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MicroRNA-128-3p Enhances the Chemosensitivity of Temozolomide in Glioblastoma by Targeting c-Met and EMT

Zhao, Chengbin, Guo, Ruiming, Guan, Fangxia, Ma, Shanshan, Li, Mu, Wu, Junru, Liu, Xianzhi, Li, Hongwei, Yang, Bo·2020·Scientific Reports
DOI 10.1038/s41598-020-65331-3PMC PMC7289811
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Abstract

Temozolomide is a first line anti-tumor drug used for the treatment of patients with Glioblastoma multiforme (GBM). However, the drug resistance to temozolomide limits its clinical application. Therefore, novel strategies to overcome chemoresistance are desperately needed for improved treatment of human GBM. Recent studies have demonstrated that miRNAs are closely related to resistance to cancer chemotherapy. This study aimed to further validate the biological role of miR-128-3p and to investigate whether miR-128-3p can enhance the chemosensitivity of glioblastoma to temozolomide (TMZ) and the underlying mechanisms. The effects of miR-128-3p and TMZ on the proliferation of glioblastoma cells were investigated by cell counting kit-8 (cck8). Transwell and intracerebral invasion assays were applied to determine the effects of the combination of miR-128-3p and TMZ on the invasion and migration of glioblastoma in vitro and in vivo. Flow cytometry was used to detect apoptosis in each group, and immunofluorescence was used to determine the expression levels of EMT-related proteins. RT-PCR and Western-blot were applied to detect EMT-transformed proteins (c-Met, PDGFRα, Notch1, and Slug) and EMT phenotype-associated proteins (Vim, CD44, and E-cadherin) at both mRNA and protein levels. Based on the microRNA.org database, we predicted the target genes of miR-128-3p. The target-relationship between miR-128-3p and c-Met and PDGFRα was verified by dual luciferase reporter gene. The tumor volume, weight and the expression levels of the proteins described above were measured in subcutaneously transplanted tumor model in nude mice. We found that the expression of miR-128-3p was down-regulated in glioblastoma tissue samples and cell lines. miR-128-3p suppressed the proliferation, migration, and invasion of GBM both in vitro and in vivo; miR-128-3p enhanced the therapeutic effect of TMZ via inhibition of proliferation, invasion and migration of glioblastoma cells and induction of apoptosis. Overexpression of miR-128-3p down-regulated the expression levels of EMT-transformed proteins (c-Met, PDGFRα, Notch1 and Slug) to enhance the effect of TMZ. In addition, we found that miR-128-3p targeted and bound c-Met. More importantly, the upregulation of c‐Met significantly prompted U87 and U251 cell proliferation. This effect could be abolished when c‐Met was silenced. The investigation in tumor bearing nude mice showed that miR-128-3p in combination with TMZ reduced tumor volume and the invasion extent, and increased the sensitivity of glioblastoma to TMZ. miR-128-3p is capable of enhancing the sensitivity of glioblastoma to TMZ through regulating c-Met/EMT.

