生长激素促泌素受体1a慢病毒载体的构建及其对直肠癌SW480细胞生长和凋亡的影响

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

目的建立靶向生长激素促泌素受体1a（GHSR1a）基因短发夹干扰RNA（shRNA）慢病毒表达载体，感染至人直肠癌细胞株SW480，观察沉默GHSR1a基因对体外SW480细胞生长和凋亡的影响。方法Western blot检测GHSR1a在人直肠癌Caco-2和SW480细胞及人正常结肠上皮细胞株NCM460中的表达；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；通过慢病毒感染建立稳定表达GHSR1a shRNA的SW480细胞（KD）、阴性对照细胞（NC）及空白对照细胞（BC），反转录-聚合酶链反应（RT-PCR）检测GHSR1a mRNA在细胞中的表达；通过Western blot技术检测细胞中GHSR1a蛋白的表达；细胞计数试剂盒（CCK-8）法测定感染GHSR1a shRNA后对SW480细胞增殖的影响；流式细胞术检测细胞凋亡。结果在体外实验中，GHSR1a蛋白在Caco-2和SW480细胞中的表达水平分别为89.24±12.95和124.82±17.67，明显高出NCM460细胞中的表达水平（23.51±4.28）；通过荧光表达及流式细胞学结果表明成功建立稳定表达GHSR1a shRNA的SW480细胞；SW480细胞感染GHSR1a shRNA后，GHSR1a蛋白在BC、NC和KD组中的表达分别为73.27±8.69、69.82±7.14和11.73±1.94，GHSR1a mRNA在BC、NC和KD组中的相对表达量分别为124.31±14.78、136.62±17.20和38.94±4.13，KD组细胞中GHSR1a蛋白及mRNA的表达明显低于BC组或NC组（P＝0.000）；沉默GHSR1a基因72 h后，KD组细胞增殖较BC组下降（32.24±3.67）%，差异有统计学意义（P＝0.002）；感染GHSR1a shRNA 72 h后，BC、NC和KD组细胞早期凋亡率分别为（2.3±0.5）%、（3.4±0.6）%和（12.4±3.5）%，KD组细胞早期凋亡率明显高于BC组或NC组（P＝0.000）。结论慢病毒介导的GHSR1a shRNA对体外人直肠癌细胞生长有明显抑制作用，并促进细胞凋亡，表明GHSR1a基因有可能具有影响直肠癌发生发展的作用。

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.