A study on communication mechanism of lung cancer cells in tumor microenvironment mediated by pleckstrin-2/miR-196a signal axis

Manli WANG; Hui CHEN; Zhi DUAN; Qimei XU; Zhen LI

doi:10.19401/j.cnki.1007-3639.2024.07.002

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A study on communication mechanism of lung cancer cells in tumor microenvironment mediated by pleckstrin-2/miR-196a signal axis

Article | 更新时间：2025-12-31

- A study on communication mechanism of lung cancer cells in tumor microenvironment mediated by pleckstrin-2/miR-196a signal axis
- China Oncology Vol. 34, Issue 7, Pages: 628-638(2024)
- 作者机构：
  
  长沙市第一医院（中南大学湘雅医学院附属长沙医院）病理科，湖南长沙 410005
- 作者简介：
- 基金信息：
- DOI：10.19401/j.cnki.1007-3639.2024.07.002
  CLC： R734.2
- Received：17 November 2023，
  
  Revised：2024-02-27，
  
  Published：30 July 2024
- 稿件说明：
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Manli WANG, Hui CHEN, Zhi DUAN, et al. A study on communication mechanism of lung cancer cells in tumor microenvironment mediated by pleckstrin-2/miR-196a signal axis[J]. China Oncology, 2024, 34(7): 628-638.
DOI：

Manli WANG, Hui CHEN, Zhi DUAN, et al. A study on communication mechanism of lung cancer cells in tumor microenvironment mediated by pleckstrin-2/miR-196a signal axis[J]. China Oncology, 2024, 34(7): 628-638. DOI： 10.19401/j.cnki.1007-3639.2024.07.002.

摘要

背景与目的：

阐明介导肿瘤相关成纤维细胞（cancer-associated fibroblasts，CAFs）与肿瘤细胞之间通讯的信号分子仍然是一个巨大的挑战，这些信号分子对癌症转移至关重要。本研究旨在探讨普列克底物蛋白2（pleckstrin-2，PLEK2）/miR-196a信号轴介导肿瘤微环境中肺癌细胞的通讯机制。

方法：

选择人肺腺癌细胞系H1299和人胚胎肺细胞MRC

-5作为研究对象。用表达PLEK2的慢病毒（PLEK2）和载体对照（Vector）转染H1299细胞，并在转染24 h后分离外泌体（Vector_exo、PLEK2_exo）。采用miR-196a模拟物或抑制剂转染MRC-5细胞。通过蛋白质印迹法（Western blot）分析PLEK2和上皮-间充质转化（epithelial-mesenchymal transition，EMT）相关蛋白水平，采用聚合酶链反应（polymerase chain reaction，PCR）分析miR-196a表达，采用transwell实验测定细胞转移和侵袭能力。将6只雌性BALB/c-nu小鼠随机分为Vector组和PLEK2组，每组3只。通过尾静脉向各组小鼠注射转染Vector或PLEK2的H1299细胞。4周后，取出肺组织进行H-E染色和免疫组织化学染色分析α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）表达。所有动物实验均经长沙市第一医院（中南大学湘雅医学院附属长沙医院）伦理委员会批准（伦理编号为EI-2021-103）。

结果：

与Vector组相比，PLEK2组小鼠肺结节数和转移灶中α-SMA表达显著增加（

0.001）。与Vector组相比，PLEK2组H1229细胞中miR-196a表达水平显著增加（

0.05），并且PLEK2_exo中miR-196a表达水平显著高于Vector_exo（

0.05）。与Vector_exo组相比，PLEK2_exo组MRC-5细胞中miR-196a、α-SMA和成纤维细胞活化蛋白（fibroblast activation protein，FAP）表达水平显著增加（

