Mechanism of LINC00601 in regulating sensitivity of hepatocellular carcinoma cells to oxaliplatin chemotherapy

Keshu HU; Wenfeng LIU; Feng ZHANG; Bing QUAN; Xin YIN

doi:10.19401/j.cnki.1007-3639.2023.01.003

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Mechanism of LINC00601 in regulating sensitivity of hepatocellular carcinoma cells to oxaliplatin chemotherapy

Article | 更新时间：2025-12-31

- Mechanism of LINC00601 in regulating sensitivity of hepatocellular carcinoma cells to oxaliplatin chemotherapy
- China Oncology Vol. 33, Issue 1, Pages: 25-35(2023)
- 作者机构：
  
  1. 复旦大学附属中山医院肝肿瘤内科，上海 200032
  2. 复旦大学附属中山医院肝癌研究所，上海 200032
- 作者简介：
  
  YIN Xin.
- 基金信息：
- DOI：10.19401/j.cnki.1007-3639.2023.01.003
  CLC： R735.7
- Received：08 September 2022，
  
  Revised：2022-10-08，
  
  Published：30 January 2023
- 稿件说明：
移动端阅览
Keshu HU, Wenfeng LIU, Feng ZHANG, et al. Mechanism of LINC00601 in regulating sensitivity of hepatocellular carcinoma cells to oxaliplatin chemotherapy[J]. China Oncology, 2023, 33(1): 25-35.
DOI：

Keshu HU, Wenfeng LIU, Feng ZHANG, et al. Mechanism of LINC00601 in regulating sensitivity of hepatocellular carcinoma cells to oxaliplatin chemotherapy[J]. China Oncology, 2023, 33(1): 25-35. DOI： 10.19401/j.cnki.1007-3639.2023.01.003.

摘要

背景与目的：

肝细胞癌（hepatocellular carcinoma，HCC）仍是当今中国乃至全世界的高发肿瘤之一，大部分HCC患者确诊时处于中晚期，对化疗耐药或介入治疗抵抗是影响此类患者生存预后的重要因素，但其中涉及的具体分子机制尚未完全明确。我们的前期研究发现长链非编码RNA LINC00601在奥沙利铂耐药的HCC细胞中高表达。本研究旨在探索LINC00601调控HCC细胞对奥沙利铂化疗耐药的作用及其机制，为临床筛选适宜接受奥沙利铂化疗的HCC患者并为中晚期HCC患者的治疗提供新的靶点和理论依据。

方法：

利用体外长期低剂量奥沙利铂刺激人HCC细胞株MHCC-97H和Hep-3B，建立奥沙利铂耐药细胞株97H-OXR和3B-OXR。采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）检测奥沙利铂耐药细胞株中LINC00601的表达情况。进一步采用细胞计数试剂盒-8（cell counting kit-8，CCK-8）实验检测

LINC

00601基因沉默或过表达对HCC细胞奥沙利铂化疗耐药的影响。通过流式细胞术和蛋白质印迹法（Western blot）检测细胞凋亡情况的变化。最后采用Western blot和免疫荧光染色检测生长阻滞和DNA损伤诱导蛋白45α（growth arrest and DNA damage-inducible protein 45α，GADD45A）的核转位，并通过RNA pull-down实验和RNA免疫共沉淀（RNA immunoprecipitation，RIP）实验验证LINC00601和GADD45A的结合。

结果：

与对照组细胞相比，奥沙利铂耐药细胞株中LINC00601的表达水平显著升高。奥沙利铂耐药细胞及过表达LINC00601的MHCC-97H细胞对奥沙利铂体外刺激的敏感性显著下降；相反，下调耐药细胞中LINC00601的表达后，其对于奥沙利铂体外刺激的敏感性明显提高。同时，在奥沙利铂的处理下，高表达LINC00601的HCC细胞株的凋亡水平被明显抑制，且与不同细胞株对奥沙利铂体外处理的敏感性水平相一致。此外，LINC00601的过表达使GADD45A的核转位水平降低，细胞核中GADD45A的表达下降。进一步研究发现LINC00601与GADD45A可以相互结合。

结论：

LINC00601可能通过调控GADD45A的核转位，进而抑制奥沙利铂处理后的细胞凋亡，导致HCC奥沙利铂耐药。

Abstract

Background and purpose:

Hepatocellular carcinoma (HCC) is still one of the most prevalent malignancies in China and throughout the world

while most of these patients are in intermediate or advanced stage when diagnosed. The resistance of advanced HCC to chemotherapy or interventional therapy is one of the crucial factors significantly limiting the survival and prognosis of patients. However

the molecular mechanisms involved have not been fully clarified. We previously found that the long non-coding RNA LINC00601 was highly expressed in oxaliplatin resistant H

CC cells. This study aimed to explore the role and specific mechanism of LINC00601 in regulating the sensitivity of HCC cells to oxaliplatin chemotherapy

to provide a novel target to identify proper candidates for oxaliplatin chemotherapy

and the theoretical basis for the treatment of patients with advanced HCC.

Methods:

Human HCC cell line MHCC-97H and Hep-3B were induced to establish oxaliplatin resistant cell lines 97H-OXR and 3B-OXR by long-term low-dose oxaliplatin treatment

in vitro

. Real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) was used to detect the expression of LINC00601 in cell lines. Cell counting kit-8 (CCK-8) assay was performed to detect the effect of LINC00601 gene silencing or overexpression on the sensitivity of HCC cells to oxaliplatin treatment. Flow cytometry and Western blot experiments were conducted to detect the alteration of cell apoptosis. Finally

Western blot and immunofluorescence staining were applied to explore the nuclear translocation of growth arrest and DNA damage-inducible protein 45α (GADD45A)

and RNA pull-down together with RNA immunoprecipitation (RIP) experiments were carried out to validate the combination of LINC00601 and GADD45A.

Results:

Compared with the control cells

LINC00601 expression level was significantly increased in oxaliplatin resistant cells. Both induced-oxaliplatin-resistant cells and LINC00601-overexpressed MHCC-97H cells were observed to be less sensitive to oxaliplatin administration. On the contrary

the down-regulation of LINC00601 expression in oxaliplatin resistant cells significantly increased the sensitivity to oxaliplatin treatment. Meanwhile

upregulated expression of LINC00601 inhibited the apoptosis level of HCC cell lines after oxaliplatin treatment

which was consistent with the sensitivity levels of different cell lines to oxaliplatin as above. In addition

the overexpression of LINC00601 reduced the nuclear translocation of GADD45A and the expression of GADD45A in t

he nucleus. Furthermore

the combination of LINC00601 and GADD45A was observed.

Conclusion:

LINC00601 might regulate the apoptotic progress of cells after oxaliplatin chemotherapy by interfering with the nuclear translocation of GADD45A

which eventually lead to the resistance to oxaliplatin.

关键词

Keywords

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