IGFBP3通过癌旁脂肪细胞促进肾癌细胞生长与转移的作用研究

IGFBP3 Enhances Proliferation and Migration Capability of Renal Carcinoma Cell via Adjacent Adipocytes

目的:探讨肾透明细胞癌的分泌蛋白IGFBP3对癌旁脂肪细胞分化的作用及通过脂肪细胞促进肾透明癌细胞生长与转移的作用。方法:通过肾细胞癌的基因数据库发现过表达的基因IGFBP3,免疫组化和RT-PCR确认IGFBP3在标本中的表达。RT-PCR和Western Blot检测IGFBP3对脂肪细胞分化成熟特征标志物表达的影响。以过表达IGFBP3的786-O细胞为模型,Western Blot检测IGFBP3的促脂肪细胞分化作用与TGFβ-smad1/5/8及TGFβ-p38MAPK信号通路的关系。将过表达IGFBP3的786-O细胞与脂肪细胞共培养得到条件培养基,通过油红染色检测条件培养的肾癌细胞中脂滴含量,迁移实验和CCK8实验分别检测脂肪细胞对肾癌细胞侵袭性及增殖的影响。结果:相较于正常组,肾癌标本中IGFBP3的表达增加(P=0.017)。IGFBP3使脂肪细胞分化成熟相关标志物PPARγ、PGC1α、c/EBPα、Prdm16、UCP1表达增加。以IGFBP3处理脂肪细胞时,可以增加TGFβ下游蛋白表达水平,30 min后p-smad1/5/8表达增加(P=0.024),60 min后p-p38MAPK表达明显增加(P=0.013)。条件培养后的786-O细胞内的脂滴形成增加(P=0.028),脂肪细胞促进786-O细胞的增殖和迁移能力。结论:IGFBP3是肾透明细胞癌中过度表达的蛋白,能够促进前脂肪细胞分化,其机制主要通过激活TGFβ通路中的smad1/5/8、p38MAPK。成熟的脂肪细胞能够促进肾癌细胞质脂滴形成,并且促进肿瘤的增殖、提高肿瘤的侵袭性。

Objective: To investigate the mechanisms of IGFBP3 in the action on adjacent adipocytes and the effect of adjacent adipocytes on the proliferation and migration capability of clear cell renal cell carcinoma(ccRCC). Methods: IGFBP3 was found to be overexpressed in the database of ccRCC, which was verified in the specimen of ccRCC. RT-PCR and Western Blot were used to detected the biomarkers of adipocytes. Renal carcinoma cell line 786-O cell overexpressing IGFBP3 was used as cellular model and western blot was used to detect the effect of IGFBP3 on TGFβ-smad1/5/8 and TGF β-p38 MAPKpathway. Conditioned medium was prepared via cocultivation of 786-O overexpressing IGFBP3 and preadipocytes. Oil red staining was used to detect the contents of lipid droplets in renal carcinoma cell. Transwell and CCK8 was used to detect the effect of adipocytes on migration capability and proliferation of renal carcinoma cell. Results: The expression of IGFBP3 markedly increased in the specimen of renal cell carcinoma(P=0.017). IGFBP3 promoted the differentiation of preadipocyte after activating smad1/5/8(P=0.024) and p38 MAPK(P=0.013). The contents of lipid droplets increased in 786-O cell cultivated in the conditioned medium(P=0.028) and migration capability and proliferation of 786-O were promoted.Conclusions: IGFBP3, overexpressed in renal cell carcinoma, promoted the expression of preadipocyte by activating smad1/5/8 and p38 MAPK. Mature adipocytes could increase the contents of lipid droplets and promoted migration capability and proliferation of renal cell carcinoma.