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Par6 Enhances Glioma Invasion by Activating MEK/ERK Pathway Through a LIN28/let-7d Positive Feedback Loop

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Abstract

The invasion of glioblastoma usually results in the recurrence and poor prognosis in patients with glioma. However, the underlying mechanisms involved in glioma invasion remains undefined. In this study, immunohistochemistry analyses of glioma specimens demonstrated that high expression of Par6 was positively correlated with malignancy and poor prognosis of patients with glioma. Par6-overexpressing glioma cells showed much more fibroblast-like morphology, suggesting that regulation of Par6 expression might be associated with tumor invasion in glioma cells. Further study indicated that Par6 overexpression subsequently increased CD44 and N-cadherin expression to enhance glioma invasion through activating MEK/ERK/STAT3 pathway, in vivo and in vitro. Moreover, we found that LIN28/let-7d axis was involved in this process via a positive feedback loop, suggesting that MEK/ERK/LIN28/let-7d/STAT3 cascade might be essential for Par6-mediated glioma invasion. Therefore, these data highlight the roles of Par6 in glioma invasion, and Par6 may serve as a potential therapeutic target for patients with glioma.

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Data Availability

All routine analysis methods are included in the “Methods” section. Sequencing data that supporting the findings of this study have been deposited in the Gene Expression Omnibus database under accession code SUB6244767. The data that support the findings of this study are presented in the paper, and all raw data are available from the corresponding author upon reasonable request. The published miRbase dataset used in this study were obtained from the publicly available database.

Abbreviations

CNS:

Central nervous system

GBM:

Glioblastoma

EMT:

Epithelial-to-mesenchymal transition

ECM:

Extracellular matrix

TJs:

Tight junctions

TCGA:

The Cancer Genome Atlas

ERK:

Extracellular signal-regulated kinase

NSCLC:

Non-small-cell lung cancer

STR:

Short tandem repeat

IHC:

Immunohistochemistry

GO:

Gene Ontology

KEGG:

Kyoto Encyclopedia of Genes and Genomes

H&E:

Hematoxylin-eosin

TGF-β:

Transforming growth factor-β

TMA:

Tissue microarray

MMP:

Matrix metalloproteinase

DEGs:

Differentially expressed genes.

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Acknowledgements

Not applicable.

Funding

This study was supported by National Natural Science Foundation of China (81872070 and 81673652), Science and Technique Foundation of Guangdong Province (210728156901639), and Natural Science Foundation of Guangdong Province (2022A1515012424).

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Author notes

  1. Yishan Huang, Pei Liu and Juanjuan Luo contributed equally to this work.

Authors and Affiliations

  1. Guangdong Provincial Key Laboratory of Infectious Disease and Molecular Immunopathology, Shantou University Medical College, Shantou, China

    Yishan Huang, Pei Liu, Juanjuan Luo, Chenchen Zhu, Chunjiao Lu & Xiaojun Yang

  2. Department of Pharmacology, College of Life Science and Biopharmaceutical of Shenyang Pharmaceutical University, Shenyang, China

    Na Zhao & Wei Cui

  3. Cell Biology Department, Wuxi School of Medicine, Jiangnan University, Wuxi, China

    Weijiang Zhao

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  1. Yishan Huang
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Contributions

YH, PL, JL, CZ, CL, and NZ performed experiments and collected data. WZ and WC reviewed and helped the manuscript writing. WC and XY supervised the project, and wrote the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Wei Cui or Xiaojun Yang.

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Ethics Approval and Consent to Participate

All animal experiments in this study were approved by the Shantou University Medical College Animal Committee. Ethical approvals were from the Medical Ethics Committee of Shantou University Medical College (no. SUMC2019-002 and SUMC2020-73).

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Supplementary Information

Fig. S1

The inhibition of MEK/ERK signaling pathways significantly inhibits Par6-mediated glioma invasion. (A,B) The determination of wound healing (A) and transwell assay (B) in Par6-OE with or without U0126 treatment (n = 3 for each group). Scale bars, 200 μm. (PNG 2345 kb)

High Resolution Image (TIF 3103 kb)

Fig. S2

TGF-β treatment induces glioma invasion through upregulating Par6 expression. (A) Par6 expression in TGF-β stimulation in different time points (0, 1, 2, 4, and 8 h) in U87MG cells. (B) TGF-β stimulation induces the upregulation of Par6 in time-dependent manner from 12 to 48 h in U87MG and U251 cells. (C) The expression of Par6, CD44, and N-cadherin in TGF-β-treated U251 and U87MG cells. (D) The expression of MEK, pMEK, ERK1/2, and pERK1/2 in U87MG and U251 cells with or without TGF-β treatment. (E) The expression of STAT3, pSTAT3 in U251 and U87MG cells with or without TGF-β treatment. (F) The representative images and quantitative analyses of glioma invasion in U251 and U87MG cells with or without TGF-β treatment, respectively (n = 3 for each group). Scale bar, 200 μm. (G) AT-1 treatment inhibit TGF-β-stimulated glioma invasion in U87MG cells. Scale bar, 200 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significance. (PNG 802 kb)

High Resolution Image (TIF 1062 kb)

Fig. S3

The Par6 expression is positively correlative with the glioma invasion and migration in primary glioma cells. (A) The expression levels of CD44, N-cadherin, MMP2, and MMP9 in Par6-OE, Par6-KD, and control groups of the cell lines from two primary glioma specimens (GBM1 and GBM2). (B) Cell invasion assays in Par6-OE, Par6-KD and control groups in two primary glioma cell lines (n = 3 random fields of view each group). Representative images of invading cells visualized by crystal violet staining, and quantification of cell invading capacity at 24 h. Scale bar, 200 μm. (C) Wound healing for cell migration in Par6-OE, Par6-KD and control groups in primary glioma cells. Representative images and quantification of cell migration into the wounded area at 0, 12, and 24 h (n = 3 for each group). Scale bar, 200 μm. *P < 0.05, **P < 0.01, ***P < 0.001. (PNG 1264 kb)

High Resolution Image (TIF 1530 kb)

Fig. S4

The validation of downstream LIN28/let-7d axis in Par6-mediated tumor invasion in primary glioma cells. (A) Western blot determination and quantification of LIN28 expression in different groups in primary glioma cells. (B,C) Wound healing and transwell assays for migration (B) and invasion (C) in Par6-OE groups in primary glioma cells with or without the inhibition of LIN28 expression (n = 3 for each group). (D) The downregulation of CD44 and N-cadherin expression in Par6-OE cells after the inhibition of LIN28 expression. (E) qPCR assays were performed to determine the levels of let-7d in Par6-OE and control cells. (F) U0126 treatment can induce the upregulation of let-7d in Par6-OE groups in primary glioma cells. (G,H) Wound healing and transwell assays for migration (G) and invasion (H) in Par6-KD groups in primary glioma cells with or without the treatment of let-7d inhibitor (n = 3 for each group). (I,J) Western blot determination and quantification of CD44, N-cadherin (I), STAT3, and pSTAT3 (J) in Par6-KD groups in primary glioma cells with or without the treatment of let-7d inhibitor. *P < 0.05, **P < 0.01, ***P < 0.001. (PNG 1179 kb)

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Huang, Y., Liu, P., Luo, J. et al. Par6 Enhances Glioma Invasion by Activating MEK/ERK Pathway Through a LIN28/let-7d Positive Feedback Loop. Mol Neurobiol 60, 1626–1644 (2023). https://doi.org/10.1007/s12035-022-03171-0

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