The effect of growth hormone secretagogue receptor 1a on the proliferation and apoptosis of rectal cancer cells in vitro

Abstract

Objective To investigate the therapeutic effects of lentivirus-mediated short hairpin RNA (shRNA) targeting of growth hormone secretagogue receptor 1a (GHSR1a) in rectal cancer cell line SW480 in vitro. Methods The expression of GHSR1a in rectal cancer cell lines Caco-2 and SW480, along with NCM460, a non-malignant colon epithelial cell line was analyzed by Western blotting. Human GHSR1a sequence was used for the design of shRNA targeting GHSR1a, which was then introduced to lentivirus, followed by transfection into SW480 cells. reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to detect the mRNA expression of GHSR1a in rectal cancer cells. The protein level of GHSR1a was detected by Western blotting. Cell counting kit-8 (CCK-8) assay was performed to detect cell proliferation. The apoptotic rate was measured by flow cytometry. Results In vitro study, the relative expression of GHSR1a protein in Caco-2, SW480 and NCM460 cells were 89.24±12.95, 124.82±17.67 and 23.51±4.28 respectively. The level of GHSR1a is over-expressed in malignant cells in comparison to normal cells. The tumor-specific lentivirus mediated shRNA targeting GHSR1a gene and GHSR1a knockdown SW480 cells were successfully constructed by fluorescence and flow-cytometry. After transfection with GHSR1a shRNA, the relative level of GHSR1a protein in blank control (BC) group, negative control (NC) group and knockdown (KD) group were 73.27±8.69, 69.82±7.14 and 11.73±1.94, respectively. The relative expression of GHSR1a mRNA in BC group, NC group and KD group were 124.31±14.78, 136.62±17.20 and 38.94±4.13, respectively. The mRNA and protein levels of GHSR1a were markedly inhibited in SW480 cells compared with the BC or NC, the difference was statistically significant(P=0.000). Furthermore, the cell growth rate of SW480 cells was inhibited by (32.24±3.67)% after infected with GHSR1a shRNA compared with negative control. Additionally, the early apoptosis rate of SW480 cells from BC group, NC group and KD group were (2.3±0.5)%, (3.4±0.6)% and (12.4±3.5)%, respectively. Apoptosis rate induced by the GHSR1a shRNA transfection was markedly higher than that of controls(P=0.000). Conclusion GHSR1a is up-regulated in human rectal cancer cells, down-regulation of GHSR1a by shRNA technology can effectively inhibit proliferation and promote apoptosis of SW480 cells in vitro, silencing GHSR1a expression could become a novel therapeutic strategy for rectal adenocarcinoma prevention and treatment in the future. Key words: Rectal cancer; Growth hormone secretagogue receptor 1a; Apoptosis