MAT2A siRNA对荷瘤鼠肝癌细胞生长的影响

Effects of MAT2A siRNA on growth of implanted human hepatoma cells in nude mice

目的探讨MAT2A小干扰RNA对荷瘤鼠肝癌细胞生长和细胞凋亡的影响。方法采用脂质体转染法将MAT2A小干扰RNA质粒表达载体转染人肝癌细胞系Bel-7402细胞，建立持续转录小干扰RNA的细胞系，并将该细胞系皮下接种裸鼠，观察肿瘤生长情况；为进一步观察裸鼠体内抑瘤性，将皮下接种稳定表达小干扰RNA的Bel-7402细胞系的裸鼠分为三组：A、盐水对照组，B、空质粒对照组，C、siRNA治疗组；于接种后第12天，每组动物行纯化质粒DNA瘤内注射，观察肿瘤大小；治疗后2周，处死所有动物，取肿瘤，测量大小；部分石蜡包埋切片、HE染色，部分进行凋亡检测。结果得到稳定转染siRNA载体、并能持续表达siRNA的Bel-7402细胞系，将稳定建系的细胞传代，持续表达siRNA细胞系肿瘤细胞生长明显缓慢。皮下接种建系的Bel-7402细胞建立荷瘤鼠模型，可以观察到siRNA组肿瘤生长缓慢，平均第13天才长出皮下可及、但肉眼看不到的肿瘤，而接种control siRNA组，平均第9天可长出肉眼可见的肿瘤；用裸鼠肿瘤模型观察siRNA的体内抑瘤效果，质粒DNA注射后，对照组瘤体10．93mm^3，空质粒对照组为11．13mm^3，而siRNA质粒转染组为4．26mm^3（P〈0.01）；HE染色显示，对照组和空质粒组质粒DNA注射后，病灶充满肿瘤细胞；而siRNA组质粒DNA注射后，病灶内有大量炎性细胞浸润，坏死明显，仅见少量肿瘤细胞；且siRNA明显诱导荷瘤鼠肝癌细胞凋亡，其凋亡指数为（28．79±2．13）％，较盐水对照组（9．54±1．89）％和空质粒治疗组（10．24±2．06）％明显升高（t=15．41，P〈0．01）。结论靶向MAT2A基因的siRNA诱导荷瘤鼠肝癌细胞凋亡，抑制肝癌细胞生长。

Objective To investigate the effects of MAT2A on growth and apoptosis of human hepatoma cells in nude mice by small interfering RNA （siRNA） targeting the gene. Methods The recombinant plasmid producing siRNA targeting the MAT2A gene was transfected into Bel-7402 cells using this construct via lipofectamine method. The effects of the siRNAs on the expression of MAT2A gene were determined by RT-PCR. Bel-7402 cells which can permanently express siRNA were implanted under the skin of nude mice and the size of tumor was determined. After the tumor grew to a definite size, the mice were randomized into the saline control group （group A）, empty plasmid group （group B） and siRNA group （group C） and the tumors were respectively injected with saline, pSilencer 2. 1-U6 and recombinant plasmid siRNA4. Two weeks later, all the mice were killed and the size, histochemical features and apoptotic index of the tumors were determined. Results The Bel-7402 cells that can permanently express siRNA targeting the MAT2A gene were acquired and the grew slower than those without transfection of siRNA. RT-PCR demonstrated that the treatment of Bel-7402 cells with siRNA resulted in the knockdown of MAT2A expression. Furthermore, in vivo, with the treatment of plasmid DNA, the size of the tumors in group C was significantly lower than those in group A and B. Histochemical analysis revealed that there were large number of inflammatory cells and dead cells and few tumor cells in group （3. However, there were large number of tumor cells in group A and B. The apoptotic index was （9.54±1.89）%, （10. 24±2. 06）% and （28. 79±2. 13） % in group A, B and C, respectively. Conclusions RNA interference-mediated silencing of MAT2A gene induces cell apoptosis and attenuates the tumor growth in the liver cancer model. MAT2A is an ideal target of gene-specific therapy for liver cancer.