环状 RNA PDE3B 通过 miR-136-5p/MAP3K2 轴调控食管鳞癌细胞的致瘤性。

CircRNA PDE3B regulates tumorigenicity via the miR-136-5p/MAP3K2 axis of esophageal squamous cell carcinoma.

机构信息

Department of Thoracic Surgery, Peking University Shenzhen Hospital, Shenzhen City, Guangdong, China.

出版信息

Histol Histopathol. 2023 Sep;38(9):1029-1041. doi: 10.14670/HH-18-567. Epub 2022 Dec 1.

DOI:10.14670/HH-18-567

PMID:36533720

Abstract

BACKGROUND

CircRNA has a covalently closed circular conformation and a stable structure. However, the exact role of circRNA in esophageal squamous cell carcinoma (ESCC) remains uncertain. The purpose of this study was to explore the role of hsa_circ_0000277 (circ_PDE3B) in ESCC.

METHODS

The expression levels of circ_PDE3B, miR-136-5p and mitogen-activated protein kinase kinase kinase 2 (MAP3K2) in ESCC tissues and cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. The proliferation ability of EC9706 and KYSE30 cells was detected by clonal formation, 5-ethynyl-2'-deoxyuridine (EdU) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assays. Flow cytometry was used to detect the apoptosis rate of cells. Transwell assay was used to detect the invasion ability of EC9706 and KYSE3 cells. The relationship between miR-136-5p and circ_PDE3B or MAP3K2 was verified by dual-luciferase reporter assay and RNA pull-down, and the effect of circ_PDE3B on tumor growth in vivo was explored through tumor transplantation experiment. Immunohistochemistry (IHC) assay was used to detect MAP3K2 and Ki67 expression in mice tumor tissues.

RESULTS

The results showed that circ_PDE3B was highly expressed in ESCC tissues and cells. Downregulated circ_PDE3B expression in ESCC cells significantly reduced cell proliferation, migration and invasion. Circ_PDE3B served as a sponge for miR-136-5p, and miR-136-5p inhibition reversed the roles of circ_PDE3B knockdown in ESCC cells. MAP3K2 was a direct target of miR-136-5p, and miR-136-5p targeted MAP3K2 to inhibit the malignant behaviors of ESCC cells. Furthermore, circ_PDE3B regulated MAP3K2 expression by sponging miR-136-5p. Importantly, circ_PDE3B knockdown inhibited tumor growth in vivo.

CONCLUSIONS

In conclusion, circ_PDE3B acted as oncogenic circRNA in ESCC and accelerated ESCC progression by adsorption of miR-136-5p and activation of MAP3K2, supporting circ_PDE3B as a potential therapeutic target for ESCC.

摘要

背景

CircRNA 具有共价闭合的环形构象和稳定的结构。然而，circRNA 在食管鳞状细胞癌（ESCC）中的确切作用仍不确定。本研究旨在探讨 hsa_circ_0000277（circ_PDE3B）在 ESCC 中的作用。

方法

采用实时定量聚合酶链反应（qRT-PCR）或 Western blot 检测 ESCC 组织和细胞中 circ_PDE3B、miR-136-5p 和丝裂原活化蛋白激酶激酶激酶 2（MAP3K2）的表达水平。采用集落形成、5-乙炔基-2'-脱氧尿苷（EdU）和 3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2-H-四唑溴盐（MTT）检测 EC9706 和 KYSE30 细胞的增殖能力。流式细胞术检测细胞凋亡率。Transwell 检测 EC9706 和 KYSE3 细胞的侵袭能力。通过双荧光素酶报告基因检测和 RNA 下拉验证 miR-136-5p 与 circ_PDE3B 或 MAP3K2 的关系，通过肿瘤移植实验探讨 circ_PDE3B 对体内肿瘤生长的影响。免疫组织化学（IHC）检测小鼠肿瘤组织中 MAP3K2 和 Ki67 的表达。

结果

结果表明，circ_PDE3B 在 ESCC 组织和细胞中高表达。下调 ESCC 细胞中的 circ_PDE3B 表达显著降低细胞增殖、迁移和侵袭。Circ_PDE3B 作为 miR-136-5p 的海绵，抑制 miR-136-5p 逆转了 circ_PDE3B 敲低对 ESCC 细胞的作用。MAP3K2 是 miR-136-5p 的直接靶标，miR-136-5p 通过靶向 MAP3K2 抑制 ESCC 细胞的恶性行为。此外，circ_PDE3B 通过吸附 miR-136-5p 调节 MAP3K2 的表达。重要的是，circ_PDE3B 敲低抑制了体内肿瘤的生长。