长链非编码 RNA HAND2-AS1 通过调节 miR-20a/PDCD4 轴抑制结直肠癌细胞对 5-氟尿嘧啶的耐药性。
LncRNA HAND2-AS1 inhibits 5-fluorouracil resistance by modulating miR-20a/PDCD4 axis in colorectal cancer.
机构信息
Department of Gastrointestinal Surgery, The Sixth Affiliated Hospital of Sun Yat-sen University, Guangdong Institute of Gastroenterology, Guangdong Provincial Key Laboratory of Colorectal and Pelvic Floor Diseases Supported by National Key Clinical Discipline, Guangzhou 510655, Guangdong, China.
Department of Gastrointestinal Surgery, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518000, Guangdong, China.
出版信息
Cell Signal. 2020 Feb;66:109483. doi: 10.1016/j.cellsig.2019.109483. Epub 2019 Nov 21.
BACKGROUND
Chemoresistance is one of the main obstacles in the therapy of human cancers, including colorectal cancer (CRC). Long non-coding RNA heart and neural crest derivatives expressed 2-antisense RNA 1 (lncRNA HAND2-AS1) has been demonstrated to be associated with CRC. However, the function of HAND2-AS1 in 5-Fluorouracil (5-FU) resistance of CRC remains unclear.
METHODS
Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of HAND2-AS1, miR-20a and programmed cell death factor 4 (PDCD4) mRNA. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was conducted to evaluate IC50 of 5-FU and cell proliferation. Flow cytometry analysis was used to determine cell apoptosis. Transwell assay was carried out to measure cell migration and invasion. Western blot assay was conducted to examine the protein levels of B-cell lymphoma-2 (Bcl-2), BCL2-Associated X (Bax), matrix metalloprotein 2 (MMP2), MMP9 and PDCD4. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull down assay were utilized to verify the combination between miR-20a and HAND2-AS1. Dual-luciferase reporter assay was used to analyze the association between miR-20a and PDCD4. Murine xenograft assay was used to confirm the function of HAND2-AS1 in vivo.
RESULTS
HAND2-AS1 and PDCD4 were downregulated and miR-20a was upregulated in 5-FU-resistant CRC tissues and cells. HAND2-AS1 suppressed 5-FU resistance, cell proliferation, migration and invasion and promoted cell apoptosis in 5-FU-resistant CRC cells. HAND2-AS1 acted as a sponge of miR-20a to regulate PDCD4 expression. Moreover, HAND2-AS1 suppressed cell progression and 5-FU resistance by upregulating PDCD4 via sponging miR-20a in 5-FU-resistant CRC cells. Besides, HAND2-AS1 inhibited tumor growth in vivo.
CONCLUSION
HAND2-AS1/miR-20a/PDCD4 axis inhibited cell progression and 5-FU resistance in 5-FU-resistant CRC cells.
背景
化疗耐药是包括结直肠癌（CRC）在内的人类癌症治疗的主要障碍之一。长链非编码 RNA 心脏和神经嵴衍生物表达 2-反义 RNA 1（lncRNA HAND2-AS1）已被证明与 CRC 有关。然而，HAND2-AS1 在 CRC 对 5-氟尿嘧啶（5-FU）耐药中的作用尚不清楚。
方法
采用实时定量聚合酶链反应（qRT-PCR）检测 HAND2-AS1、miR-20a 和程序性细胞死亡因子 4（PDCD4）mRNA 的表达。3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2-H-四唑溴盐（MTT）测定法评估 5-FU 的 IC50 和细胞增殖。流式细胞术分析用于测定细胞凋亡。Transwell 测定法用于测量细胞迁移和侵袭。Western blot 测定法用于检测 B 细胞淋巴瘤-2（Bcl-2）、Bcl2 相关 X（Bax）、基质金属蛋白酶 2（MMP2）、MMP9 和 PDCD4 的蛋白水平。双荧光素酶报告基因检测、RNA 免疫沉淀（RIP）检测和 RNA 下拉检测用于验证 miR-20a 与 HAND2-AS1 的结合。双荧光素酶报告基因检测用于分析 miR-20a 与 PDCD4 的相关性。小鼠异种移植实验用于体内证实 HAND2-AS1 的功能。
结果
5-FU 耐药 CRC 组织和细胞中 HAND2-AS1 和 PDCD4 下调，miR-20a 上调。HAND2-AS1 抑制 5-FU 耐药、细胞增殖、迁移和侵袭，促进 5-FU 耐药 CRC 细胞凋亡。HAND2-AS1 作为 miR-20a 的海绵体调节 PDCD4 的表达。此外，HAND2-AS1 通过海绵 miR-20a 上调 PDCD4 抑制 5-FU 耐药 CRC 细胞的细胞进展和 5-FU 耐药。此外，HAND2-AS1 抑制体内肿瘤生长。
结论
HAND2-AS1/miR-20a/PDCD4 轴抑制 5-FU 耐药 CRC 细胞的细胞进展和 5-FU 耐药。
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