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Figures

Figure 1 Expression of miR-128-3p in glioma cell lines and tissues and in vitro and in vivo functions. (A) TCGA analysis of the expression of miR-128-3p and prognosis (n = 620, p = 0.0026); (B) RT-PCR detection of expression of Hsa-miR-128-3p gene in glioma cell lines U251, SHG44, A172, U87, LN229 and HA1800. U6 were taken as an internal reference gene, compared with HA1800 (C) The relative expression level of hsa-miR-128-3p in glioma tissues (tumor) and their matched adjacent normal tissues was examined by RT-PCR. The date were presented as fold change. normalized by U6, n = 24. Compared with the control group, * means P < 0.05, *** means P < 0.001.
Figure 1 Expression of miR-128-3p in glioma cell lines and tissues and in vitro and in vivo functions. (A) TCGA analysis of the expression of miR-128-3p and prognosis (n = 620, p = 0.0026); (B) RT-PCR detection of expression of Hsa-miR-128-3p gene in glioma cell lines U251, SHG44, A172, U87, LN229 and HA1800. U6 were taken as an internal reference gene, compared with HA1800 (C) The relative expression level of hsa-miR-128-3p in glioma tissues (tumor) and their matched adjacent normal tissues was examined by RT-PCR. The date were presented as fold change. normalized by U6, n = 24. Compared with the control group, * means P < 0.05, *** means P < 0.001.CC BY · PubMed Central
Figure 2 miR-128-3p suppressed the proliferation, migration and invasion of GBM in vitro and in vivo. (A) RT-PCR was used to verify the expression of miR-128-3p in stably transfected U87 and U251 cells; (B) cck-8 detects cell viability; (C,D) Transwell detects invasion, migration and statistical analysis, (E,F) wound healing experiments and statistical analysis, (G) RT-PCR was used to verify the expression of miR-128-3p gene, U6 was taken as an internal reference gene; (H) tumor-bearing results in nude mice, (I) tumor growth curve analysis; and (J) statistical analysis of tumor weight. Each sample was repeated in triplicate. Data are expressed as mean ± standard error. Compared with the LV-miR-NC group, * means P < 0.05, ** means P < 0.01, and *** means P < 0.001, # means P < 0.05.
Figure 2 miR-128-3p suppressed the proliferation, migration and invasion of GBM in vitro and in vivo. (A) RT-PCR was used to verify the expression of miR-128-3p in stably transfected U87 and U251 cells; (B) cck-8 detects cell viability; (C,D) Transwell detects invasion, migration and statistical analysis, (E,F) wound healing experiments and statistical analysis, (G) RT-PCR was used to verify the expression of miR-128-3p gene, U6 was taken as an internal reference gene; (H) tumor-bearing results in nude mice, (I) tumor growth curve analysis; and (J) statistical analysis of tumor weight. Each sample was repeated in triplicate. Data are expressed as mean ± standard error. Compared with the LV-miR-NC group, * means P < 0.05, ** means P < 0.01, and *** means P < 0.001, # means P < 0.05.CC BY · PubMed Central
Figure 3 miR-128-3p increases the effect of TMZ in vitro. TMZ: Temozolomide (A) cck-8 for cell viability, (B,C) Transwell for cell migration, invasion, and statistical analysis (D,E) wound healing assay for migration and statistical analysis, (F,G) Detection of apoptosis and statistical analysis with flow cytometry. Each sample was repeated in triplicate. Data are expressed as mean ± standard error. Compared with the LV-miR-NC group, * means P < 0.05, ** means P < 0.01, and *** means P < 0.001.
Figure 3 miR-128-3p increases the effect of TMZ in vitro. TMZ: Temozolomide (A) cck-8 for cell viability, (B,C) Transwell for cell migration, invasion, and statistical analysis (D,E) wound healing assay for migration and statistical analysis, (F,G) Detection of apoptosis and statistical analysis with flow cytometry. Each sample was repeated in triplicate. Data are expressed as mean ± standard error. Compared with the LV-miR-NC group, * means P < 0.05, ** means P < 0.01, and *** means P < 0.001.CC BY · PubMed Central
Figure 4 miR-128-3p increases the effect of TMZ in vivo. (A) RT-PCR to verify miR-128-3p gene expression, U6 as an internal reference gene; (B) Photographs of the dissected tumors. (C) tumor growth curve analysis; (D) tumor weight statistical analysis; (E) U251 brain invasion test, (1) Stereotactic injection of mixed U251 cells (LV-miR-NC group (DIL-red) + LV-miR-128-3p group (DIO-green)) (2) intracerebral injection sites; (3) and (5) same sagittal plane fluorescence imaging, (4) and (6) same cross-sectional fluorescence imaging, The red circle represents the intracerebral invasion range of the LV-miR-NC group, and the green circle represents the intracerebral invasion range of the LV-miR-128-3p group, (7) and (8) Sagittal and cross-sectional merge imaging; (F) statistical analysis of intracerebral invasion experiments, each sample was repeated three times. Data are expressed as mean ± standard error. Compared with the TMZ + LV-miR-NC group, * means P < 0.05, ** means P < 0.01, and *** means P < 0.001.
