靶向ABCG2的RNA干扰逆转乳腺癌球囊细胞化疗耐药的研究

RNA interference targeting ABCG2 reverses chemotherapy resistance in mamosphere cell of breast cancer

目的通过靶向ABCG2的RNA干扰逆转乳腺癌球囊细胞化疗耐药。方法以ABCG2为靶点,设计ABCG2-siRNA,构建ABCG2干扰慢病毒载体并包装好病毒,稳定转染到筛选好的MCF-7球囊细胞中,Western Blot方法检测ABCG2的表达情况,MTT法检测RNAi沉默ABCG2基因对球囊细胞的增殖抑制作用,同时流式细胞术检测RNAi沉默ABCG2对MCF-7球囊细胞在耐药情况下的细胞周期变化和凋亡状况。结果 ABCG2干扰慢病毒载体成功构建并包装病毒,建立起ABCG2稳定沉默的MCF-7乳腺癌球囊细胞系。Western Blot显示球囊细胞中高表达的ABCG2,在RNAi沉默后表达下降;MTT结果显示,在5-Fu的作用下的MCF-7贴壁细胞抑制率高于MCF-7球囊细胞,而沉默ABCG2的MCF-7球囊细胞其抑制率会随着5-Fu量的增加而增高;流式细胞仪也显示球囊细胞大多处于G0/G1期,加入5-Fu和沉默ABCG2后,处于G0/G1期细胞数会增加,凋亡率会明显增加。结论乳腺癌球囊细胞高表达ABCG2蛋白,其5-Fu耐药性高于亲本MCF-7细胞,沉默ABCG2后能够增加球囊细胞在5-Fu的作用下的细胞抑制率及凋亡率,从而逆转对5-Fu的耐药性。

Objective To investigate the effect of RNA interference targeting ABCG2 gene on reverse chemotherapy resistance of mamosphere cell of breast cancer. Methods ABCG2-siRNA sequence was designed and inserted into lentiviral vector and virus was packaged, then a stable ABCG2-silenced MCF-7 mamosphere cell lines was estabhshed. The expression level of ABCG2 was detected by Western blot. The proliferation was measured by MTTassay in MCF-7 mamosphere cells after RNA interference. Flow cytometry was performed to assess the cell cycle and apoptotic rate. Results Western Blot showed that ABCG2 was highly expressed in the mamosphere cells, and the expression decreased concurrently after RNAi silenced ABCG2. MTr showed that inhibition rate of MCF-7 adherent cells was higher than that of MCF-7 mamosphere cells of in the presence of 5-Fu, and the inhibition rate increased as the concentration of 5-FU increased in ABCG2-silenced mamosphere cells. Flow cytometry also showed that most mamosphere cells were in Go/G1 phase, and the number of mamosphere cells in G0/G1 phase and apoptosis were elevated with increasing concentration of 5-Fu or ABCG2 gene blockage. Conclusion ABCG2 protein highly expressed in breast cancer mamosphere cells. The inhibition of ABCG2 gene expression could significantly increase the cell inhibition rate and the apoptosis rate in mamosphere cell, and reverse its resistance to 5-Fu.