0.05）。与阴性对照（negative control，NC）相比，miR-196a转染的MRC-5细胞中α-SMA和FAP表达水平显著增加（

0.05）。相反，通过miR-196a抑制剂（si-miR-196a#1、si-miR-196a#）转染，α-SMA和FAP表达水平被显著抑制（

0.05）。与NC-CM组相比，miR-196a-CM组H1299细胞的转移、侵袭细胞数和波形蛋白（vimentin）表达均显著增加（

0.001），E-钙黏蛋白（E-cadherin）表达显著降低（

0.001）。此外，与Vector_exo-CM组相比，PLEK2_exo-CM组H1299细胞的转移、侵袭细胞数和vimentin表达均显著增加（

0.01），E-cadherin表达显著降低（

0.001）。

结论：

PLEK2上调能够增强肺癌细胞来源的外泌体miR-196a水平，从而促进CAFs激活。激活的CAFs能够进一步增强肺癌细胞的侵袭能力。

Abstract

Background and purpose:

It is still a great challenge to clarify the signal molecules that mediate the communication between cancer-associated fibroblasts (CAFs) and tumor cells. These signal molecules are very important for cancer metastasis. The purpose of this study was to explore the communication mechanism of pleckstrin-2/miR-196a signal axis mediated by lung cancer cells in tumor microenvironment.

Methods:

Human lung adenocarcinoma cell line H1299 and human embryonic lung cell MRC-5 were selected as the research objects. H1299 cells wer

e transfected with lentivirus (PLEK2) expressing PLEK2 and Vector control

and exosomes (Vector_exo

PLEK2_exo) were isolated after 24 h of transfection. MRC-5 cells were transfected with miR-196a mimetic or inhibitor. The expressions of PLEK2 and epithelial-mesenchymal transition (EMT)-related proteins were analyzed by Western blot. The expression of miR-196a was analyzed by polymerase chain reaction (PCR)

and the metastasis and invasion ability of cells were determined by transwell assay. Six female BALB/c-nu mice were randomly divided into Vector group and PLEK2 group

with 3 mice in each group. Mice in each group were injected with H1299 cells transfected with Vector or PLEK2 through the tail vein. After 4 weeks

lung tissue was taken out for H-E staining and immunohistochemical staining to analyze the expression of α-smooth muscle actin (α-SMA). All animal experiments were approved by the ethics committee of First Hospital of Changsha City (Changsha Hospital

Xiangya School of Medicine

Central South University) (ethics number: EI-2021-103).

Results:

Compared with the Vector group

the number of pulmonary metastatic nodules and the expression of α-SMA in metastatic cancer in PLEK2 group increased significantly (

0.001). Compared with Vector group

the expression level of miR-196a in H1229 cells in PLEK2 group increased significantly (

0.05)

and the expression level of miR-196a was significantly higher in PLEK2_exo than in Vector_exo (

0.05). Compared with Vector_exo group

the expression levels of miR-196a

α-SMA and fibroblast activation protein (FAP) in MRC-5 cells in PLEK2_exo group increased significantly (

0.05). Compared with the negative control (NC)

the expression levels of α-SMA and FAP in MRC-5 cells transfected with miR-196a increased significantly (

0.05). On the contrary

by transfection with miR-196a inhibitors (si-miR-196a#1 and si-miR-196a#)

the expression levels of α-SMA

and FAP were significantly inhibited (

0.05). Compared with NC-CM group

the number of metastatic cells

invasive cells and the expression of vimentin in miR-196a-CM group increased significantly (

0.001)

and the expression of E-cadherin decreased significantly (

0.001). In addition

compared with Vector_exo-CM group

PLEK2_exo-CM group had significant increase in number of metastatic and invasive cells and the expression of vimentin (

0.01)

and significant decrease in the expression of E-cadherin (

0.001).

Conclusion:

Upregulation of PLEK2 can enhance the level of exosomes miR-196a derived from lung cancer cells

thereby promoting the activation of CAFs. The activated CAFs can further enhance the invasive ability of lung cancer cells.

关键词

Keywords

references

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