Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jan 27:13:773-785.
doi: 10.2147/CMAR.S281969. eCollection 2021.

Inhibition of Bone Morphogenetic Protein 2 Suppresses the Stemness Maintenance of Cancer Stem Cells in Hepatocellular Carcinoma via the MAPK/ERK Pathway

Juncheng Guo  1 , Min Guo  1 , Jinfang Zheng  1
Affiliations

Inhibition of Bone Morphogenetic Protein 2 Suppresses the Stemness Maintenance of Cancer Stem Cells in Hepatocellular Carcinoma via the MAPK/ERK Pathway

Juncheng Guo et al. Cancer Manag Res. .

Abstract

Background: Hepatocellular carcinoma (HCC) remains a life-threatening malignant tumor. Cancer stem cells (CSCs) harbor tumor-initiating capacity and can be used as a therapeutic target for human malignancies. Bone morphogenetic proteins (BMPs) play a regulatory role in CSCs. This study investigated the role and mechanism of BMP2 in CSCs in HCC.

Methods: BMP2 expression in HCC tissues and cells, and CSCs from HepG2 cells and SMMC7721 cells (HepG2-CSCs and SMMC7721-CSCs) was measured. The association between BMP2 expression and prognosis of HCC patients was analyzed. CSCs were interfered with BMP2 to evaluate the abilities of colony and tumor sphere formation, levels of stemness-related markers, epithelial-mesenchymal transition (EMT), and invasion and migration. Levels of MAPK/ERK pathway-related proteins in HepG2-CSCs were detected after BMP2 knockdown. The effect of the activated MAPK/ERK pathway on HepG2-CSCs was assessed. Finally, the effect of BMP2 inhibition on CSCs in HCC was verified in vivo.

Results: BMP2 showed obvious upregulation in HCC tissues and cells and was further upregulated in CSCs in HCC, with its higher expression indicative of worse prognosis. Silencing BMP2 inhibited colony and tumor sphere formation, levels of stemness-related markers, as well as EMT, invasion and migration of HepG2-CSCs and SMMC7721-CSCs. The MAPK/ERK pathway was suppressed after BMP2 knockdown, and its activation reversed the inhibitory effect of shBMP2 on hepatic CSCs. BMP2 accelerated tumor growth and EMT of CSCs in HCC in vivo.

Conclusion: We concluded that BMP2 knockdown inhibited the EMT, proliferation and invasion of CSCs in HCC, thereby hindering the stemness maintenance via suppressing the MAPK/ERK pathway.

Keywords: MAPK/ERK pathway; bone morphogenetic protein 2; cancer stem cells; epithelial-mesenchymal transition; hepatocellular carcinoma; stemness.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflicts of interest for this work.

