RasGRF1在结直肠癌细胞中的表达及其对癌细胞生物学行为的影响

Expression of RasGRF1 in colorectal cancer cells and its effect on the cancer cell biological behavior

摘要:

目的 探究RasGRF1在结直肠癌细胞中的表达及对癌细胞生物学功能的影响。方法 采用实时荧光定量聚合酶链反应（qRT-PCR）检测不同结直肠癌细胞系DiFi、SNU175、HT-29、HCT-15细胞中RasGRF1 mRNA；体外培养人结肠癌细胞，随机分为si-RasGRF1-NC组（阴性对照）、si-RasGRF1组（沉默RasGRF1表达）、NG组（空白对照）。采用qRT-PCR检测各组RasGRF1 mRNA，CCK-8法检测细胞存活情况，AnnexinV/PITC双染法检测细胞凋亡，Western blotting检测细胞PI3K/Akt通路蛋白。结果 DiFi细胞中RasGRF1 mRNA相对表达量最高（P <0.05），选择DiFi细胞进行后续实验。si-RasGRF1组光密度值低于NG组、si-RasGRF1-NC组（P <0.05）。si-RasGRF1组细胞凋亡率高于NG组和si-RasGRF1-NC组（P <0.05）。si-RasGRF1组p-PI3K/PI3K、p-Akt/Akt蛋白相对表达量低于NG组和si-RasGRF1-NC组（P <0.05），Caspase-3蛋白相对表达量高于NG组和si-RasGRF1-NC组（P <0.05）。结论 RasGRF1在结直肠癌细胞中异常高表达，抑制RasGRF1表达可抑制DiFi细胞增殖并诱导细胞凋亡，且该作用是通过抑制DiFi细胞PI3K/Akt通路活化来实现的。

Abstract:

Objective To determine the expression of RasGRF1 in colorectal cancer cells and to investigate its effect on the cancer cell biological behavior.Methods The mRNA expressions of RasGRF1 in different colorectal cancer cell lines including DiFi, SNU175, HT-29 and HCT-15 were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Human colorectal cancer cells were cultured in vitro and randomly divided into si-RasGRF1-NC group, si-RasGRF1 group and NG group. The expression level of RasGRF1 mRNA in different groups was detected by qRT-PCR. The cell survival was detected by CCK-8 assay and cell apoptosis was detected by Annexin V/PITC. The protein expression levels of molecules associated with PI3K/Akt pathway were detected by Western blotting.Results The mRNA expression level of RasGRF1 was highest in the DiFi cells (P <0.05), which were selected for subsequent experiments. The OD value in the si-RasGRF1 group was lower than that in the NG group and the si-RasGRF1-NC group (P <0.05). The apoptosis rate in the si-RasGRF1 group was higher than that in the NG group and the si-RasGRF1-NC group (P <0.05). Compared with the NG group and the si-RasGRF1-NC group, the protein expression levels of p-PI3K/PI3K and p-Akt/Akt were lower, while the protein expression level of Caspase-3 was higher in the si-RasGRF1 group (P < 0.05).Conclusions The expression of RasGRF1 is abnormally high in colorectal cancer cells, and the inhibition of the expression of RasGRF1 could suppress the proliferation of DiFi cells and induce apoptosis, possibly by restricting the activation of PI3K/Akt pathway in DiFi cells.