Ma D J, Cao Z, Wang B S, Sun Y L
School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan 250062, China.
Department of Gastrointestinal Cancer Surgery, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan 250117, China.
Zhonghua Zhong Liu Za Zhi. 2020 May 23;42(5):362-368. doi: 10.3760/cma.j.cn112152-112152-20191106-00714.
To investigate the effect of silencing hepatocyte growth factor receptor (c-Met) expression on the biological characteristics of HCT116 colon cancer cells. Cellular model of c-Met transient transfection was established by using small interfering RNA (siRNA), the expression of c-Met in colon cancer cells was detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and western blot. The apoptosis assay, cell invasion assay, cell migration and other experiments were conducted to observe the effects of silencing c-Met on the biological characteristics of colon cancer cells. RT-qPCR results showed that the relative expression levels of c-Met mRNA in siRNA-Met group, blank control group and siRNA negative control (siRNA-NC) group were 0.32±0.26, 1.01±0.03 and 1.05±0.23, respectively, and the difference was statistically significant (<0.05). Western blot analysis showed that the expression level of c-Met protein in the siRNA-Met group was 0.24±0.03, significantly lower than 1.23±0.06 in the blank control group and 1.18±0.11 in the siRNA-NC group (<0.05). The cell counting kit-8 (CCK8) results showed that the 72-hour absorbance (A) values of the siRNA-Met group, blank control group and the siRNA-NC group were 1.13±0.05, 1.48±0.08 and 1.53±0.07, respectively, and the difference was statistically significant (<0.01). Cell cycle results showed that the proportion of cells in G(2)/M phase was (14.65±1.41)% in siRNA-Met group , (5.07±0.70)% in blank control group and (5.63±0.71)% in siRNA-NC group, and the difference was statistically significant (<0.05). The expression levels of cell cycle regulatory proteins Cdc25c and cyclin B1 in siRNA-Met group were significantly decreased. The apoptotic rate in siRNA-Met group was (5.85±0.35)%, significantly higher than (1.00±0.17)% in blank control group and (0.91±1.14)% in siRNA-NC group (<0.05). The expression level of apoptosis-related protein Bcl-2 in the siRNA-Met group was significantly decreased while Bcl-2 associated X protein (BAX) expression level was significantly increased. The cell scratching result showed that the cell migration abilities of the siRNA-Met group, blank control group and the siRNA-NC group were (51.33±8.62)%, (100.00±3.72)% and (102.33±6.43)%, respectively, and the difference was statistically significant (<0.05). The number of cell penetrating into the basement membrane of the siRNA-Met group, blank control group and the siRNA-NC group were 47.50±10.60, 100.00±5.33 and 102.50±10.61, respectively, and the difference was statistically significant (<0.05). The expressions of invasion related proteins including MMP-2 and MMP-9 in siRNA-Met group were decreased significantly. c-Met plays an important role in maintaining the biological characteristics of colon cancer cells. Inhibition of c-Met may have important values in the treatment of colon cancer.
探讨沉默肝细胞生长因子受体（c-Met）表达对HCT116结肠癌细胞生物学特性的影响。采用小干扰RNA（siRNA）建立c-Met瞬时转染细胞模型，通过实时荧光定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法检测结肠癌细胞中c-Met的表达。进行细胞凋亡检测、细胞侵袭实验、细胞迁移等实验，观察沉默c-Met对结肠癌细胞生物学特性的影响。RT-qPCR结果显示，siRNA-Met组、空白对照组和siRNA阴性对照组（siRNA-NC）中c-Met mRNA的相对表达水平分别为0.32±0.26、1.01±0.03和1.05±0.23，差异具有统计学意义（<0.05）。蛋白质免疫印迹分析显示，siRNA-Met组中c-Met蛋白表达水平为0.24±0.03，显著低于空白对照组的1.23±0.06和siRNA-NC组的1.18±0.11（<0.05）。细胞计数试剂盒-8（CCK8）结果显示，siRNA-Met组、空白对照组和siRNA-NC组72小时的吸光度（A）值分别为1.13±0.05、1.48±0.08和1.53±0.07，差异具有统计学意义（<0.01）。细胞周期结果显示，siRNA-Met组中G(2)/M期细胞比例为（14.65±1.41）%，空白对照组为（5.07±0.70）%，siRNA-NC组为（5.63±0.71）%，差异具有统计学意义（<0.05）。siRNA-Met组中细胞周期调节蛋白Cdc25c和细胞周期蛋白B1的表达水平显著降低。siRNA-Met组的凋亡率为（5.85±0.35）%，显著高于空白对照组的（1.00±0.17）%和siRNA-NC组的（0.91±1.14）%（<0.05）。siRNA-Met组中凋亡相关蛋白Bcl-2的表达水平显著降低，而Bcl-2相关X蛋白（BAX）表达水平显著升高。细胞划痕结果显示，siRNA-Met组、空白对照组和siRNA-NC组的细胞迁移能力分别为（51.33±8.62）%、（100.00±3.72）%和（102.33±6.43）%，差异具有统计学意义（<0.05）。siRNA-Met组、空白对照组和siRNA-NC组穿透基底膜的细胞数分别为47.50±10.60、100.00±5.33和102.50±10.61，差异具有统计学意义（<0.05）。siRNA-Met组中包括基质金属蛋白酶-2（MMP-2）和基质金属蛋白酶-9（MMP-9）在内的侵袭相关蛋白表达显著降低。c-Met在维持结肠癌细胞生物学特性中起重要作用。抑制c-Met可能在结肠癌治疗中具有重要价值。
