促肝细胞再生磷酸酶-3基因对大肠癌细胞侵袭和失巢凋亡的调控

Effects of phosphatase of regenerating liver cell-3 gene on invasion and anoikis of human colon cancer cell

目的探讨促肝细胞再生磷酸酶(phosphatase of regenerating liver cell-3,PRL-3)基因对大肠癌细胞侵袭的影响及可能机制。方法应用PRL-3基因小干扰RNA(small interfering RNA,siRNA)转染处理人大肠癌HCT116细胞后,分别采用荧光实时定量RT-PCR和Western blot检测PRL-3 mRNA和蛋白水平,分别采用软琼脂集落培养试验和Boyden小室模型试验检测癌细胞的锚着不依赖性增殖和侵袭能力,采用琼脂糖凝胶电泳和TUNEL检测癌细胞失巢凋亡情况。结果转染组癌细胞PRL-3 mRNA和蛋白水平明显被抑制,且与时间和浓度相关。与对照组比较,转染组细胞所形成的软琼脂集落数和穿膜细胞数明显减少,且呈时间和浓度依赖性。琼脂糖凝胶电泳和TUNEL结果显示,转染组细胞出现明显的凋亡现象:凋亡指数增加,出现明显的DNA条带。结论PRL-3基因siRNA转染可明显抑制大肠癌细胞的锚着不依赖性增殖和恶性侵袭,其机制可能与诱导失巢凋亡有关。

Objective To explore the effects of phosphatase of regenerating liver cell-3（PRL-3） gene on human colon cancer cell. Methods After human colon cancer HCT116 cells were transfeeted by PRL-3 small interfering RNA（siRNA）, we detected the expression of PRL-3 mRNA and protein by using real-time PCR and Western blot assays, respectively. Anchorage-independent growth of the cancer cells was measured using clony forming in soft agar. Invasion of the cancer cells was measured using boyden chamber model. And Anoikis was measured using DNA fragmentation assay and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling （TUNEL） assay. Results The PRL-3 mRNA and protein level in HCT116 cells decreased markedly in a time-and dose-dependent manner, so did the colonies formed in soft agar and cells traversed membrane, compared with those not transfeeted cells. The results from agarose gel eleetrophoresis and TUNEL showed significant increasing apoptosis index and DNA ladder in a dose-dependent manner. Conclusions Knock-downing PRL-3 with siRNA might inhibit growth and invasion ability of colon cancer through induing anoikis.