NRSN2介导的Wnt/β-catenin信号通路在骨肉瘤细胞增殖、分化中的作用及其分子机制

The effect of NRSN2-mediated Wnt/β-catenin signaling pathway on proliferation and differentiation of osteosarcoma cells and its molecular mechanism

目的:探讨神经膜蛋白2(neurensin 2,NRSN2)与骨肉瘤发生之间的相关性和NRSN2在骨肉瘤细胞增殖、分化中的相关分子机制。方法:采用免疫组织化学染色法和实时荧光定量PCR(real-time fluorescence quantitative PCR,RT-qPCR)实验,检测我院2016年01月至2020年01月收集的30例经病理学明确诊断的经典型骨肉瘤患者骨肉瘤组织标本和对应的正常组织标本中NRSN2的mRNA和蛋白表达水平。采用RT-qPCR和Western blot实验,分别检测MG63、Saos2、U2OS三种骨肉瘤细胞系中NRSN2的mRNA和蛋白水平。三种骨肉瘤细胞系中选择NRSN2相对高表达和低表达的骨肉瘤细胞系,利用RNA干扰技术构建沉默和过表达NRSN2的质粒,利用慢病毒转染法转染到骨肉瘤细胞系,构建沉默和过表达NRSN2的骨肉瘤细胞系。采用Western blot实验检测沉默NRSN2的U2OS骨肉瘤细胞系和过表达NRSN2的MG63骨肉瘤细胞系中Wnt/β-catenin信号通路相关蛋白(p-GSK3β、GSK3β、nu-β-catenin)的表达水平。采用CCK8法检测过表达NRSN2的MG63骨肉瘤细胞系中分别加入0μmol/L、5μmol/L的Wnt/β-catenin抑制剂IWR-1-endo后24 h、48 h、72 h、96 h时骨肉瘤细胞的增殖能力。结果:在骨肉瘤组织标本中NRSN2的阳性表达率明显高于正常组织标本;NRSN2的mRNA表达水平明显高于正常组织标本(P<0.01)。三种骨肉瘤细胞系中NRSN2的mRNA和蛋白表达水平均显著高于成骨细胞hFOB1.19(P<0.01);U2OS和Saos2骨肉瘤细胞系中NRSN2的mRNA和蛋白表达水平高于MG63骨肉瘤细胞系(P<0.05)。沉默NRSN2的U2OS骨肉瘤细胞系中p-GSK3β、nu-β-catenin的蛋白表达水平明显低于未转染组和重新上调NRSN2组的蛋白表达水平;过表达NRSN2的MG63骨肉瘤细胞系中p-GSK3β、nu-β-catenin的蛋白表达水平明显高于空质粒组;未加入IWR-1-endo的过表达NRSN2组细胞增殖能力在48 h、72 h和96 h均高于加入IWR-1-endo组(均P<0.01);未加入IWR-1-endo的空质粒NRSN2组骨肉瘤细胞增殖能力在48 h、72 h和96 h均高于加入IWR-1-endo抑制剂组(均P<0.05)。结论:本研究探讨了NRSN2与骨肉瘤发生之间的相关性,且NRSN2通过调控Wnt/β-catenin信号通路促进骨肉瘤细胞增殖、分化。

Objective:To investigate the correlation between NRSN2 and osteosarcoma and to study the molecular mechanism of NRSN2 in the proliferation and differentiation of osteosarcoma cells.Methods:To investigate the expression differences of NRSN2 in tumor tissues and normal tissues,the expression of NRSN2 mRNA and protein level was detected by real-time quantitative PCR(RT-qPCR)and IHC in 30 cases of osteosarcoma.To study the expression differences of NRSN2 in osteosarcoma cell lines,the expression of NRSN2 mRNA and protein level in U2OS,Saos2 and MG63 cell lines were detected by RT-qPCR and Western blot.Select osteosarcoma cell lines with relatively high expression and low expression of NRSN2,use RNA interference technology to construct a silencing/overexpression NRSN2 plasmid,and use lentiviral transfection method to transfect into osteosarcoma cell lines to construct silence/overexpresses of NRSN2.Western blot were used to detect the protein expression levels of p-GSK3β,GSK3βandβ-catenin in the silenced NRSN2 U2OS osteosarcoma cell line and the overexpressed NRSN2 MG63 osteosarcoma cell line.The CCK8 was used to detect the proliferation ability of osteosarcoma cells 24 h,48 h,72 h and 96 h after adding Wnt/β-catenin inhibitor IWR-1-endo to the overexpressing NRSN2 MG63 osteosarcoma cell line.Results:The mRNA and protein expression of NRSN2 in 30 cases of osteosarcoma tumor tissues were significantly higher than normal tissues(P<0.01).The levels of NRSN2 mRNA and protein in U2OS,Saos2 and MG63 osteosarcoma cells were higher than hFOB1.19 cells.U2OS and Saos2 were relatively higher than MG63(P<0.05).The protein expression levels of p-GSK3βand nu-β-catenin in the U2OS osteosarcoma cell line silencing NRSN2 were significantly lower than those in the untransfected and re-upregulated groups.In the MG63 osteosarcoma cell line overexpressing NRSN2,the protein expression levels of p-GSK3βand nu-β-catenin were significantly higher than those in the empty plasmid group.The cell proliferation ability of the overexpression NRSN2 group without IWR-1-endo was higher than that in the IWR-1-endo group at 48 h,72 h and 96 h.The difference between the two groups was statistically significant(P<0.01).The proliferation ability of osteosarcoma cells in the empty plasmid NRSN2 group without IWR-1-endo was higher than that with IWR-1-endo inhibitor group at 48 h,72 h and 96 h,and the difference between the two groups was statistically significant(P<0.05).Conclusion:This study explored the correlation between NRSN2 and osteosarcoma.NRSN2 promotes the proliferation and differentiation of osteosarcoma cells by regulating the Wnt/β-catenin signaling pathway.