Figures

Figure 1
Figure 1
BMP2 is highly expressed in HCC tissues and cells. (A) The expression of BMP2 in HCC and normal liver tissues was analyzed using online visualization data from Starbase (http://starbase.sysu.edu.cn/panCancer.php) in TCGA; (B) The relationship between the expression of BMP2 in Starbase and the prognosis of HCC patients; (C) qRT-PCR was used to detect the mRNA expression of BMP2 in HCC cells and normal liver cells; (D) WB was used to detect the protein level of BMP2 in five HCC cells and normal liver cells. The cell experiment was repeated three times, and the data were expressed as mean ± standard deviation. Data in panels (C and D) were analyzed using one-way ANOVA and Tukey’s multiple comparisons test. *p < 0.05.
Figure 2
Figure 2
BMP2 expression is further increased in CSCs in HCC. (A) HepG2-CSCs and SMMC7721-CSCs were isolated and identified using flow cytometry; (B) The sphere-forming rate of HepG2 cells, SMMC7721 cells, HepG2-CSCs and SMMC7721-CSCs was detected using sphere formation assay; (C) The colony-forming ability of HepG2 cells, SMMC7721 cells, HepG2-CSCs and SMMC7721-CSCs was detected using colony formation assay; (D) The mRNA expression of CSC markers (CD44 and ALDH1) and pluripotent transcription factors (Bmil, SOX2 and OCT4) was detected using qRT-PCR; (E) CD44 protein level was detected using WB; (F) The mRNA expression of BMP2 in HepG2 cells, SMMC7721 cells, HepG2-CSCs and SMMC7721-CSCs was detected using qRT-PCR; (G) The protein level of BMP2 in HepG2 cells, SMMC7721 cells, HepG2-CSCs and SMMC7721-CSCswas detected using WB. The experiment was repeated three times, and the data were expressed as mean ± standard deviation. Data in panels (A and C) were analyzed using one-way ANOVA, and data in panels (E-G) were analyzed using two-way ANOVA, followed by Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.01.
Figure 3
Figure 3
BMP2 knockdown inhibits the stemness of CSCs in HCC. (A) The relative mRNA expression of BMP2 was detected using qRT-PCR; (B) The relative protein level of BMP2 was detected using WB; (C) The influence of inhibition of BMP2 on the colony-forming ability of HepG2-CSCs and SMMC7721-CSCs was detected using colony formation assay; (D) The effect of inhibition of BMP2 on the sphere-forming ability of HepG2-CSCs and SMMC7721-CSCs was detected using sphere formation assay; (E) The proliferation ability of transfected cells was measured using CCK-8 assay; (F) qRT-PCR was used to detect the expression of Nanog, SOX2, OCT4, CD13, CD44 and EpCAM in HepG2-CSCs and SMMC7721-CSCs after BMP2 knockdown. The cell experiment was repeated three times, and the data were expressed as mean ± standard deviation. Data in panels (A-D) were analyzed using independent t-test, and data in panel (E and F) were analyzed using two-way ANOVA, followed by Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.01.
Figure 4
Figure 4
BMP2 knockdown inhibits the EMT and the invasion and migration of CSCs in HCC. (A) WB was used to detect the levels of E-cadherin, Vimentin, N-cadherin and Snail in CSCs in HCC; (B) Transwell assay was used to detect the effect of BMP2 silencing on the migration and invasion ability of CSCs in HCC. The cell experiment was repeated three times, and the data were expressed as mean ± standard deviation. Data in the panel (B) were analyzed using independent t-test, and data in the panel (A) were analyzed using two-way ANOVA, followed by Tukey’s multiple comparisons test. * p < 0.05.
Figure 5
Figure 5
BMP2 knockdown inhibits the proliferation, migration and invasion of CSCs in HCC via inhibiting the MAPK/ERK pathway. (A) WB was used to detect the levels of p-ERK1/2, ERK1/2, MEK1/2 and p-MEK1/2; (B) The effect of MAPK/ERK pathway inhibitor on the colony-forming ability of liver CSCs was detected using colony formation assay; (C) The effect of MAPK/ERK pathway inhibitor on the sphere-forming rate of liver CSCs was detected using sphere formation assay; (D) The effect of the MAPK/ERK pathway on the colony-forming ability of liver CSCs was detected using the colony formation assay; (C) The effect of the MAPK/ERK pathway on the sphere-forming ability of hepatic CSCs was detected using sphere formation assay. The cell experiment was repeated three times, and the data were expressed as mean ± standard deviation. Data in panels (D and E) were analyzed using independent t-test, data in panels (B and C) were analyzed using one-way ANOVA and data in the panel (A) were analyzed using two-way ANOVA, followed by Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.01.
Figure 6
Figure 6
BMP2 knockdown inhibits tumor growth and the EMT of CSCs in HCC in nude mice. (A) Tumor volume of nude mice; (B) Tumor xenograft image and tumor weight; (C) qRT-PCR was used to detect the mRNA expression of CSC markers CD44 and ALDH1; (D) WB was used to detect the levels of E-cadherin, Vimentin, N-cadherin and Snail. The data were expressed as mean ± standard deviation. Data in the panel (B) were analyzed using independent t-test, and data in panels (A, C, D) were analyzed using two-way ANOVA, followed by Tukey’s multiple comparisons test. *p < 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kim DW, Talati C, Kim R. Hepatocellular carcinoma (HCC): beyond sorafenib-chemotherapy. J Gastrointest Oncol. 2017;8(2):256–265. doi:10.21037/jgo.2016.09.07 - DOI - PMC - PubMed
    1. Mathew S, Ali A, Abdel-Hafiz H, et al. Biomarkers for virus-induced hepatocellular carcinoma (HCC). Infect Genet Evol. 2014;26:327–339. doi:10.1016/j.meegid.2014.06.014 - DOI - PubMed
    1. Wang EA, Stein JP, Bellavia RJ, Broadwell SR. Treatment options for unresectable HCC with a focus on SIRT with Yttrium-90 resin microspheres. Int J Clin Pract. 2017;71(11):e12972. doi:10.1111/ijcp.12972 - DOI - PubMed
    1. Giannelli G, Koudelkova P, Dituri F, Mikulits W. Role of epithelial to mesenchymal transition in hepatocellular carcinoma. J Hepatol. 2016;65(4):798–808. doi:10.1016/j.jhep.2016.05.007 - DOI - PubMed
    1. Eun K, Ham SW, Kim H. Cancer stem cell heterogeneity: origin and new perspectives on CSC targeting. BMB Rep. 2017;50(3):117–125. doi:10.5483/bmbrep.2017.50.3.222 - DOI - PMC - PubMed

Grants and funding

This work was supported by the Key Research and Development Plan of Hainan Province (2019RC373) and Supported by Hainan Natural Science Foundation (818MS161).

LinkOut - more resources