胃癌细胞系SGC7901通过间充质干细胞原纤毛调控钙信号对成骨分化的影响

Effect of gastric cancer cell line SGC7901 on osteoblast differentiation by regulating calcium signaling through primary cilia in mesenchymal stem cells

目的探究胃癌细胞系SGC7901如何影响间充质干细胞(mesenchymal stem cells,MSCs)成骨分化。方法分离SD大鼠乳鼠股骨,获取原代MSCs。将原代MSCs和经血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)处理的原代MSCs分别与胃黏膜细胞系GES-1和胃癌细胞系SGC7901共培养,并以成骨诱导培养基(OS培养基)诱导72 h,提取MSCs总蛋白和总RNA。对总RNA进行RNA-seq检测并对显著差异基因进行京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)和真核直系同源群(euKaryotic Ortholog Groups,KOG)可视化分析;以GAPDH为内参,Western blot检测成骨标志蛋白,钙信号相关蛋白和原纤毛结构相关蛋白表达;对共培养后获取的MSCs总蛋白进行碱性磷酸酶(alkaline phosphatase,ALP)活性检测。结果MSCs原代培养细胞呈多角形或梭形;经过成骨诱导培养基(OS培养基)诱导,茜素红染色矿化结节呈红色;以SGC7901为对照,MSCs细胞高表达CD44,低表达CD34。与GES-1共培养相比,SGC7901共培养的MSCs中骨钙蛋白(osteocalcin,OCN)和Runx2表达均降低(P<0.01),ALP酶活力显著降低(P<0.001)。SGC7901共培养上调了MSCs中乙酰化α-微管蛋白和γ-微管蛋白表达(P<0.01)。RNA-seq分析显著差异基因共3712个,2756个基因表达上调,956个基因表达下调;SGC7901共培养的MSCs中ADRB3和PLN表达均降低(P<0.001)。经过AngⅡ处理后,SGC7901共培养抑制的Runx2和骨桥蛋白(osteopontin,OPN)表达水平增加(P<0.05),ALP酶活力增强(P<0.001)。结论胃癌细胞SGC7901通过促进MSCs原纤毛生长,抑制钙信号从而抑制MSCs成骨分化。

Objective To investigate the effects of gastric cancer cell line SGC7901 on the osteoblast differentiation of mesenchymal stem cells(MSCs).Methods Femur of SD rats were isolated and primary MSCs were obtained.The primary MSCs and angiotensinⅡ(AngⅡ)treated MSCs were co-cultured with gastric mucosa cell line GES-1 or gastric cancer cell line SGC7901,respectively,and induced with osteogenic induction medium(OS medium)for 72 hours.The total protein and total RAN of MSCs were extracted.Total RNA was detected by RNA-seq,Kyoto Encyclopedia of Genes and Genomes(KEGG)and euKaryotic Ortholog Groups(KOG)visualization analysis were performed for significantly different genes.With GAPDH as an internal reference,the expression levels of osteoblast marker proteins,calcium signaling related proteins and structure-related proteins of primary cilia were analyzed by Western blot.The alkaline phosphatase(ALP)activity of MSCs was detected in the total protein obtained after co-culture.Results The primary culture cells of MSCs was polygonal or fusiform,the red color of alizarin red staining mineralized nodules was induced by osteogenesis induction medium(OS medium),MSCs cells with high expression of CD44 and low expression of CD34 with SGC7901 as a control.Compared to GES-1,MSCs cocultured with SGC7901 showed reduced expression of osteocalcin(OCN)and Runx2(P<0.01)and significantly reduced ALP activity(P<0.001).SGC7901 coculture up-regulated the expression of acetylatedα-tubulin andγ-tubulin in MSCs(P<0.01).There were 3712 significantly different genes by RNA-seq analysis,of which 2756 genes were up-regulated and 956 genes were down-regulated.ADRB3 and PLN expression were decreased in MSCs co-cultured with SGC7901(P<0.001).After AngⅡtreatment,the expression levels of Runx2 and osteopontin(OPN)were increased(P<0.05)and ALP activity was enhanced(P<0.001)in SGC7901 cocultured MSCs.Conclusion Gastric cancer cell line SGC7901 inhibits osteoblast differentiation by promoting the growth of primary cilia and inhibiting calcium signal in MSCs.