泛素结合酶2C通过Wnt/β-catenin信号通路促进肾恶性横纹肌样瘤的恶性进展

Ubiquitin-conjugating enzyme 2C promotes malignant progression of malignant rhabdoid tumor of the kidney via the Wnt/β-catenin signaling pathway

摘要:

目的 探讨泛素结合酶2C（ubiquitin-conjugating enzyme 2 C，UBE2C）对肾恶性横纹肌样瘤（malignant rhabdoid tumor of the kidney，MRTK）的影响及作用机制。方法 采用蛋白免疫印迹（Western blot，WB）以及免疫荧光法在收集的MRTK临床标本以及细胞系G401细胞中验证UBE2C的表达情况。从TARGET数据库下载MRTK的基因表达数据进行验证，Kaplan-Meier法（KM法）评估UBE2C与预后的关系。采用小干扰RNA（small interfering RNA，siRNA）抑制UBE2C在G401细胞中的表达。通过CCK-8检测转染后G401细胞增殖情况，流式细胞术检测细胞凋亡能力，划痕实验和Transwell实验分别检测细胞迁移和侵袭能力的改变。采用基因集富集分析（Gene Set Enrichment Analysis，GSEA）探索UBE2C调控的相关通路，并通过WB验证通路蛋白的表达。结果 在MRTK临床标本中，UBE2C表达量是癌旁对照组的（3.189±1.900）倍（P=0.033）。G401细胞系中UBE2C表达量是HEK293细胞的（2.092±0.231）倍（P=0.000），KM生存分析显示，高表达UBE2C的患者预后更差（P=0.019），并且UBE2C在4期患者（680.9±167.7）中高于早期患者（560.5±166.9），差异有统计学意义（P=0.021）。采用siRNA成功将UBE2C的表达敲低至（0.446±0.058）倍（P=0.000），并且发现敲低UBE2C抑制了G401细胞增殖、侵袭、迁移以及促进了细胞凋亡（P=0.000）。GSEA富集分析发现UBE2C与Wnt/β-catenin信号通路相关（P=0.000），敲低UBE2C可以抑制Wnt/β-catenin信号通路及上皮间质转换（epithelial-mesenchymal transition，EMT）（P=0.000）。结论 UBE2C在MRTK中高表达与不良预后相关，参与调控Wnt/β-catenin信号通路，抑制UBE2C可以抑制MRTK的增殖、迁移、侵袭，EMT，促进其凋亡。

Abstract:

Objective To investigate the influence of ubiquitin-conjugating enzyme 2C（UBE2C） on malignant rhabdoid tumor of the kidney（MRTK） and its mechanism of action.Methods Western blot and immunofluorescence assay were used to verify the expression of UBE2C in MRTK clinical specimens and G401 cells. Gene expression data of MRTK were downloaded from the TARGET database for validation，and the Kaplan-Meier method was used to assess the association between UBE2C and prognosis. Small interfering RNA（siRNA） was used to inhibit the expression of UBE2C in G401 cells. CCK-8 assay was used to observe the proliferation of G401 cells after transfection，flow cytometry was used to observe cell apoptosis，and scratch assay and Transwell assay were used to observe the changes in cell migration and invasion abilities. Gene Set Enrichment Analysis（GSEA） was used to explore the pathways regulated by UBE2C，and Western blot was used to verify the expression of pathway proteins.Results The expression of UBE2C in MRTK clinical specimens was 3.189±1.900 times that in adjacent control samples（P=0.033）. The expression of UBE2C in G401 cells was 2.092±0.231 times that in HEK293 cells（P=0.000）. The Kaplan-Meier survival analysis showed that the patients with high UBE2C expression tended to have a worse prognosis（P=0.019），and the patients with stage 4 MRTK had a significantly higher expression of UBE2C than the patients in the early stage（680.9±167.7 vs. 560.5±166.9，P=0.021）. Our research group successfully knocked down the expression of UBE2C to 0.446±0.058 folds（P=0.000） by using siRNA，and it was found that UBE2C knockdown inhibited the proliferation，invasion，and migration of G401 cells and promoted the apoptosis of G401 cells（P=0.000）. GSEA enrichment analysis revealed that UBE2C was associated with the Wnt/β-catenin signaling pathway（P=0.000），and UBE2C knockdown inhibited the Wnt/β-catenin signaling pathway and epithelial-mesenchymal transition（EMT）（P=0.000）.Conclusion The high expression of UBE2C in MRTK is associated with poor prognosis and is involved in the regulation of the Wnt/β-catenin signaling pathway，and inhibition of UBE2C can inhibit proliferation，migration，invasion，and EMT and promote apoptosis in MRTK.