Circ_0000274 通过调控 miR-338-3p/NUCB2 轴和 JAK1/STAT3 通路促进肾细胞癌进展。

Circ_0000274 contributes to renal cell carcinoma progression by regulating miR-338-3p/NUCB2 axis and JAK1/STAT3 pathway.

机构信息

Department of Urology Surgery, Linyi Central Hospital, Linyi 276400, Shandong Province, China.

Department of Urology, Yidu Central Hospital, Weifang 262500, Shandong Province, China.

出版信息

Transpl Immunol. 2022 Oct;74:101626. doi: 10.1016/j.trim.2022.101626. Epub 2022 May 13.

DOI:10.1016/j.trim.2022.101626

PMID:35569717

Abstract

BACKGROUND

Kidney transplant recipients (KTRs) are at increased risk of developing renal cell carcinoma (RCC). Accumulating evidence has demonstrated that circular RNAs (circRNAs) are essential players in tumor advancement. However, the functions of circ_0000274 in renal cell carcinoma (RCC) are barely explored.

METHODS

The primary RCC cell lines 786-O and A498 were used in this study. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was employed for the RNA levels of circ_0000274, microRNA-338-3p (miR-338-3p) and nucleobindin 2 (NUCB2). RNase R assay was conducted to analyze the feature of circ_0000274.Cell Counting Kit-8 (CCK-8) assay, colony formation assay, transwell assay, tube formation assay and flow cytometry analysis were conducted for cell viability, colony formation, metastasis, angiogenesis and apoptosis, respectively. Western blot assay was utilized for protein levels. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were adopted to analyze the associations of circ_0000274 RNA, miR-338-3p RNA and NUCB2 protein. Murine xenograft model was established to explore the function of circ_0000274 RNA in vivo. Immunohistochemistry (IHC) assay was used to analyze NUCB2 protein level in xenograft tumors.

RESULTS

Compared to normal tissues and cells, circ_0000274 RNA level was elevated in RCC tissues and cells. Knockdown of circ_0000274 RNA suppressed cell viability, colony formation, metastasis and tube formation and promoted apoptosis in RCC cells in vitro. Circ_0000274 RNA sponged miR-338-3p RNA to positively regulate NUCB2 protein in RCC cells. Inhibition of miR-338-3p reversed the impacts of circ_0000274 knockdown on RCC cell malignant behaviors. MiR-338-3p RNA overexpression repressed the malignant phenotypes of RCC cells, while NUCB2 protein elevation could abrogate the effect. Moreover, circ_0000274 RNA knockdown blocked tumorigenesis in vivo. Besides, circ_0000274 RNA knockdown inactivated the JAK1/STAT3 protein signaling pathway.

CONCLUSION

Circ_0000274 RNA functioned as an oncogene in RCC development by regulating miR-338-3p RNA/NUCB2 protein axis and activating the JAK1/STAT3 protein signaling pathway.

摘要

背景

肾移植受者（KTR）发生肾细胞癌（RCC）的风险增加。越来越多的证据表明，环状 RNA（circRNA）是肿瘤进展的重要参与者。然而，circ_0000274 在肾细胞癌（RCC）中的功能仍鲜为人知。

方法

本研究采用人肾透明细胞癌细胞系 786-O 和 A498。采用实时定量聚合酶链反应（qRT-PCR）检测 circ_0000274、微小 RNA-338-3p（miR-338-3p）和核结合蛋白 2（NUCB2）的 RNA 水平。采用核糖核酸酶 R 试验分析 circ_0000274 的特征。采用细胞计数试剂盒-8（CCK-8）试验、集落形成试验、Transwell 试验、管形成试验和流式细胞术分析分别检测细胞活力、集落形成、转移、血管生成和细胞凋亡。采用 Western blot 检测蛋白水平。采用双荧光素酶报告基因检测和 RNA 免疫沉淀（RIP）检测分析 circ_0000274 RNA、miR-338-3p RNA 和 NUCB2 蛋白之间的关联。建立小鼠异种移植模型，探讨 circ_0000274 RNA 在体内的功能。采用免疫组织化学（IHC）检测异种移植瘤中 NUCB2 蛋白水平。

结果

与正常组织和细胞相比，RCC 组织和细胞中的 circ_0000274 RNA 水平升高。体外敲低 circ_0000274 RNA 抑制 RCC 细胞的细胞活力、集落形成、转移和管形成，并促进细胞凋亡。circ_0000274 RNA 海绵吸附 miR-338-3p RNA，正向调节 RCC 细胞中的 NUCB2 蛋白。抑制 miR-338-3p RNA 逆转了 circ_0000274 RNA 敲低对 RCC 细胞恶性表型的影响。miR-338-3p RNA 过表达抑制 RCC 细胞的恶性表型，而 NUCB2 蛋白升高则能消除其作用。此外，circ_0000274 RNA 敲低抑制体内肿瘤发生。此外，circ_0000274 RNA 敲低还能抑制 JAK1/STAT3 蛋白信号通路的激活。