同源盒基因2在黑色素瘤细胞对于曲美替尼耐药中的作用

Role of MSX2 in melanoma cells for trametinib resistance

目的:研究同源盒基因2(MSX2)在黑色素瘤细胞A375对于曲美替尼耐药中的作用机制。方法:构建曲美替尼耐药的A375细胞株(A375-AR),曲美替尼干预A375和A375-AR后采用CCK-8检测细胞活力,流式细胞术检测细胞凋亡率,蛋白免疫印迹法(Western blot)检测细胞中Bcl-2、Bax、Caspase-3、Caspase-9的表达以及MSX2的表达。采用小干扰RNA(siRNA)沉默A375-AR中MSX2基因,设计A375、A375-AR、A375-AR-MSX2,曲美替尼干预后,CCK-8检测细胞活力,流式细胞术检测细胞凋亡率,Western blot检测细胞中Bcl-2、Bax、Caspase-3、Caspase-9的表达以及MSX2的表达。构建MSX2过表达的A375细胞系(A375-OE),设置A375、A375-OE和A375-AR组,曲美替尼干预后,CCK-8检测细胞活力,流式细胞术检测细胞凋亡率,Western blot检测细胞中Bcl-2、Bax、Caspase-3、Caspase-9的表达以及MSX2的表达。结果:A375-AR对 1.8 nmol/L的曲美替尼耐药,采用 1.8 nmol/L曲美替尼干预后,A375细胞活力和细胞凋亡率显著高于A375-AR,A375细胞中Bcl-2基因表达水平低于A375-AR,而Bax、Caspase-3、Caspase-9的表达水平高于A375-AR。A375-AR沉默MSX2后,细胞对于曲美替尼敏感性显著增高,且细胞活力下调,凋亡率上调,细胞中Bcl-2表达下调,Bax、Caspase-3、Caspase-9的表达上调。A375过表达MSX2后,细胞对于曲美替尼敏感性显著下调,细胞活力上调,凋亡率下调,细胞中Bcl-2表达上调,Bax、Caspase-3、Caspase-9的表达下调。结论:MSX2基因可以诱导黑色素瘤细胞对于曲美替尼的耐药,是治疗黑色素瘤耐药的潜在靶点。

AIM: To study the role of homeobox gene MSX2 in the melanoma cell line A375 of trametinib resistance. METHODS: To construct trametinib resistant A375 cell line (A375-AR). After trametinib intervented A375 and A375-AR,the cell viability was detected by CCK-8, the cell apoptosis rate was detected by flow cytometry, and the expression of Bcl-2, Bax, Caspase-3, Caspase-9 and MSX2 in the cells were detected by Western blot. Small interfering RNA (siRNA) was used to silence MSX2 gene in A375-AR. After trametinib intervented A375 and A375-AR,the cell viability was detected by CCK-8, the cell apoptosis rate was detected by flow cytometry, and the expression of Bcl-2, Bax, Caspase-3, Caspase-9 and MSX2 in the cells were detected by Western blot. To construct MSX2 over-expressing A375 cell line (A375-OE), and A375, A375-OE and A375-AR groups were set up. After trametinib intervented A375 and A375-AR,the cell viability was detected by CCK-8, the cell apoptosis rate detected by flow cytometry, and the expression of Bcl-2, Bax, Caspase-3, Caspase-9 and MSX2 in the cells were detected by Western blot. RESULTS:A375-AR was resistant to trametinib of 1.8 nmol/L, the activity of A375 cells and the rate of cell apoptosis were significantly higher than that of A375-AR, and the expression level of Bcl-2 gene in A375 cells was lower than that of A375-AR, while the expression level of Bax, Caspase-3 and Caspase-9 was higher than that of A375-AR. After A375-AR silenced of MSX2, the cells were significantly increased to the sensibility of trametinib, the apoptosis rate was up-regulated, the expression of Bcl-2 in the cells was down-regulated, and the expression of Bax, Caspase-3 and Caspase-9 was up-regulated. After A375 overexpression of MSX2, the cells were down regulated to the sensibility of trametinib, the cell viability was up-regulated, the apoptosis rate was down-regulated, the expression of Bcl-2 in the cells was up-regulated, and the expression of Bax, Caspase-3 and Caspase-9 was down regulated. CONCLUSION: MSX2 gene can induce the resistance of melanoma cells to trametinib. It is a potential target for the treatment of melanoma resistance.