Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jul 20;12(7):e0180844.
doi: 10.1371/journal.pone.0180844. eCollection 2017.

MicroRNA-200c inhibits epithelial-mesenchymal transition, invasion, and migration of lung cancer by targeting HMGB1

Po-Len Liu  1 , Wei-Lun Liu  2   3 , Jia-Ming Chang  4   5 , Yung-Hsiang Chen  6   7 , Yu-Peng Liu  8 , Hsuan-Fu Kuo  9 , Chong-Chao Hsieh  10 , Yu-Sian Ding  4   5 , Wei-Wei Chen  4   5 , Inn-Wen Chong  1   11
Affiliations

MicroRNA-200c inhibits epithelial-mesenchymal transition, invasion, and migration of lung cancer by targeting HMGB1

Po-Len Liu et al. PLoS One. .

Abstract

MicroRNAs (miRs) play critical roles in cancer development, proliferation, epithelial-mesenchymal transition (EMT), invasion, and migration through regulating the expression of oncogenes and tumour suppressor genes. Previous studies have indicated that miR-200c acts as a tumour suppressor in various cancers by downregulating high-mobility group box 1 (HMGB1) and thereby suppressing EMT and metastasis. In addition, miR-200c was reported to be downregulated and correlated with poor outcomes in non-small cell lung cancer (NSCLC). However, its functional role in HMGB1 regulation in NSCLC is still unclear. This study aimed to clarify whether miR-200c acts as a tumour suppressor in NSCLC by downregulating HMGB1, which is associated with EMT, invasion, cytoskeleton rearrangement, and migration in vitro and in vivo. In order to demonstrate HMGB1 downregulation by miR-200c, the NSCLC cell line A549 was transfected with miR-200c mimic or inhibitor. The mimic significantly reduced HMGB1 expression and suppressed EMT, invasion, and migration, while the inhibitor generated the opposite effects. Additionally, using xenograft mouse models, we confirmed that HMGB1 overexpression increased tumour EMT. In summary, our results demonstrated that miR-200c could suppress EMT, invasion, and migration of NSCLC cells by downregulating HMGB1.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The expression of HMGB1 and epithelial-mesenchymal transition (EMT)-associated proteins in NSCLC patients.
Representative photomicrographs are shown that demonstrate increased HMGB1, β-catenin, vimentin, and α-SMA expression in specimens of patients with non—small cell lung cancer (NSCLC). Lung samples (tumor and corresponding normal adjacent lung tissues) were collected and subjected to immunohistochemical staining with antibodies against HMGB1, β-catenin, vimentin, and α-SMA (3,3ʹ-diaminobenzidine staining and hematoxylin counterstaining). For negative controls, the antibody was replaced by control IgG. Scale bar = 100 μm.
Fig 2
Fig 2. Overexpression or silencing of HMGB1 in lung cancer cells.
Overexpression or silencing of HMGB1 expression in A459 NSCLC cells by cloning (LV-HMGB1) or siRNA transfection, respectively, was examined by real-time polymerase chain reaction (PCR) (A and B), western blotting (C and D), and immunocytochemical staining (E). *p <0.05 as compared to the control group. α-Tubulin was used as a loading control. Scale bar = 50 μm.
Fig 3
Fig 3. HMGB1 induces the expression of EMT-associated proteins in A549 cells.
Overexpression or silencing of HMGB1 in A549 cells regulates α-SMA, vimentin, and β-catenin mRNA and protein expression as determined by real-time PCR (A), western blotting (B), and immunocytochemical staining (C). *p <0.05 as compared to the control group. α-Tubulin was used as a loading control. Scale bar = 50 μm.
Fig 4
Fig 4. Effects of HMGB1 on migration, invasion, and cytoskeletal rearrangement in lung cancer cells.
Overexpression or silencing of HMGB1 was performed in A459 cells for 48h and then the metastatic and invasive abilities of the cells were measured by migration assay (A and B) and invasion assay (C), respectively. Cytoskeletal actin reorganization and cell adhesion dynamics were measured by immunocytochemical staining (D) and field emission scanning electron microscopy (E)*p<0.05 as compared to the control group. α-Tubulin was used as a loading control.
Fig 5
Fig 5. miR-200c regulates HMGB1 expression in lung cancer cells.
