Mechanism of long non-coding RNA DLX6-AS1 in the regulation of laryngeal cancer cells

Munire·Jiapaer; Ayiheng·Qukuerhan; YONG Jun; Pilidong·Kuyaxi; Muzhapaier·Mierzhakemu

doi:10.11659/jjssx.01E022102

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Mechanism of long non-coding RNA DLX6-AS1 in the regulation of laryngeal cancer cells

Basic Research | 更新时间：2023-06-13

- Mechanism of long non-coding RNA DLX6-AS1 in the regulation of laryngeal cancer cells
- JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY Vol. 32, Issue 6, Pages: 487-493(2023)
- 作者机构：
  
  新疆医科大学第一附属医院耳鼻喉科，新疆乌鲁木齐 830000
- 作者简介：
- 基金信息：
- DOI：10.11659/jjssx.01E022102
  CLC： R739.8
- Published：25 June 2023，
  
  Received：29 January 2022，
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穆妮热·贾帕尔 ,阿依恒·曲库尔汗 ,雍军等.长链非编码RNA DLX6-AS1调控喉癌细胞的机制研究[J].局解手术学杂志,2023,32(06):487-493.

Munire·Jiapaer Jun,Ayiheng·Qukuerhan Jun,YONG Jun,et al.Mechanism of long non-coding RNA DLX6-AS1 in the regulation of laryngeal cancer cells[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2023,32(06):487-493.
穆妮热·贾帕尔 ,阿依恒·曲库尔汗 ,雍军等.长链非编码RNA DLX6-AS1调控喉癌细胞的机制研究[J].局解手术学杂志,2023,32(06):487-493. DOI： 10.11659/jjssx.01E022102.

Munire·Jiapaer Jun,Ayiheng·Qukuerhan Jun,YONG Jun,et al.Mechanism of long non-coding RNA DLX6-AS1 in the regulation of laryngeal cancer cells[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2023,32(06):487-493. DOI： 10.11659/jjssx.01E022102.

摘要

目的

分析长链非编码RNA（lncRNA） DLX6-AS1通过调节miR-144诱导喉癌巨噬细胞M2极化对喉癌细胞的影响。

方法

从健康志愿者中分离外周血单核细胞PBMCs，从喉癌患者中分离肿瘤相关巨噬细胞TAMs，qRT-PCR检测PBMCs和TAMs中白细胞介素10（IL-10）、精氨酸酶1（Arg-1）、lncRNA DLX6-AS1与miR-144的表达。用白细胞介素4(IL-4）诱导人单核细胞白血病细胞THP-1巨噬细胞M2极化，qRT-PCR检测lncRNA DLX6-AS1、IL-10和Arg-1与CD163表达。下调lncRNA DLX6-AS1在Hep-2细胞中的表达量后，Hep-2细胞与THP-1细胞(M2型巨噬细胞)共培养48 h，CCK-8实验检测Hep-2细胞增殖活力，Transwell实验检测细胞侵袭数目，Western blot实验检测细胞上皮间质转化能力。生物信息学软件miRcode预测lncRNA DLX6-AS1与miR-144的结合位点，并进行双荧光素酶活性检测。过表达lncRNA DLX6-AS1与miR-144后，分别用CCK-8、Transwell和Western blot检测Hep-2细胞增殖、侵袭和上皮间质转化能力。

结果

与PBMCs相比，TAMs中IL-10和Arg-1的mRNA表达明显升高（

0.01），lncRNA DLX6-AS1表达显著升高（

0.01），miR-144表达显著降低（

0.01）。与Control组相比，IL-4组THP-1巨噬细胞IL-10、Arg-1和CD163表达显著上调（

0.01）；与IL-4+si-NC组相比，IL-4+si-DLX6-AS1组THP-1巨噬细胞IL-10、Arg-1和CD163表达则显著下调（

0.01）。与IL-4+si-NC组相比，IL-4+si-DLX6-AS1组Hep-2细胞活力显著下降（

0.01），侵袭数目明显减少（

0.01），N-cadherin表达显著下调（

0.01），E-cadherin表达显著上调（

0.01）。生物信息学分析发现，lncRNA DLX6-AS1与miR-144之间存在潜在的结合位点；双荧光素酶报告系统检测显示，lncRNA DLX6-AS1与miR-144之间存在直接相互作用。与pcDNA-3.1(+)+mimics NC组相比，pcDNA-DLX6-AS1+mimics NC组THP-1巨噬细胞内IL-10、Arg-1和CD163表达显著上调（