Figure 4 miR-128-3p increases the effect of TMZ in vivo. (A) RT-PCR to verify miR-128-3p gene expression, U6 as an internal reference gene; (B) Photographs of the dissected tumors. (C) tumor growth curve analysis; (D) tumor weight statistical analysis; (E) U251 brain invasion test, (1) Stereotactic injection of mixed U251 cells (LV-miR-NC group (DIL-red) + LV-miR-128-3p group (DIO-green)) (2) intracerebral injection sites; (3) and (5) same sagittal plane fluorescence imaging, (4) and (6) same cross-sectional fluorescence imaging, The red circle represents the intracerebral invasion range of the LV-miR-NC group, and the green circle represents the intracerebral invasion range of the LV-miR-128-3p group, (7) and (8) Sagittal and cross-sectional merge imaging; (F) statistical analysis of intracerebral invasion experiments, each sample was repeated three times. Data are expressed as mean ± standard error. Compared with the TMZ + LV-miR-NC group, * means P < 0.05, ** means P < 0.01, and *** means P < 0.001.CC BY · PubMed Central
Figure 5 miR-128-3p increases TMZ chemosensitivity by inhibiting EMT and related pathways. (A) immunofluorescence analysis of α-Tubulin and F-actin expression; (B) immunofluorescence analysis of EMT-related protein VIM and E-cadherin expression; (C) immunofluorescence detection of subcutaneous tumor-bearing model EMT-related protein in nude mice VIM and E-cadherin expression; (D) Western-blot detection of EMT-related protein VIM, CD44, E-cadherin expression, GAPDH as an internal reference; (E) RT-PCR detection of EMT-related genes VIM, CD44, E-cadherin expression; (F) Western-blot detection of the expression of PDGFRα, Notch1 and Slug protein, GAPDH was taken as the internal reference, (G) Western-blot statistical analysis; (H) RT-PCR detection of the mRNA expression levels of PDGFRα, Notch1 and Slug genes. Each sample was repeated in triplicate. The data is expressed as mean ± standard error. Compared with the miR-NC group, * means P < 0.05, ** means P < 0.01, and *** means P < 0.001.
Figure 5 miR-128-3p increases TMZ chemosensitivity by inhibiting EMT and related pathways. (A) immunofluorescence analysis of α-Tubulin and F-actin expression; (B) immunofluorescence analysis of EMT-related protein VIM and E-cadherin expression; (C) immunofluorescence detection of subcutaneous tumor-bearing model EMT-related protein in nude mice VIM and E-cadherin expression; (D) Western-blot detection of EMT-related protein VIM, CD44, E-cadherin expression, GAPDH as an internal reference; (E) RT-PCR detection of EMT-related genes VIM, CD44, E-cadherin expression; (F) Western-blot detection of the expression of PDGFRα, Notch1 and Slug protein, GAPDH was taken as the internal reference, (G) Western-blot statistical analysis; (H) RT-PCR detection of the mRNA expression levels of PDGFRα, Notch1 and Slug genes. Each sample was repeated in triplicate. The data is expressed as mean ± standard error. Compared with the miR-NC group, * means P < 0.05, ** means P < 0.01, and *** means P < 0.001.CC BY · PubMed Central
Figure 6 miR-128-3p directly targets c-Met. (A) MET and PDGFRα 3′UTR and Has-miR-128-3p targeted binding site; (B) c-Met dual luciferase statistical analysis; (C) Statistical analysis of PDGFRα dual luciferase; (D) Analysis of the relationship between miR-128-3p and c-Met and PDGFRα expression in TCGA database; (E) Western-blot detection of the expression of c-Met protein; (F) Western-blot statistical analysis; (G) RT-PCR was used to detect the expression level of c-Met mRNA (H,I) cck-8 for cell viability. Data are expressed as mean ± standard error. Compared with the miR-NC group, * means P < 0.05, ** means P < 0.01, and *** means P < 0.001.
Figure 6 miR-128-3p directly targets c-Met. (A) MET and PDGFRα 3′UTR and Has-miR-128-3p targeted binding site; (B) c-Met dual luciferase statistical analysis; (C) Statistical analysis of PDGFRα dual luciferase; (D) Analysis of the relationship between miR-128-3p and c-Met and PDGFRα expression in TCGA database; (E) Western-blot detection of the expression of c-Met protein; (F) Western-blot statistical analysis; (G) RT-PCR was used to detect the expression level of c-Met mRNA (H,I) cck-8 for cell viability. Data are expressed as mean ± standard error. Compared with the miR-NC group, * means P < 0.05, ** means P < 0.01, and *** means P < 0.001.CC BY · PubMed Central

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Metadata

Authors
Zhao, C., Guo, R., Guan, F., Ma, S., Li, M., Wu, J., Liu, X., Li, H., & Yang, B.
ORCID
Guan, Fangxia
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2020-06-11
Volume
10
Issue
1
ISSN
2045-2322
DOI
10.1038/s41598-020-65331-3
PMC
PMC7289811
Cited by
51
References
2
Language
en
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