WIF-1对人骨肉瘤MG-63细胞中β-catenin表达的作用研究

Effects of WIF-1 on the expression of β-catenin in human osteosarcoma MG-63 cells

目的 WIF-1是Wnt信号通路中最常见抑制因子之一,本实验研究WIF-1对人骨肉瘤MG-63细胞Wnt信号通路中β-catenin表达量的影响。方法细胞培养人骨肉瘤MG-63细胞,0 ng/ml浓度的WIF-1作为空白对照组,分别以0、48、60、80、120 ng/ml浓度的WIF-1刺激MG-63细胞,分别用FQ-PCR及Western blot法从基因水平、蛋白水平测定细胞中β-catenin的表达量;细胞培养人骨肉瘤MG-63细胞,用WIF-1刺激细胞,分别在第0、12、24、48、72、96 h对各组细胞进行计数,观察空白组及WIF-1刺激组细胞数目的变化情况。结果不同浓度(0、48、60、80、120ng/ml)WIF-1刺激下,FQ-PCR法显示β-catenin mRNA的相对表达总量2-ΔΔCT分别为1、0.55±0.30、0.44±0.24、0.35±0.15、0.24±0.21。与空白组相比较,WIF-1刺激下β-catenin mRNA表达量均降低(P<0.05)。随着WIF-1浓度的增加,β-catenin mRNA表达量下降,但组间的抑制作用无明显差别(P>0.05);Western blot法显示β-catenin/β-actin蛋白相对表达量分别为1.40±0.11、0.86±0.10、0.51±0.11、0.45±0.06、0.18±0.03;与空白组相比较,差异有统计学意义(P<0.05);空白组及WIF-1刺激组细胞数目有差异,空白组细胞增殖情况优于WIF-1刺激组。结论 WIF-1可降低人骨肉瘤MG-63细胞Wnt信号通路中β-catenin表达量,并抑制人骨瘤MG-63细胞的增殖。

ObjectiveWIF-1（Wnt inhibitory factor-1） was one of the most common antagonists of wnt signal pathway, this experiment was to study the expression of β-catenin interfered with WIF-1 in human osteosarcoma MG-63 cells.MethodsHuman MG-63 cells interfered with WIF-1 in the concentration of 0 ng/ml,48 ng/ml,60 ng/ml,80 ng/ml,120 ng/ml was cultured, the concentration of 0 ng/ml was considered as the blank group. The expression of β-catenin was retrospectively detected by the methods of real-time fluorescent quantitative polymerase chain reaction（FQ-PCR） and Western blot at the genetic and protein level. Culturing human MG-63 cells, interfered with the same concentration of WIF-1, the quantity of cells was partly noted in the hour of 0, 12, 24, 48, 72, 96 h. The number was compared between in the blank group and experiment group. Results Interfered with different concentrations（0, 48, 60, 80, 120 ng/ml） of WIF-1. It was showed by FQ-PCR that the relative fold-chages ofβ-catenin mRNA was retrospectively recorded as 1, 0.55±0.30, 0.44±0.24, 0.35±0.15, 0.24±0.21. Compared with the blank group, the stimulating groups were all found to have a lower expression（P〈0.05）. With the improvement of concentrations of WIF-1, the expressions of β-catenin presents a decreasing trend .However, these was no significant differences in the stimulating groups themselves; Western blot showed us that the relative fold-chages ofβ-catenin/β-actin was separately memorized as 1.40±0.11, 0.86±0.10, 0.51±0.11, 0.45±0.06, 0.18±0.03; compared with the blank group, differences were statistically significant（P〈0.05）. Compared with the stimulating group, the blank group was found to have a higher proliferation. ConclusionWIF-1 could decrease the expressions of β-catenin in the canonical wnt signal pathway of human osteosarcoma MG-63 cells and inhibit the proliferation of human osteosarcoma MG-63 cell.