Exosomes loaded with miR-665 inhibit the progression of osteosarcoma in vivo and in vitro

Abstract

Objective: Osteosarcoma (OS) is the most common primary malignant bone tumor and has a poor prognosis. Recent research has suggested that miR-665 affects the progression of OS. Moreover, an exosome delivery system presents better targeting effects, higher permeability, and lower immunogenicity than other nano-delivery systems do. The purpose of this study is to explore whether an exosome loaded with the miR-665 delivery system can inhibit OS development.

Methods: The miR-665 expression was detected through a quantitative real-time polymerase chain reaction assay. Transmission electron microscopy, nano-particle size analysis, and fluorescence microscope were utilized to observe exosomes. Cell growth was estimated by cell counting kit 8 and ethynyl deoxyuridine analyses. Assays of flow cytometry and Terminal-deoxynucleotidyl Transferase Mediated Nick End Labeling were introduced to test apoptosis in vitro or in vivo, respectively. Cell migration and invasion were measured using scratch and transwell assays. Engineered exosomes were prepared using electroporation. H&E staining was employed to observe necrotic cells and the function of heart, liver, spleen, lung and kidney. The expression of proteins was estimated by immunoblot analysis.

Results: This work documented that the expression of miR-665 was down-regulated in OS tissues. Additionally, we proved that the over-expression of miR-665 inhibited OS proliferation. Besides, we found that exosomes loaded with miR-665 had similar tumor-inhibiting effects in vivo and in vitro. Furthermore, we verified that the exosome delivery system exhibited good safety and target efficiency.

Conclusion: This work proved that exosomes loaded with miR-665 inhibited the progression of OS in vivo and in vitro in a safe manner.

Keywords: Osteosarcoma; exosomes; miR-665.

Figure 1

MiR-665 expression was down-regulated in the OS tissues and cell lines. A: Integrated OS data from miRCancer, GEO and Genecards database and found 6 OS-associated overlapping microRNAs; B: Q-PCR results showed that the expression of miR-665 was significantly down-regulated in the OS tissues compared to the normal tissues. ***P<0.001 vs. Normal; C: Q-PCR results revealed that the expression level of miR-665 in these osteosarcoma cell lines was significantly lower than that in normal osteoblasts. *P<0.05, ***P<0.001 vs. hFOB; D: Q-PCR results indicated that miR-665 was successfully overexpressed in osteosarcoma cell lines. ***P<0.001 vs. NC mimic.

Figure 2

Tracing of exosomes derived from OS cells. A: q-PCR results showed that when exosomes loaded with miR-665 were extracted, the expression of miR-665 in exosomes increased significantly; B: Transmission electron microscopy (TEM) results showed that the negative-stained exosomes were in the shape of saucers with a diameter of about 100 nm; C: The results of nano-particle size analysis showed that the particle sizes peak of exosomes derived from pericytes were at about 100 nm; D: Immunoblot results showed that exosomes derived from OS cells specifically expressed CD63 and TSG101, while the culture medium after exosome extraction did not express such molecules; E: PKH26 labeled exosomes were injected into experimental mice by tail vein injection, and then the red fluorescent substances in the OS site were observed under a fluorescence microscope as exosomes; B: ***P<0.001 vs. PBS.

Figure 3

MiR-665 inhibited the proliferation of OS cells. A: Expression level of miR-665 in different groups. ***P<0.001 vs. NC mimic; ###P<0.001 vs. unload Exo; B: In the CCK-8 test, cell viability was measured by OD value after MG63 cells were transfected with NC mimic, miR-665 mimic, unloaded exosomes, or exosomes loaded with miR-665 for 24 h, 48 h, and 72 h. **P<0.01 vs. NC mimic; ###P<0.001 vs. unload Exo; C: EdU assay detection showed that miR-665 mimic and exosomes loaded with miR-665 induced more apoptotic cells. **P<0.01 vs. NC mimic; ##P<0.01 vs. unload Exo; D: FACS for cell distribution analysis showed that miR-665 mimic and exosomes loaded with miR-665 induced more apoptotic cells. ***P<0.001 vs. NC mimic; ###P<0.001 vs. unloaded Exo; E: Immunoblot analysis showed that miR-665 mimic and exosomes loaded with miR-665 significantly increased the levels of the pro-apoptotic protein of Bax, cleaved caspase-3 and cleaved caspase-9, but decreased levels of anti-apoptotic protein of Bcl-2. ***P<0.001 vs. NC mimic; ###P<0.001 vs. unload Exo.

Figure 4

MiR-665 inhibited the migration and invasion of OS cells. Scratch (A) and transwell (B) chamber assays showed that the miR-665 mimic and exosomes loaded with miR-665 markedly inhibited migration and invasion of OS cells; (C) Western blot assays revealed that the protein levels of MMP-2 and MMP-9 were decreased by miR-665 in OS cells. *P<0.05, **P<0.01, ***P<0.001 vs. NC mimic; ###P<0.001 vs. unloaded Exo.

Figure 5

The exosomes loaded with miR-665 exerted tumor-inhibition effects on OS in vivo. A: In vivo bioluminescence imaging was performed on the treated mice after four-week treatment to measure the volume of the transplanted tumor. The results showed that the exosomes loaded with miR-665 significantly inhibited the growth of gliomas; B, C: Size and weight analysis of OS showed that exosomes loaded with miR-665 had strong tumor-inhibiting effects on OS; D: H&E staining indicated that the exosomes loaded with miR-665 group induced more necrotic cells than that in the unloaded-Exo group; E: TUNEL assay indicated that miR-665-Exo induced more apoptotic cells. IHC test showed that miR-665-Exo reduced the protein level of PCNA; F: q-PCR detection suggested that miR-665 expression levels in exosomes significantly increased. *P<0.05, **P<0.01, ***P<0.001 vs. Ad-NC mimic; ##P<0.01, ###P<0.001 vs. unloaded Exo.

Figure 6

The evaluation of the safety of exosome treatment. A: Body weight detection showed that there was no significant difference between the unloaded-Exo and miR-665-Exo groups; B: Liver function of the mice in three groups by biochemical blood test showed that there were no significant differences; C: The functional tests of heart, liver, spleen, lung and kidney showed no significant difference among the three groups.