过表达TRIM2对肾透明细胞癌增殖、迁移和干性的影响

Overexpression of TRIM2 suppresses proliferation, migration and stemness of clear cell renal cell carcinoma cells in vitro

目的探究三结构域蛋白2 (tripartite motif-containing protein 2,TRIM2)对肾透明细胞癌增殖、迁移和干性的影响。方法从TCGA数据库中预测TRIM2对肾透明细胞癌患者预后的影响,免疫组化检测TRIM2在肾透明细胞癌及癌旁组织的表达,Western blot实验选出TRIM2表达相对低的两个肾透明细胞癌细胞株,并用TRIM2过表达质粒转染肾透明细胞癌细胞株,Western blot以及荧光显微镜观测转染效率。成功转染TRIM2过表达质粒后,用CCK-8实验、划痕实验和细胞成球实验检测细胞增殖、迁移和成球能力,并用q-PCR和Western blot检测TRIM2对细胞干性相关基因的影响,从COEXPEDIA数据库构建一个在肾癌中与TRIM2相关的基因共表达网络,进一步探索TRIM2在肾透明细胞癌发生、发展中的作用机制。结果 TRIM2高表达的肾透明细胞癌患者的预后较TRIM2低表达患者好(P<0.001),免疫组化结果显示,与癌旁组织比较,TRIM2在肾透明细胞癌组织中低表达。TRIM2过表达后,肾透明细胞癌细胞增殖、迁移和成球能力均明显下降(P<0.01),且干性基因(C-MYC、NANOG、OCT4)的表达也明显下调(P<0.01)。构建的基因共表达网络提示,与TRIM2相关程度较高的基因有NR3C2、ESRRG、COL4A3和CTDSPL。结论 TRIM2过表达后能抑制肾透明细胞癌增殖、迁移和成球能力,且下调干性基因的表达,与TRIM2相关程度较高的基因可能参与肾透明细胞癌的发生、发展。

Objective To explore the effect of tripartite motif-containing protein 2(TRIM2)on the proliferation,migration and stemness of clear cell renal clear carcinoma.Methods We performed a survival analysis of the patients with clear cell renal cell carcinoma using TCGA database to assess how TRIM2 expression affected the clinical outcomes of the patients.We also collected the clinical specimens of clear cell renal cell carcinoma tissues and paired adjacent tissues for testing the expression of TRIM2 using immunohistochemistry.From different clear cell renal clear carcinoma cells,we selected 2 cell lines that expressed low levels of TRIM2 based on the results of Western blotting,and tested the effect of TRIM2 overexpression on the cell proliferation,migration and sphere-forming ability using CCK8 assay,wound healing assay and cell sphere formation assay;the effect of TRIM2 overexpression on the stemness of the cells was evaluated using quantitative real-time PCR and Western blotting.Based on the data from COEXPEDIA database,we constructed a gene co-expression network associated with TRIM2 in renal cancer to explore the role of TRIM2 in the tumorigenesis and progression of clear cell renal cell carcinoma.Results Analysis of the TCGA database showed that in patients with clear cell renal cell carcinoma,a high expression of TRIM2 was associated with a higher survival possibility(P<0.001).The results of immunohistochemistry showed that TRIM2 was lowly expressed in renal clear cell carcinoma tissues as compared with the adjacent tissues.In clear cell renal cell carcinoma cells with low TRIM2 expression,overexpression of TRIM2 significantly suppressed the cell proliferation(P<0.01),migration(P<0.01)and cell sphere formation(P<0.01)and obviously lowered the expressions of the genes(C-MYC,NANOG,and OCT4)associated with the cell stemness(P<0.01).Conclusion Overexpression of TRIM2 in clear cell renal cell carcinoma cells can inhibit the cell proliferation,migration and sphere formation and down-regulate the expressions of the stemness genes.The genes(NR3 C2,ESRRG,COL4 A3 and CTDSPL)that are closely related with TRIM2 may all participate in the occurrence and progression of clear cell renal cell carcinoma.