RNAi沉默ADM表达对人骨肉瘤细胞中β-catenin的作用及对成骨分化的影响

Effect of ADM siRNA on the expression of β-catenin and terminal differentiation of osteosarcoma cells

目的探讨通过RNAi方法沉默肾上腺髓质素(ADM)的表达对骨肉瘤细胞中β-catenin蛋白表达的作用及对骨肉瘤成骨终末分化的影响。方法细胞免疫组化方法观察ADM siRNA转染骨肉瘤细胞系F5M2后,β-catenin在0、48、72h的表达情况;应用Western blot检测ADM siRNA对F5M2细胞中β-catenin、GSK3β及其磷酸化蛋白的作用。HE染色观察ADM siRNA处理前后F5M2细胞形态的变化。观察碱性磷酸酶(ALP)染色、骨钙蛋白(OCN)的表达情况,了解ADM siRNA诱导F5M2细胞成骨终末分化的作用。结果 RNAi靶向敲低ADM的表达后,应用细胞免疫组化法观察到β-catenin蛋白从胞质逐渐向胞核内转移;Western blot结果显示ADM siRNA使磷酸化β-catenin蛋白表达降低,而磷酸化GSK3β表达升高。ADM siRNA作用骨肉瘤细胞后,细胞形态向良性分化,ALP染色加深,OCN蛋白表达升高。结论沉默ADM基因表达能诱导骨肉瘤细胞F5M2中β-catenin的活性增加及向核内转移。体外实验中,ADM siRNA通过活化β-catenin通路诱导骨肉瘤细胞的终末分化。

Objective To observe the effect of silencing the expression of ADM using P, NA interfering technique on the expression of β-catenin and terminal differentiation of osteosarcoma cells. Methods After the intervention of 0 h, 48 h and 72 h by ADM siRNA, we observed the change of distribution of β-catenin in FSM2 using immunocytochemistry staining. The expression levels of total and phosphorylated β-catenin and GSK3β were detected by Western blot after the intervention by ADM siRNA. The FSM2 cells treated with ADM siRNA were subjected to HE staining, alkaline phosphatase （ALP） assay and immunocytochemistry staining to investigate the biological effects of ADM siRNA on the morphology and terminal differentiation of FSM2 cells. Results After the intervention of 0 h, 48 h and 72 h by ADM siRNA, the distribution of β-catenin was transferred from the cytoplasm to the nucleus. ADM siRNA downregulated the expression level of P-β-catenin and upregulated P-GSK3β detected by Western blot. HE staining revealed that the configuration of F5M2 had undergone restorational changes similar to those of normal cells after ADM siRNA treatment. ALP assay and immunocytochemistry staining showed that the expression of the earlier and later molecular biomarkers of terminal differentiation, including ALP and osteocalcin were strongly positive. Gonclusion Silencing the expression of ADM can activate β-catenin to transfer to the nucleus from the cytoplasm, and induce osteosarcoma cells to make terminal differentiation through activating β-catenin signaling pathway.