0.01）；与pcDNA-3.1(+)+miR-144 mimics组相比，pcDNA-DLX6-AS1+miR-144 mimics组THP-1巨噬细胞内IL-10、Arg-1和CD163表达显著上调（

0.01）。与pcDNA-3.1(+)+miR-144 mimics组相比，pcDNA-DLX6-AS1+miR-144 mimics组Hep-2细胞活力及细胞侵袭数目显著增加（

0.01），N-cadherin表达显著上调（

0.01），E-cadherin表达显著下调（

0.01）。

结论

lncRNA DLX6-AS1可通过调节miR-144诱导喉癌巨噬细胞M2极化，并促进喉癌细胞发展。

Abstract

Objective

To analyze the effect of long non-coding RNA （lncRNA） DLX6-AS1 on laryngeal cancer cells by regulating the M2 polarization of laryngeal cancer macrophages induced by miR-144.

Methods

Peripheral blood mononuclear cells PBMCs were isolated from healthy volunteers， and tumor-associated macrophages TAMs were isolated from laryngeal cancer patients. The expression of interleukin-10 （IL-10），Arginase 1 （Arg-1），lncRNA DLX6-AS1 and miR-144 in PBMCs and TAMs were detected by qRT-PCR. Interleukin-4 （IL-4） was used to induce M2 polarization of human monocytic leukemia cell THP-1 macrophages， and the expression of lncRNA DLX6-AS1， IL-10， Arg-1 and CD163 was detected by qRT-PCR. After down-regulating the expression of lncRNA DLX6-AS1 in Hep-2 cells，Hep-2 cells were co-cultured with THP-1 cells （M2-type macrophages） for 48 hours. The proliferative activity of Hep-2 cells was detected by CCK-8 assay，the number of invasive cells was detected by Transwell assay，and the ability of epithelial mesenchymal transformation was detected by Western blot. Bioinformatics software miRcode was used to predict the binding site of lncRNA DLX6-AS1 and miR-144，and dual luciferase activity was detected. After overexpression of lncRNA DLX6-AS1 and miR-144，the proliferation， invasion and ability of epithelial mesenchymal transformation of Hep-2 cells were detected by CCK-8，Transwell and Western blot，respectively.

Results

Compared with PBMCs， the mRNA expression of IL-10 and Arg-1 in TAMs were significantly increased （

0.01），the expression of lncRNA DLX6-AS1 was significantly increased （

0.01），and the expression of miR-144 was significantly decreased （

0.01）. Compared with the Control group，the expression of IL-10，Arg-1 and CD163 of THP-1 macrophages in the IL-4 group were significantly up-regulated （

0.01）. Compared with the IL-4+si-NC group，the expression of IL-10，Arg-1 and CD163 of THP-1 macrophages in the IL-4+si-DLX6-AS1 group were significantly down-regulated （

0.01）. Compared with the IL-4+si-NC group， the Hep-2 cell activity in the IL-4+si-DLX6-AS1 group was significantly decreased （

0.01），the number of invasive cells was significantly reduced （

0.01），the expression of N-cadherin was significantly down-regulated （

0.01），and the expression of E-cadherin was significantly up-regulated （

0.01）. Bioinformatics analysis revealed a potential binding site between lncRNA DLX6-AS1 and miR-144. The dual luciferase reporting system showed that there was a direct interaction between lncRNA DLX6-AS1 and miR-144. Compared with the pcDNA-3.1（+）+mimics NC group， the expression of IL-10， Arg-1 and CD163 of THP-1 macrophages in the pcDNA-DLX6-AS1+mimics NC group were significantly up-regulated （

0.01）. Compared with the pcDNA-3.1（+）+miR-144 mimics group， the expression of IL-10， Arg-1 and CD163 of THP-1 macrophages in the pcDNA-DLX6-AS1+miR-144 mimics group were significantly up-regulated （

0.01）. Compared with the pcDNA-3.1（+）+miR-144 mimics group， the Hep-2 cell activity and the number of invasive cells in the pcDNA-DLX6-AS1+miR-144 mimics group were significantly increased （

0.01）， the expression of N-cadherin was significantly up-regulated （

0.01）， and the expression of E-cadherin was significantly down-regulated （

0.01）.

Conclusion

LncRNA DLX6-AS1 can induce M2 polarization of laryngeal cancer macrophages and promote the development of laryngeal cancer cells by regulating miR-144.

关键词

长链非编码RNAlncRNA DLX6-AS1miR-144人单核细胞白血病细胞喉癌增殖

Keywords

long non-coding RNAlncRNA DLX6-AS1miR-144human monocytic leukemia celllaryngeal cancerproliferation

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