微小RNA-494过表达与RNA干扰抑制Survivin基因对前列腺癌移植瘤生长的比较

Effect of microRNA-494 over-expression and RNA interference-mediated Survivin gene inhibition on xenograft prostate cancer growth

目的比较过表达微小RNA（microRNA）-494与采用RNA干扰抑制前列腺癌PC．3细胞中Survivin基因的表达，对前列腺癌移植瘤生长的影响。方法将过表达mieroRNA-494的腺病毒载体Ad494及负载Survivin短发卡RNA（shRNA）腺病毒载体Ad-sur分别或联合感染PC-3细胞后，将相应PC-3细胞接种于裸鼠右前肢皮下，建立前列腺癌移植瘤模型。并以磷酸盐缓冲液（PBS）组及空载体组（Ad-GFP）作为对照。动态测量肿瘤体积。55d后处死各组裸鼠，瘤体称重计算抑瘤率。Westernblot检测肿瘤组织中Survivin表达变化。免疫组织化学测定Survivin、B淋巴细胞／白血病-2（bcl-2）、bcl-2相关x蛋白（bax）及半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-3表达变化。结果Ad-sur组、Ad-494组、Ad-sur＋Ad-494组裸鼠前列腺癌移植瘤的生长明显被抑制。第35天时即表现差异。其中Ad-sur＋Ad-494组瘤体积最小，为（40．69±0．69）mm3，Ad-sur、Ad-494组瘤体体积分别为（102．11±5．32）mm3、（99．03±3．50）mm3，3组肿瘤体积均明显小于PBS组（572．72±10．46）mm3、Ad．GFP组（544．85±10．28）mm3（P〈0．05）。但Ad-sur组与Ad_494组间瘤体大小差异无统计学意义（P〉0．05）。55d后，Ad-sur、Ad-494、Ad-sur＋Ad-494组对移植瘤抑制率分别64．62％、65．98％、86．67％，Ad-sur＋Ad-494组具有抑瘤增效的相加作用（Q=0．99）。同对照组比较，实验各组的Survivin基因均表达下降，差异有统计学意义（P〈0．05）。其中，Ad-494组与Ad-sur组间差异无统计学意义（P〉0．05），Ad．sur＋Ad-494组较Ad-494、Ad-sur组更为显著；实验各组中的bax、Caspase-3表达上调，bcl-2表达下调，且Ad-sur＋Ad-494组效果优于Ad-sur、Ad-494组。结论过表达microRNA-494与RNA干扰方法均可抑制前列腺癌裸鼠移植瘤Survivin基因表达，从而抑制移植瘤的生长，且两者联合效果更加显著。

Objective To investigate the effect of microRNA-494 over-expression and RNA inter- ference-mediated Survivin gene inhibition on on xenograft prostate cancer growth in nude mice. Methods Two adenovirus vectors, Ad-494 which can overexpressed microRNA-494, and Ad-sur loaded with Survivin short hairpin RNA, were transfected into PC-3 cells alone or in combination, and the following groups were established： PBS group （blank control）, Ad-GFP group （negative control）, Ad-sur group, Ad-494 group, Ad-sur ＋ Ad-494 group. Then a total of 1 - 107 transfeeted cells were injected to the right armpit of one nude mouse to establish xenograft prostate cancer model. The gross tumor volumes were measured periodi- cally. Fifty-five days after cell injection, the mice were sacrificed and tumor volume was measured. The expression of Survivin gene in tumor sample was detected by using Western blotting and immunohistochem- istry. The expression of B lymphoeytes/leukemia-2 （bel-2）, bcl-2 associated X protein （bax）, and cystei- nyl aspartate-specifie protease （Caspase）-3 in tumor samples was examined by using immunohistochemis- try. Results The tumor growth was inhibited dramatically in Ad-sur group, Ad-494 group and Ad-sur ＋Ad-494 group as compared with control groups （ P 〈 0.05 ）. Thirty-five days after tumor cell injection, the difference in the tumor volume was found in each group. The average volume in Ad-sur ＋ Ad-494 group wasthe smallest （40. 69 ± 0. 69） mm3 as compared to that in either Ad-sur group （102. 11 ±5.32） mm3 or Ad-94 group （99.03 ± 3.50） mm3 （P 〈 0.05）. Fifty-five days after tumor cell injection, the tumor inhi- bition rate in Ad-sur group, Ad-494 group and Ad-sur ＋ Ad-494 group was 64.62%, 65.98% and 86. 67%, respectively, suggesting the synergetic anti-tumor effect of Ad-sur ＋ Ad494 （ Q = 0. 99）. Both Western blotting and immunohistochemistry revealed that Survivin gene expression was down-regulated in all experimental groups as compared with control groups, more obviously in Ad-sur ＋ Ad-49g group （P 〈 0. 05 ）. The expression of bax and Caspase-3 was up-regulated and that of bcl-2 was down-regulated in Ad- sur group, Ad494 group and Ad-sur ＋ Ad49g group, more significantly in Ad-sur ＋ Ad494 group. Con- elusion Both microRNA494 overexpression and RNA interference can inhibit Survivin gene expression in xenograft prostate cancer tumor tissue and then retard the growth of xenograft prostate cancer. The com- bined use of Ad-sur ＋ Ad-94 exerts the synergetic effeccts.