小干扰RNA沉默MAT1基因治疗胰腺癌的实验研究

Experimental study of MAT1 gene silencing mediated by siRNA in pancreatic cancer

目的了解体外转录法合成的小干扰 RNA(siRNA)沉默 MAT1基因对胰腺癌的体内体外抗癌效应。方法用 siPORT-脂质体(lipid)中性阳离子脂质体转染由体外转录法合成、Cy3荧光标记的双链 siRNA 人胰腺癌 BxPC3细胞,观察对胰腺癌细胞生长和细胞周期曲线的影响,检测 MAT1基因被 siRNA 沉默后 mRNA 和蛋白质的表达水平。在裸鼠胰腺癌皮下移植瘤瘤体内直接注射 siRNA观察抑瘤效应。结果合成的4条 MAT1-siRNA 序列中有一半可显著抑制 BxPC3细胞的生长。体外转染 MAT1-siRNA 72 h 后,BxPC3细胞生长抑制率达34.9%(P<0.01);83.9%的 BxPC3细胞滞留于G_0/G_1期,而空白对照组仅59.86%(P<0.01)。体外转染 MAT1-siRNA 48 h 后 MAT1-mRNA 水平下调了80.12%,72 h 后 MAT1蛋白质水平下调了50.12%,与各对照组相比差异均有统计学意义(均 P<0.01)。MAT1-siRNA 注射组裸鼠移植瘤重量和体积均明显低于对照组(均 P<0.05),抑瘤率达42.53%。结论 siRNA 介导的 MAT1基因沉默在体外实验中显著抑制了胰腺癌细胞的生长,在体内实验中对裸鼠胰腺癌皮下移植瘤产生明显的抑瘤效应。

Objective To investigate the inhibitory effect of gene silencing mediated by MAT1- siRNA constructed in vitro transcription for pancreatic cancer in vivo and in vitro. Methods 21-nt double strand siRNA targeting MAT1 gene was constructed and labeled with Cy3 fluorescent labeling reagent. Human pancreatic cancer cells of the line BxPC3 were cultured and divided into 4 groups： MAT1-siRNA transfected group, negative siRNA control group, lipid control group, and blank control group. The rate of cell duplication was determined by counting the cells for three consecutive days. Cell cycle profiles were determined by flow cytometry. Semi-quantitative analysis of the level of MAT1-mRNA expression was performed using the RT-PCR technique. The level of MAT1 protein expression was analyzed by Western- blotting. 18 nude mice were injected subcutaneously with BxPC3 cells to establish mouse tumor models, and then divided randomly into 3 equal groups： MAT1-siRNA group undergoing injection of MAT1-siRNA directly into the tumors 2 times a week for 4 weeks, blank control group, and negative MAT1-siRNA group. 4 weeks later the mice were killed to observe the weight and size of tumor and to calculate the tumor inhibition rate. Results Two of the 4 designed MAT1-siRNAs significantly suppressed the growth of the BxPC3 cells. 72 h after transfection the cell duplication was inhibited by 34.9% in the MAT1-siRNA transfection group. The cell cycle profile showed 83.9% of the MAT1-siRNA transfected cells were in the G0/G1 phase, a rate significantly higher than that in the blank control group （59.86%, P 〈0.01 ）. 48 h later the content of MAT1-mRNA of the MAT1-siRNA transfected cells was significantly reduced by 80. 12%, and 72 h after the transfection the content of MAT1 protein was reduced by 50. 12%, a rate significantly higher than those of the 2 control groups （ both P 〈 0.01 ）. The weight and volume of the transplant tumors in the MATI1-siRNA injected nude mice were significantly reduced compare with the negative siRNA injected control nude mice and blank control nude mice （ both P 〈 0.05 ）. The inhibition rate was 42.53%. Conclusion MAT1 gene silencing mediated by siRNA significantly suppresses the growth of pancreatic cancer cells in vitro, and significantly achieves an anti-tumor effect on the subcutaneously transplanted pancreatic tumor in vivo.