Human Bone Marrow Mesenchymal Stem Cell (hBMSCs)-Derived miR-29a-3p-Containing Exosomes Impede Laryngocarcinoma Cell Malignant Phenotypes by Inhibiting PTEN

Abstract

Although microRNA-29a-3p was reported to inhibit laryngocarcinoma progression, the potential mechanisms have not been explored clearly. Laryngocarcinoma tissues were collected for analyzing the levels of miR-29a-3p and phosphatase and tensin homolog (PTEN). The miR mimics or inhibitor was transfected into laryngocarcinoma cell lines M4E and Hep2 for the investigation of the biological functions (proliferative, invasion, migratory rates, and apoptotic rates) of this miRNA. The exosomes (Exo) from human bone marrow mesenchymal stem cells (hBMSCs) after the transfection of miR mimics/inhibitor/si-PTEN were isolated and used to stimulate M4E and Hep2 cells. The in vivo mouse model was constructed to verify our findings. The miR-29a-3p level was decreased, and PTEN level was elevated in laryngocarcinoma tissues and the cancer cell lines. MiR mimics could inhibit proliferative, invasive migratory rates while promoting apoptotic rates of M4E and Hep2 cells. MiR-29a-3p was enriched in hBMSC-derived Exo, and the Exo from miR-29a-3p mimics transfected hBMSCs could inhibit laryngocarcinoma cell malignant phenotypes in vitro and prevent tumor progression in vivo. In addition, the direct binding relationship between miR-29a-3p and PTEN in this disease was determined. In conclusion, hBMSC-derived Exo with upregulated miR-29a-3p inhibited laryngocarcinoma progression via regulating PTEN, providing a potential diagnostic and therapeutic target in this disease.

Figure 1

The relationship between miR-29a-3p and PTEN in laryngocarcinoma. (a–b) QRT-PCR analysis of miR-29a-3p in laryngocarcinoma tissues (a) and cancer cell lines (b). (c–d) QRT-PCR analysis of PTEN in laryngocarcinoma tissues (c) and cancer cell lines (d). (e) Correlation analysis between miR-29a-3p and PTEN level in laryngocarcinoma tissues. (f) Base sequence of miR-29a-3p and the wild-type as well as the mutant PTEN 3′UTR by Targetscan. (g) Luciferase activity detection after transfection in M4E cells. (h) The enrichment analysis of miR-29a-3p in M4E cells using biotin labelled PTEN probe. (i) QRT-PCR analysis of PTEN in M4E cells after transfected with miR mimics. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 2

MiR-29a-3p upregulation reduced laryngocarcinoma cell malignant progression. M4E and Hep2 cells were transfected with miR-29a-3p mimics or miR-NC. (a) QRT-PCR analysis of miR-29a-3p. (b) CCK-8 assay. (c) Colony formation assay. (d) Transwell assay for detecting cell invasive and migratory rates. (e) Flow cytometry analysis of apoptotic rate. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 3

Identification of hBMSC-derived exosomes. (a) The morphology of hBMSCs by inverted microscope. Magnification × 400. (b) The ultrastructure of hBMSC-derived Exo by a TEM (scale bar = 200 nm). (c) Western blot analysis of Exo surface markers. (d) Flow cytometry analysis of hBMSCs' surface markers.

Figure 4

HBMSC-derived Exo reduced laryngocarcinoma cell malignant progression. M4E and Hep2 cells were treated with hBMSC-derived Exo for 48 h. (a) CCK-8 assay. (b) Colony formation assay. (c) Transwell assay for detecting cell invasive and migratory rates. (d) Flow cytometry analysis of apoptotic rate. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 5

HBMSC-derived Exo with upregulated miR-29a-3p inhibited laryngocarcinoma cell malignant progression. (a) QRT-PCR analysis of miR-29a-3p in hBMSC-derived Exo, M4E, and Hep2 cells. (b) QRT-PCR analysis of miR-29a-3p in hBMSC-derived Exo after the transfection of miR mimics and miR-NC. (c–f) The Exo derived from miR mimics or miR-NC-transfected hBMSCs were used to stimulate M4E and Hep2 cells for 48 h. (c) CCK-8 assay. (d) Colony formation assay. (e) Transwell assay for detecting cell invasive and migratory rates. (f) Flow cytometry analysis of apoptotic rate. (g) Western blot analysis of PTEN in two cell types. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 6

The impacts of Exo miR-29a-3p inhibitor and Exo si-PTEN in laryngocarcinoma cell progression. (a–b) MiR-29a-3p inhibitor and si-PTEN were transfected into hBMSCs, and the Exo was isolated. QRT-PCR analysis of the levels of miR-29a-3p (a) and PTEN (b). (c–f) The Exo derived from inhibitor NC, miR inhibitor, si-PTEN, or miR inhibitor + si-PTEN transfected hBMSCs were used to stimulate M4E and Hep2 cells for 48 h. (c) CCK-8 assay. (d) Colony formation assay. (e) Transwell assay for detecting cell invasive and migratory rates. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 7

The impacts of Exo miR mimics and Exo inhibitor in tumor progression in vivo. (a) Representative images of tumors. (b) Tumor volume. (c) Tumor weight. (d) IHC assay using anti-Ki67 antibody in tumor tissues. Scale bar = 100 μm. (e) Western blot analysis of PTEN in tumor tissues. ^∗p < 0.05 and ^∗∗p < 0.01.