慢病毒介导的GHSR1a shRNA对结直肠癌细胞生长的影响

Lentivirus-mediated shRNA interference of ghrelin receptor blocks growth of colorectal cancer cells

目的:构建生长激素促泌素受体1a(growth hormone secretagogue receptor 1a,GHSR1a)基因短发夹干扰RNA(short hairpin RNA,shRNA)慢病毒载体,感染人结直肠癌细胞系SW480,观察沉默GHSR1a基因对SW480细胞生长的影响,并探讨其作用机制。方法:构建特异性靶向GHSR1a基因的shRNA慢病毒表达载体和阴性对照序列慢病毒载体;建立稳定表达GHSR1a shRNA的SW480细胞、阴性对照细胞(NC)及空白对照细胞(BC),RTPCR检测GHSR1a的mRNA在细胞中的表达;通过Western blot技术检测细胞中GHSR1a、ghrelin、PTEN、p-AKT及p53蛋白的水平;CCK-8法测定GHSR1a shRNA对SW480细胞活力的影响;建立裸鼠结直肠癌皮下移植瘤模型,实验结束后处死裸鼠并测量各组裸鼠肿瘤重量;免疫组织化学法检测肿瘤组织中Ki-67和PTEN的阳性表达。结果:在体外实验中,GHSR1a在人结直肠癌细胞株Caco-2及SW480中的表达明显高于人正常结肠上皮细胞株NCM460的表达;成功建立了稳定表达GHSR1a shRNA的SW480细胞;GHSR1a shRNA能明显抑制SW480细胞GHSR1a mRNA及蛋白的表达;沉默GHSR1a基因后SW480细胞活力明显减弱;与NC组相比较,GHSR1a shRNA组细胞中PTEN mRNA及蛋白水平上调,AKT磷酸化被抑制,p53的表达明显增加;在体内实验中,与NC组相比较,GHSR1a shRNA组裸鼠结直肠癌皮下移植瘤重量明显减轻,同时Ki-67表达下调,PTEN表达上调。结论:慢病毒介导的GHSR1a shRNA对体内、外人结肠癌细胞的生长有明显抑制作用,该作用可能通过上调PTEN及其下游PI3K/AKT通路实现。

AIM： To investigate the therapeutic effects of lentivirus-mediated shRNA targeting growth hormone secretagogue receptor 1a（ GHSR1a） on colorectal cancer cell line SW480 both in vitro and in vivo. METHODS： Human GHSR1a sequence was used for the design of shRNA targeting GHSR1a,which was introduced to lentivirus,followed by transfection into SW480 cells. CCK-8 assay was performed to detect cell viability. The mRNA expression of GHSR1a and PTEN in colorectal cancer cells was detected by RT-PCR. The protein levels of GHSR1a,ghrelin,PTEN,p-AKT and p53 were determined by Western blot. Stable GHSR1a silencing in SW480 xenografts in nude mice was established. After the mice were sacrificed and weighted,immunohistochemistry was used to detect the positive expression of Ki-67 and PTEN in the tumors. RESULTS： GHSR1a was over-expressed in the malignant cells in comparison with the normal cells in vitro.The tumor specific lentivirus-mediated shRNA targeting GHSR1a gene and GHSR1a knockdown SW480 cells were successfully constructed. After transfection with GHSR1a shRNA,the expression of GHSR1a at mRNA and protein levels was markedly inhibited in the SW480 cells. Furthermore,GHSR1a silencing by specific shRNA showed increased PTEN,inhibition of AKT phosphorylation and promotion of p53 release in the SW480 cells. In vivo results demonstrated that down-regulation of GHSR1a in the SW480 cells significantly decreased the expression of Ki-67 and increased the expression of PTEN in the tumor tissues,leading to a marked reduction in tumor weight in comparison to blank control or negative control tumors. CONCLUSION： Down-regulation of GHSR1a by shRNA technique inhibits the growth of colorectal cancer cell line and xenograft tumor through activation of the PTEN/PI3 K/AKT signaling pathways.