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

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-re-gulation 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/PI3K/AKT signaling pathways.