Objective To investigate the effect of staphylococcal nuclease and tudor domain containing 1(SND1) on the biological function of osteosarcoma cells and decipher the mechanism of SND1 in regulating ferroptosis in osteosarcoma cells via SLC7A11. Methods Human osteoblasts hFOB1.19 and osteosarcoma cell lines Saos-2,U2OS,HOS,and 143B were cultured,in which the expression level of SND1 was determined.Small interfering RNA was employed to knock down the expression of SND1(si-SND1) in the osteosarcoma cell line HOS and 143B.The CCK8 assay kit,colony formation assay,and Transwell assay were employed to examine the effect of SND1 expression on the biological function of osteosarcoma cells.Furthermore,we altered the expression of SND1 and SLC7A11 in osteosarcoma cells to investigate the effect of SND1 on osteosarcoma ferroptosis via SLC7A11. Results The mRNA and protein levels of SND1 in Saos-2,U2OS,HOS,and 143B cells were higher than those in hFOB1.19 cells(all P<0.01).Compared with the control group,transfection with si-SND1 down-regulated the expression level of SND1 in HOS and 143B cells(all P<0.01),decreased the viability of HOS and 143B cells,reduced the number of colony formation,and inhibited cell invasion and migration(all P<0.001).The ferroptosis inducer Erastin promoted the apoptosis of HOS and 143B cells,while the ferroptosis inhibitor Ferrostatin-1 improved the viability of HOS and 143B cells(all P<0.001).After SND-1 knockdown,Erastin reduced the viability of HOS and 143B cells,while Ferrostatin-1 restored the cell viability(all P<0.001).After treatment with Erastin in the si-SND1 group,the levels of iron and malondialdehyde were elevated,and the level of glutathione was lowered(all P<0.001).The results of in vivo experiments showed that SND1 knockdown inhibited the mass of the transplanted tumor in 143B tumor-bearing nude mice(P<0.001).Knocking down the expression of SND1 resulted in down-regulated SLC7A11 expression(all P<0.001) and increased ferroptosis in HOS and 143B cells(P<0.001,P=0.020). Conclusions SND1 presents up-regulated expression in osteosarcoma cells.It may inhibit ferroptosis by up-regulating the expression of SLC7A11,thereby improving the viability of osteosarcoma cells.
目的 探讨葡萄球菌核酸酶样结构蛋白1(SND1)对骨肉瘤细胞生物学功能的影响,及其通过SLC7A11调控骨肉瘤细胞铁死亡的作用机制。方法 检测人成骨细胞hFOB1.19以及骨肉瘤细胞系Saos-2、U2OS、HOS和143B中SND1的表达水平。采用小干扰RNA敲减骨肉瘤细胞HOS和143B中SND1的表达(si-SND1),采用CCK8法、细胞克隆形成实验、细胞迁移和侵袭实验探究SND1的表达对骨肉瘤细胞生物学功能的影响;调控骨肉瘤细胞中SND1以及SLC7A11基因的表达,探究SND1通过SLC7A1基因对骨肉瘤铁死亡介导的肿瘤细胞凋亡的影响。结果 骨肉瘤细胞Saos-2、U2OS、HOS和143B中SND1 mRNA和蛋白的表达水平显著高于人成骨细胞hFOB1.19(P均<0.01)。与对照组比较,si-SND1转染显著降低HOS和143B细胞中SND1的表达水平(P均<0.01),且细胞活性显著降低,克隆形成数量显著减少,细胞迁移和侵袭能力显著降低(P均<0.001)。铁死亡诱导剂Erastin促进骨肉瘤HOS和143B细胞凋亡,而抑制剂Ferrostatin-1刺激上调细胞活性(P均<0.001)。敲减SND-1后使用Erastin可进一步降低骨肉瘤HOS和143B细胞活性,而使用Ferrostatin-1刺激后可显著恢复细胞活性(P均<0.001);Erastin处理后,si-SND1组细胞中铁离子和丙二醛表达增高,谷胱甘肽表达降低(P均<0.001)。体内实验结果显示,敲减SND1可以明显抑制143B裸鼠移植瘤的瘤体质量(P<0.001)。敲减SND1后骨肉瘤HOS和143B细胞中SLC7A11的表达水平显著减少(P均<0.001),且铁死亡水平升高(P<0.001,P=0.020)。结论 骨肉瘤细胞中SND1表达显著增高,其可能通过上调SLC7A11的表达抑制铁死亡,进而促进骨肉瘤细胞活性。.
Keywords: ferroptosis; osteosarcoma; staphylococcal nuclease and tudor domain containing 1.