长链非编码 RNA DANCR 与 miR-125b-5p 在 HCC 细胞进展中的相互作用。

Crosstalk between lncRNA DANCR and miR-125b-5p in HCC cell progression.

机构信息

Organ Transplantation Center, The Third Xiangya Hospital of Central South University, Changsha, Hunan, China.

Internal Medicine, Hunan Chest Hospital, Changsha, Hunan, China.

出版信息

Tumori. 2021 Dec;107(6):504-513. doi: 10.1177/0300891620977010. Epub 2020 Dec 3.

DOI:10.1177/0300891620977010

PMID:33272103

Abstract

OBJECTIVE

To investigate the mechanism of long noncoding RNA (lncRNA) DANCR on the progression of hepatocellular carcinoma (HCC) cells.

METHODS

The expression levels of DANCR and miR-125b-5p were measured in normal hepatocytes (LO2) and HCC cell lines by quantitative reverse transcription polymerase chain reaction. HepG2 and Huh-7 cells were transfected with sh-DANCR, the negative control (sh-NC), miR-125b-5p mimic, or mimic NC or cotransfected with sh-DANCR and miR-125b-5p inhibitor. HCC cell proliferation was assessed through CCK8 and plate colony formation assay. Western blot quantified the expression levels of Bcl-2, Bax, caspase-3, and cleaved-caspase-3. Apoptotic rate was detected as well as migratory and invasive capacities. The implication of the MAPK signal pathway was assessed by detecting the expression levels of p38, ERK1/2, JNK, p-p38, p-ERK1/2, and p-JNK. Interactions between DANCR and miR-125b-5p were detected by dual luciferase reporter assay.

RESULTS

In HCC cells, DANCR was highly expressed and miR-125b-5p was decreased. sh-DANCR or miR-125b-5p mimic stimulation reduced HepG2 or Huh-7 cell progression while promoted cell apoptosis evidenced by increased apoptotic rate, elevated levels of Bax and cleaved-caspase-3, and decreased Bcl-2. Moreover, the migration rate and invasiveness of HCC cells were also inhibited by sh-DANCR and miR-125b-5p mimic. Levels of p-p38/p38, p-ERK1/2/ERK1/2, and p-JNK/JNK were suppressed by sh-DANCR and miR-125b-5p mimic. LncRNA DANCR negatively targeted and directly bound to miR-125b-5p. Knockdown of miR-125b-5p could reverse the inhibitory effects of sh-DANCR on HCC cells.

CONCLUSION

In HCC cells, lncRNA DANCR sponges miR-125b-5p and activates MAPK pathway, thus facilitating HCC cell progression.

摘要

目的

探讨长链非编码 RNA（lncRNA）DANCR 对肝癌（HCC）细胞进展的作用机制。

方法

采用实时定量逆转录聚合酶链反应检测正常肝细胞（LO2）和 HCC 细胞系中 DANCR 和 miR-125b-5p 的表达水平。用 sh-DANCR、阴性对照（sh-NC）、miR-125b-5p 模拟物或 mimic NC 转染 HepG2 和 Huh-7 细胞，或共转染 sh-DANCR 和 miR-125b-5p 抑制剂。通过 CCK8 和平板集落形成实验评估 HCC 细胞增殖。Western blot 定量检测 Bcl-2、Bax、caspase-3 和 cleaved-caspase-3 的表达水平。检测凋亡率以及迁移和侵袭能力。通过检测 p38、ERK1/2、JNK、p-p38、p-ERK1/2 和 p-JNK 的表达水平来评估 MAPK 信号通路的影响。通过双荧光素酶报告基因检测检测 DANCR 和 miR-125b-5p 之间的相互作用。

结果

在 HCC 细胞中，DANCR 表达升高，miR-125b-5p 表达降低。sh-DANCR 或 miR-125b-5p 模拟物刺激降低 HepG2 或 Huh-7 细胞的进展，同时促进细胞凋亡，表现为凋亡率增加、Bax 和 cleaved-caspase-3 水平升高以及 Bcl-2 水平降低。此外，sh-DANCR 和 miR-125b-5p 模拟物还抑制 HCC 细胞的迁移率和侵袭力。sh-DANCR 和 miR-125b-5p 模拟物抑制 p-p38/p38、p-ERK1/2/ERK1/2 和 p-JNK/JNK 的水平。lncRNA DANCR 负向靶向并直接结合 miR-125b-5p。下调 miR-125b-5p 可逆转 sh-DANCR 对 HCC 细胞的抑制作用。