Alignment of the sequence of miR-200c with that of the 3′untranslated region of HMGB1 showing the putative binding sites (A). Real-time PCR analysis of miR-200c expression in A459 cells after transfection with a miR-200c mimic or inhibitor (B). After transfection, HMGB1 expression was analysed by real-time PCR (C), western blotting (D), and immunocytochemical staining (E). *p <0.05 as compared to the control group. α-Tubulin was used as a loading control.
Fig 6
Fig 6. miR-200c suppresses the expression of EMT-associated proteins in A549 cells.
A miR-200c mimic or inhibitor was applied to A459 cells for 24h and then the mRNA and protein expression levels of α-SMA, vimentin, and β-catenin were measured by real-time PCR (A), western blotting (B), and immunocytochemical staining (C). *p <0.05 as compared to the control group. α-Tubulin was used as a loading control.
Fig 7
Fig 7. miR-200c inhibits lung cancer cell invasion and metastasis.
A miR-200c mimic or inhibitor was applied to A459 cells for 24h and then their metastatic and invasive abilities were measured by migration assay (A and B) and invasion assay (C), respectively. Cytoskeletal actin reorganization and cell adhesion dynamics was measured by immunocytochemical staining (D) and field emission scanning electron microscopy (E). *p <0.05 as compared to the control group.
Fig 8
Fig 8. Overexpression of HMGB1 in NSCLC xenografts model.
A549 cells and LV-HMGB1 cells were inoculated into the right flank of severe combined immunodeficiency mice. Tumor volume was measured every 3 days with slide calipers starting from day 7, and a growth curve was plotted (A and B). *p <0.05 as compared to the A549 group. Normal/tumour tissue transfected with or without LV-HMGB1were collected and the expression of miR-200c was measured by real-time PCR (C and D). *p <0.05 as compared to the normal (NR) group.
Fig 9
Fig 9. Overexpression of HMGB1 increases the EMT of NSCLC xenografts in vivo.
The mRNA and protein expression levels of HMGB1 and EMT markers were measured by real-time PCR (A and B), western blotting (C and D), and immunohistochemistry staining (E). *p <0.05 as compared to the A549 group.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Osugi J, Muto S, Matsumura Y, Higuchi M, Suzuki H, Gotoh M. Prognostic impact of the high-sensitivity modified Glasgow prognostic score in patients with resectable non-small cell lung cancer. Journal of Cancer Research and Therapeutics. 2016;12(2):945 doi: 10.4103/0973-1482.176168 - DOI - PubMed
    1. Bianchi ME, Agresti A. HMG proteins: dynamic players in gene regulation and differentiation. Current opinion in genetics & development. 2005;15(5):496–506. - PubMed
    1. Salgado J, Villalaín J, Gómez-Fernández JC. Effects of platelet-activating factor and related lipids on dielaidoylphosphatidylethanolamine by DSC, FTIR and NMR. Biochimica et Biophysica Acta (BBA)-Biomembranes. 1993;1145(2):284–92. - PubMed
    1. Xu Y-F, Ge F-J, Han B, Yang X-Q, Su H, Zhao A-C, et al. High-mobility group box 1 expression and lymph node metastasis in intrahepatic cholangiocarcinoma. World J Gastroenterol. 2015;21(11):3256–65. - PMC - PubMed
    1. Wang X, Xiang L, Li H, Chen P, Feng Y, Zhang J, et al. The role of HMGB1 signaling pathway in the development and progression of hepatocellular carcinoma: a review. International journal of molecular sciences. 2015;16(9):22527–40. doi: 10.3390/ijms160922527 - DOI - PMC - PubMed

MeSH terms

Grants and funding

This study was supported in part by grants from the Taiwan Ministry of Science and Technology (MOST 104-2314-B-037-087, MOST 104-2314-B-037-071-MY2, MOST 101-2314-B-037-065-MY2, MOST 102-2314-B-037-067, and MOST 103-2314-B-037-051), the Chi Mei Medical Center, Liouying (CLFHR10423), and the Chi-Mei Medical Center and Kaohsiung Medical University Research Foundation (103CM-KMU-08). The animal study was supported by grants from the Core Service Platform Project for Animal Pharmacology, National Research Program, Ministry of Science and Technology, Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

LinkOut - more resources