Silencing circular RNA circ_0054537 and upregulating microRNA-640 suppress malignant progression of renal cell carcinoma via regulating neuronal pentraxin-2 (NPTX2)

Hsa_circ_0054537 (circ_0054537) is a novel tumor-related circular RNA in renal cell carcinoma (RCC), and we intended to ascertain its dysregulation and functions in RCC malignant progression, as well as the underlying mechanism via serving as competing endogenous RNA (ceRNA). In this research, using real-time quantitative PCR, we found circ_0054537 was upregulated in RCC tissues and cells, and distributed throughout the cytoplasm. Then, functional effects of circ_0054537 in RCC were detected using cell counting kit-8, transwell, flow cytometry and glycolysis stress test and adenosine Triphosphate (ATP) assays. The results uncovered that circ_0054537 knockdown inhibited cell proliferation, migration, invasion, autophagy and glycolysis, but promoted apoptosis in RCC cells. Notably, circ_0054537 was identified as a ceRNA for microRNA (miR)-640, and miR-640 could target neuronal pentraxin-2 (NPTX2), as evidenced by dual-luciferase reporter assay and RNA immunoprecipitation assay. Besides, miR-640 downregulation or NPTX2 overexpression partly overturned the tumor suppressor function of circ_0054537 silence and miR-640 overexpression in RCC cells. Additionally, RCC cell growth <i>in vivo</i> was retarded by circ_0054537 silence. In conclusion, circ_0054537/miR-640/NPTX2 ceRNA pathway regulated RCC malignant progression <i>in vitro</i> and curbed RCC tumor growth <i>in vivo</i>, which could be a potential diagnosis and therapeutic target of RCC.

Hsa_circ_0054537（circ_0054537）是肾细胞癌（renal cell carcinoma, RCC）中一种新型肿瘤相关环状RNA。本研究旨在明确其在肾细胞癌恶性进展中的表达失调特征与生物学功能，并探究其作为竞争性内源RNA（competing endogenous RNA, ceRNA）发挥作用的潜在分子机制。本研究通过实时定量PCR（real-time quantitative PCR）检测发现，circ_0054537在肾细胞癌组织及细胞系中呈高表达状态，且主要分布于细胞质内。随后，本研究采用细胞计数试剂盒8（Cell Counting Kit-8, CCK-8）、Transwell实验、流式细胞术、糖酵解应激试验以及三磷酸腺苷（adenosine Triphosphate, ATP）检测，分析了circ_0054537对肾细胞癌细胞的功能影响。实验结果显示，沉默circ_0054537可抑制肾细胞癌细胞的增殖、迁移、侵袭、自噬及糖酵解过程，同时促进细胞凋亡。值得注意的是，本研究证实circ_0054537可作为微小RNA（microRNA, miR）-640的ceRNA，且miR-640可靶向调控神经戊糖蛋白2（neuronal pentraxin-2, NPTX2），上述结论通过双荧光素酶报告基因实验与RNA免疫沉淀实验得以验证。此外，抑制miR-640表达或过表达NPTX2，可部分逆转沉默circ_0054537以及过表达miR-640对肾细胞癌细胞产生的抑瘤效应。同时，沉默circ_0054537可在体内（in vivo）阻滞肾细胞癌细胞的生长。综上，circ_0054537/miR-640/NPTX2 ceRNA调控轴可在体外（in vitro）调控肾细胞癌的恶性进展，并在体内（in vivo）抑制肾细胞癌的肿瘤生长，该通路有望成为肾细胞癌潜在的诊断标志物与治疗靶点。