结果CC组zr-75-30细胞荧光强度较弱,凋亡数量较少,LC组、MC组及HC组细胞的细胞核出现核固缩的概率显著高于CC组(均P<0.05),其中,HC组与LC组、MC组相比,HC组细胞凋亡细胞数量最多(均P<0.05)。通过Western blot法,对4组zr-75-30细胞内的survivin蛋白进行免疫印迹,灰度显示,HC组zr-75-30细胞中survivin蛋白表达最低,CC组zr-75-30细胞survivin蛋白表达高于LC组、MC组及HC组(均P<0.05);LC组细胞survivin蛋白表达高于MC组、HC组(均P<0.05)。在zr-75-30细胞中RASSF2阳性染色,呈现紫色颗粒。HC组zr-75-30细胞中RASSF2阳性数较多,而在CC组zr-75-30细胞中RASSF2阳性数较少;HC组与LC组、MC组相比,HC组zr-75-30细胞中RASSF2阳性数明显较高(均P<0.05)。HC组zr-75-30细胞中的survivin mRNA表达量最低,CC组zr-75-30细胞中表达量最高;HC组与LC组、MC组相比,survivin mRNA含量明显较低(均P<0.05),RASSF2 mRNA表达含量与survivin相反。结论甲基连心碱对乳腺癌(zr-75-30)细胞的凋亡具有促进作用,且与浓度呈正相关,在20 mg/kg浓度下促进效果最佳,其作用原理可能与甲基连心碱抑制细胞中survivin表达及上调RASSF2表达有关。

Objective To explore the regulation of survivin and RASSF2 genes and apoptosis of breast cancer cells by neferine.Methods The breast cancer cell line Zr-75-30 purchased from ATCC cell bank was divided into cancer cell group(CC group),low concentration group(LC group),medium concentration group(MC group)and high concentration group(HC group).In CC group,Zr-75-30 cells were cultured normally without any other treatment.In LC group,Zr-75-30 cells were mixed with 5 mg/kg neferine.In MC group,Zr-75-30 cells were mixed with 10 mg/kg neferine.In HC group,Zr-75-30 cells were mixed with 20 mg/kg neferine.Flow cytometry,Hoechst 33258 fluorescence staining,Western blot,immunohistochemistry and qRT-PCR were used to detect the apoptosis,survivin protein expression,RASSF2 expression,survivin and RASSF2 mRNA expression of Zr-75-30 cells.Results In CC group,the fluorescence intensity of Zr-75-30 cells was weak and the number of apoptotic cells was less.The probability of nuclear pyknosis in LC group,MC group and HC group was significantly higher than that in CC group(all P<0.05).Compared with LC group and MC group,the number of apoptotic cells in HC group was the most(all P<0.05).The expression of survivin protein in Zr-75-30 cells of HC group was the lowest.The expression of survivin protein in Zr-75-30 cells of CC group was higher than that of LC group,MC group and HC group(all P<0.05).The expression of survivin protein in LC group was higher than that in MC group and HC group(all P<0.05).RASSF2 was positive in Zr-75-30 cells and showed purple granules.The number of rassf2 positive cells in HC group was higher than that in CC group.The number of rassf2 positive cells in HC group was significantly higher than that in LC and MC groups(all P<0.05).The expression of survivin mRNA was the lowest in HC group and the highest in CC group.Compared with LC group and MC group,the expression of survivin mRNA in HC group was significantly lower(all P<0.05),and the expression of RASSF2 mRNA in HC group was opposite to that in MC group.Conclusion Neferine(Nef)has a promoting effect on the apoptosis of breast cancer(zr-75-30)cells,and it has a positive correlation with the concentration.The promoting effect is the best at a concentration of 20 mg/kg,and its principle of action It may be related to Nef inhibiting survivin expression and up-regulating RASSF2 expression in cells.