Mechanism of lncRNA LINC01612 promoting malignant growth of esophageal squamous cell carcinoma cells

QIAO Bo-wei; YANG Yao-qing

doi:10.11659/jjssx.07E023011

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Mechanism of lncRNA LINC01612 promoting malignant growth of esophageal squamous cell carcinoma cells

Basic Research | 更新时间：2024-09-29

- Mechanism of lncRNA LINC01612 promoting malignant growth of esophageal squamous cell carcinoma cells
- JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY Vol. 33, Issue 9, Pages: 788-795(2024)
- 作者机构：
  
  空军军医大学第二附属医院胸外科，陕西西安 710038
- 作者简介：
- 基金信息：
- DOI：10.11659/jjssx.07E023011
  CLC： R34;R394.3
- Published：25 September 2024，
  
  Received：04 July 2023，
- 稿件说明：
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乔勃伟,杨尧庆.lncRNA LINC01612促进食管鳞状细胞癌细胞恶性生长的机制研究[J].局解手术学杂志,2024,33(09):788-795.

QIAO Bo-wei,YANG Yao-qing.Mechanism of lncRNA LINC01612 promoting malignant growth of esophageal squamous cell carcinoma cells[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2024,33(09):788-795.
乔勃伟,杨尧庆.lncRNA LINC01612促进食管鳞状细胞癌细胞恶性生长的机制研究[J].局解手术学杂志,2024,33(09):788-795. DOI： 10.11659/jjssx.07E023011.

QIAO Bo-wei,YANG Yao-qing.Mechanism of lncRNA LINC01612 promoting malignant growth of esophageal squamous cell carcinoma cells[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2024,33(09):788-795. DOI： 10.11659/jjssx.07E023011.

摘要

目的

探讨lncRNA LINC01612（LINC01612）对食管鳞状细胞癌（ESCC）细胞生物学功能的影响及机制。

方法

收集42例ESCC患者的肿瘤组织和癌旁组织，采用RT-qPCR法分别检测ESCC肿瘤组织、癌旁组织及ESCC细胞系和正常食管上皮细胞（Het-1A细胞）中LINC01612相对表达水平。将KYSE30细胞分为对照组（不进行转染）、shRNA-NC组（转染shRNA-NC）、sh-LINC01612组（转染LINC01612 shRNA）、Vector组（转染Vector）、ALKBH5组（转染ALKBH5过表达载体）、IGF2BP1组（转染IGF2BP1过表达载体）和ALKBH5+LINC01612组（共转染ALKBH5和LINC01612过表达载体）。甲基化RNA免疫沉淀（MeRIP）分析评估LINC01612的m6A修饰水平；RIP分析评估m6A修饰的LINC01612与ALKBH5或IGF2BP1的结合；放线菌素D实验检测LINC01612的稳定性；CCK-8和Transwell实验检测细胞的增殖和侵袭能力，流式细胞术分析细胞凋亡率。

结果

与癌旁组织相比，LINC01612在ESCC组织中表达显著上调（

0.01）。此外，ESCC细胞LINC01612表达水平较人正常食管上皮细胞Het-1A显著上调（

0.01）。沉默LINC01612显著抑制KYSE30细胞增殖和侵袭，诱导细胞凋亡和Caspase-3活性升高（

0.05）。ALKBH5通过与LINC01612结合去除LINC01612的m6A修饰，从而下调LINC01612的表达。过表达ALKBH5抑制细胞增殖和侵袭，诱导细胞凋亡，而过表达LINC01612抵消了ALKBH5过表达对KYSE30细胞的影响（

0.05）。RIP分析结果显示，IGF2BP1通过识别m6A修饰的LINC01612并与其结合，增强LINC01612稳定性，促进其表达。

结论

LINC01612在ESCC中的表达显著上调。ALKBH5-m6A-IGF2BP1以m6A依赖性介导LINC01612上调，促进ESCC细胞增殖和侵袭，抑制细胞凋亡。

Abstract

Objective

To investigate the effect of lncRNA LINC01612 （LINC01612） in the biological function of esophageal squamous cell carcinoma （ESCC） cells and its mechanism.

Methods

Tumor tissues and adjacent tissues from 42 ESCC patients were collected， and the relative expression levels of LINC01612 in ESCC tissues， adjacent tissues， ESCC cell lines and normal esophageal epithelial cells （Het-1A cell） were detected by RT-qPCR， respectively. KYSE30 cells were divided into the control group （without transfection）， the shRNA-NC group （transfected with shRNA-NC）， the sh-LINC01612 group （transfected with LINC01612 shRNA）， the Vector group （transfected with vector）， the ALKBH5 group （transfected with ALKBH5 overexpression vector）， the IGF2BP1 group （transfected with IGF2BP1 overexpression vector） and ALKBH5+LINC01612 group （co-transfected with ALKBH5 and LINC01612 overexpression vectors）. Methylated RNA immunoprecipitation （MeRIP） assay was used to evaluate the m6A modification level of LINC01612； RIP analysis was used to evaluate the binding of m6A modified LINC01612 to ALKBH5 or IGF2BP1； actinomycin D experiment was used to detect the stability of LINC01612； CCK-8 assay and Transwell assay were used to detect the cell proliferation and invasion abilities； and the flow cytometry was used to detect the cell apoptosis rate.

Results

The expression of LINC01612 was significantly upregulated in the ESCC tissues compared with the adjacent tissues （

0.01）. Moreover， the expression levels of LINC01612 in ESCC cells were significantly higher than that in human normal esophageal epithelial cells of Het-1A （

0.01）. Silencing LINC01612 significantly inhibited the proliferation and invasion of KYSE30 cells， induced cell apoptosis and increased Caspase-3 activity （

0.05）. ALKBH5 abrogated m6A modification of LINC01612 by binding to LINC01612， thereby downregulating the expression of LINC01612. Overexpression of ALKBH5 inhibited cell proliferation and invasion， and induced cell apoptosis （

0.01）， while overexpression of LINC01612 counteracted the effects of ALKBH5 overexpression on KYSE30 cells （

0.05）. RIP analysis showed that IGF2BP1 enhanced the stability of LINC01612 and promoted its expression by recognizing and binding LINC016122 modified by m6A.

Conclusion

LINC01612 was significantly upregulated in ESCC. ALKBH5-m6A-IGF2BP1 mediated LINC01612 upregulation in an m6A dependent manner， thereby promoting the proliferation and invasion of ESCC cell， and inhibiting cell apoptosis.

关键词

食管鳞状细胞癌lncRNA LINC01612ALKBH5IGF2BP1m6A修饰

Keywords

esophageal squamous cell carcinomalncRNA LINC01612ALKBH5IGF2BP1m6A modification

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