Knock-down of long non-coding RNA GK-IT1 inhibits proliferation and promotes apoptosis in esophageal carcinoma cells

Xin YANG; Li CHEN

doi:10.16016/j.1000-5404.202008083

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Knock-down of long non-coding RNA GK-IT1 inhibits proliferation and promotes apoptosis in esophageal carcinoma cells

Basic Medicine | 更新时间：2026-01-09

- Knock-down of long non-coding RNA GK-IT1 inhibits proliferation and promotes apoptosis in esophageal carcinoma cells
- Journal of Third Military Medical University Vol. 43, Issue 2, Pages: 146-155(2021)
- 作者机构：
  
  400016 重庆，重庆医科大学附属第一医院心胸外科
- 作者简介：
- 基金信息：
- DOI：10.16016/j.1000-5404.202008083
  CLC： R394.3;R730.23;R735.1
- Received：10 August 2020，
  
  Revised：2020-10-29，
  
  Published：30 January 2021
- 稿件说明：
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Xin YANG, Li CHEN. Knock-down of long non-coding RNA GK-IT1 inhibits proliferation and promotes apoptosis in esophageal carcinoma cells[J]. Journal of Third Military Medical University, 2021, 43(2): 146-155.
DOI：

Xin YANG, Li CHEN. Knock-down of long non-coding RNA GK-IT1 inhibits proliferation and promotes apoptosis in esophageal carcinoma cells[J]. Journal of Third Military Medical University, 2021, 43(2): 146-155. DOI： 10.16016/j.1000-5404.202008083.

摘要

目的

探讨长链非编码RNA(long non-coding RNA

lncRNA)GK-IT1在食管鳞状癌细胞株中的表达及对食管鳞状癌细胞株增殖行为的影响。

方法

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27对食管鳞状癌组织和癌旁正常组织中lncRNA GK-IT1的表达。针对lncRNA GK-IT1设计构建小干扰RNA(small interference RNA

siRNA)，在TE1细胞中转染构建GK-IT1敲低模型。采用细胞计数试剂盒(cell counting kit-8

CCK-8)检测细胞增殖能力，流式细胞仪检测细胞周期的变化

Western blot实验验证细胞周期相关蛋白的表达变化。

结果

食管鳞状癌组织中lncRNA GK-IT1的表达量明显高于癌旁组织(

0.01)。在TE1和TE10细胞中，通过转染，成功构建了敲低lncRNA GK-IT1的细胞表达载体。平板克隆实验、EDU实验以及CCK-8实验结果显示，与对照组相比，敲低lncRNA GK-IT1能够显著抑制细胞的增殖(

0.01)。流式细胞检测结果发现敲低lncRNA GK-IT1能够增加细胞的凋亡(

0.01)。使用荧光原位杂交实验探究lncRNA GK-IT1的亚细胞定位，发现GK-IT1主要分布在细胞质。流式细胞检测结果发现敲低lncRNA GK-IT1能够引起TE-1、TE-10细胞G

/S期阻滞。通过生物信息学分析，lncRNA GK-IT1可能与CDK4、CCND1蛋白结合，Western blot实验验证结果显示，lncRNA GK-IT1基因敲除后，CDK4和CCND1蛋白表达降低。

结论

敲低lncRNA GK-IT1可以导致细胞周期阻滞，抑制食管鳞状癌细胞增殖并且促进细胞凋亡，可能作为食管鳞状细胞治疗的潜在靶点。

Abstract

Objective

To investigate the expression of long non-coding (lnc) RNA GK-IT1 in esophageal squamous cell carcinoma (ESCC) and explore its effect on the proliferation and apoptosis of ESCC cell lines.

Methods

Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was carried out to detect the expression of lncRNA GK-IT1 in 27 pairs of ESCC tissues and adjacent normal tissues from the ESCC patients who had not received chemotherapy yet

and in different ESCC cell lines (KYSE-150

KYSE-510

Eca-109

TE-1 and TE-10) and normal human esophageal epithelioid cell line Het-1a

respectively. After small interference RNA (siRNA) of GK-IT1 was designed and constructed

and then transfected into TE-1 and TE-10 cells respectively to knockdown GK-IT1. Plate colony formation assay

EDU analysis and cell counting kit (CCK-8) assay were used to determine the proliferation of the cells. Flow cytometry was employed to detect the variations of apoptosis and cell cycle. Fluorescence

in situ

hybridization (FISH) was used

to explore the subcellular localization of GK-IT1

and Western blotting was performed to verify the expression of cell cycle-related proteins.

Results

The expression of GK-IT1 was significantly higher in the ESCC tissues when compared to para-cancerous tissues (

0.000 1). GK-IT1 knockdown cells were successfully constructed in TE-1 and TE-10 cells

and knock-down of GK-IT1 could significantly inhibit the proliferation (

0.01) and increase the apoptotic rate of the cells (

0.01). The results of FISH assay indicated that GK-IT1 was mainly distributed in the cytoplasm. Flow cytometry showed that GK-IT1 knockdown induced the cells arrested in G

/S phase. Finally

bioinformatics analysis suggested that GK-IT1 may bind to CDK4/CCND1 protein

and Western blotting results revealed that the expression of CDK4 and CCND1 was decreased after GK-IT1 knockdown (

0.05).

Conclusion

Knockdown of GK-IT1 can induce cell cycle arrest

inhibit proliferation and promote apoptosis in ESCC cells. It may be used as a potential therapeutic target in treatment for ESCC.

关键词

Keywords

references

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