沉默Bax Inhibitor-1基因对子宫肌瘤细胞凋亡的影响及机制研究

The effect and mechanism of Bax inhibitor-1 gene silencing on apoptosis of uterine leiomyoma cells

目的在子宫平滑肌瘤中探究Bax抑制因子1(Bax inhibitor-1,BI-1)的表达及其对子宫平滑肌瘤细胞的作用与相关机制。方法应用RT-PCR及Western blot实验检测20对子宫平滑肌瘤组织样本与瘤旁组织中BI-1的mRNA及蛋白表达;分离培养原代子宫平滑肌瘤细胞并应用α-Actin免疫细胞染色进行鉴定;应用siRNA技术将靶向沉默BI-1的si-BI-1及阴性对照序列si-NC转染入上述分离培养的子宫平滑肌瘤细胞中,并将细胞分为3组,Control组、si-BI-1组和si-NC组;分别应用CCK-8、Annexin V-FITC及JC-1与流式细胞术分析沉默BI-1对子宫肌瘤细胞增殖、凋亡、线粒体膜电位及细胞中Ca^(2+)浓度的影响,最后应用Western blot实验检测沉默BI-1对子宫肌瘤细胞中Cyt-C及凋亡相关蛋白Bcl-2、Bax及Cleaved caspase-3表达的影响。结果与瘤旁组织相比,BI-1在子宫肌瘤中的mRNA及蛋白表达均显著增加(P<0.05);成功分离培养纯度较高的原代子宫肌瘤细胞;与Control组细胞相比,沉默BI-1基因表达不影响子宫平滑肌瘤细胞的增殖能力(P>0.05),但显著促进si-BI-1组细胞的凋亡率(P<0.05),并促进细胞线粒体膜电位降低且胞质及线粒体中Ca^(2+)浓度的增加(P<0.05);Western blot实验检测结果显示,与Control组细胞相比,沉默BI-1基因表达使si-BI-1组细胞中的Bcl-2蛋白表达显著降低(P<0.05),Bax、Cleaved caspase-3及Cyt-C蛋白表达明显增加(P<0.05),Bcl-2/Bax比值明显降低(P<0.05)。结论实验发现BI-1在子宫肌瘤组织中的表达显著增加,沉默子宫肌瘤细胞中BI-1基因可能通过改变Bcl-2/Bax比值,促进胞质及线粒体中Ca^(2+)浓度的增加,从而引起线粒体膜电位的下降及损伤所诱导细胞发生凋亡。

Objective To investigate the expression of Bax inhibitor-1(BI-1)in uterine leiomyoma and its effect on uterine leiomyoma cells.Methods RT-PCR and Western blot were used to detect the expression of BI-1 mRNA and protein in 20 pairs of uterine leiomyoma samples and adjacent tissues;the primary uterine leiomyoma cells were isolated and identified byα-actin immunocytochemistry;the si-BI-1 and negative control sequence si-NC were transfected into the above-mentioned isolated and cultured uterine leiomyoma cells by si-RNA technology,and the cells were divided into three groups:control group,si-BI-1 group and si-NC group;CCK-8,Annexin V-FITC,JC-1 and flow cytometry were used to analyze the effects of BI-1 silencing on the proliferation,apoptosis,mitochondrial membrane potential and Ca2+concentration of myoma cells.Finally,Western blot was used to detect the effects of BI-1 silencing on the expression of Cyt-C,Bcl-2,Bax and cleaved Caspase-3 in myoma cells;Results Compared with the adjacent tissues,the expression of BI-1 mRNA and protein in uterine leiomyoma increased significantly(P<0.05);high purity primary uterine myoma cells were successfully isolated and cultured.Compared with the control group,the silencing of BI-1 gene expression did not affect the proliferation ability of uterine leiomyoma cells(P>0.05),but significantly promoted the apoptosis rate of si-BI-1 cells(P<0.05),decreased the mitochondrial membrane potential,and increased the Ca2+concentration in cytoplasm and mitochondria(P<0.05).The results of Western blot showed that compared with the control group,the expression of Bcl-2 protein decreased significantly(P<0.05),the expression of Bax,cleaved Caspase-3 and Cyt-C protein increased significantly(P<0.05),and the ratio of Bcl-2/Bax decreased significantly(P<0.05).Conclusion The expression of BI-1 increased significantly in uterine leiomyoma tissue.The silencing of BI-1 gene in uterine leiomyoma cells may promote the increase of Ca2+concentration in cytoplasm and mitochondria by changing the ratio of Bcl-2/Bax,thus leading to the decrease of mitochondrial membrane potential and apoptosis induced by injury.