miRNA-221 of exosomes originating from bone marrow mesenchymal stem cells promotes oncogenic activity in gastric cancer

Figures & data

Table 1 Sequences of synthesized miR-221 oligonucleotides

Oligonucleotides	Sequences (5′ to 3′)
miR-221 mimics	AGC UAC AUU GUC UGC UGG GUU UC
miR-221 inhibitor	GAA ACC CAG CAG ACA AUG UAG CU
Negative control	UUC UCC GAA CGU GUC ACG UdTdT

Abbreviation: miR-221, microRNA-221.

Figure 1 The expression of miR-221 in exosomes of the peripheral blood is closely associated with the prognosis of GC.

Notes: (A) The expression of the biomarkers CD9 and CD63, as examined by Western blotting, indicated the successful extraction of exosomes from the peripheral blood. β-actin was used as the internal reference. (B) From January 2016 to April 2016, peripheral blood samples were obtained from 40 GC patients and 20 normal controls who were recruited for this research. miR-221 expression in exosomes of the peripheral blood of GC patients, as assessed by qRT-PCR, was generally higher than that of the normal controls. U6 was used as the internal control.
Abbreviations: miR-221, microRNA-221; GC, gastric cancer; qRT-PCR, quantitative real-time polymerase chain reaction.

Table 2 Correlation of GC clinicopathologic features and miR-221 expression of exosomes in peripheral blood (n=40)

Parameters	Cases	Relative level to U6	P-value
Sex			0.80
Male	22	2.46 (0.60–4.20)
Female	18	2.48 (1.10–5.30)
Age			0.47
<60	19	2.22 (1.00–4.20)
≥60	21	2.69 (0.60–5.30)
CEA grading			0.46
Normal	18	2.47 (0.60–4.20)
High	22	2.46 (1.10–5.30)
CA19-9 grading			0.73
Normal	31	2.41 (0.60–5.30)
High	9	2.67 (1.10–4.20)
CRP grading			0.49
Normal	25	2.37 (0.60–5.30)
High	15	2.62 (1.50–4.20)
Tumor location			0.25
Gastric fundus and cardiac	7	2.32 (1.50–4.20)
Gastric body	11	2.26 (1.10–4.10)
Gastric antrum	22	2.39 (0.60–5.30)
Tumor size			0.59
<4.0 cm	20	2.57 (0.60–5.30)
≥4.0 cm	20	2.37 (1.00–4.20)
Lauren type			0.94
Intestinal type	19	2.44 (1.10–4.20)
Diffuse type	9	2.41 (1.50–4.10)
Mixed type	12	2.55 (0.60–5.30)
Borrmann type			0.82
I	3	2.63 (1.50–4.10)
II	17	2.35 (0.60–5.30)
III	20	2.54 (1.00–4.20)
Presence of vascular and nerve invasion			0.16
No	16	2.14 (1.10–3.40)
Yes	24	2.68 (0.60–5.30)
Histological grade			0.48
Well differentiated	2	3.25 (3.20–3.30)
Moderately differentiated	6	2.33 (1.00–4.20)
Poorly differentiated	28	2.37 (0.60–4.20)
Undifferentiated	4	2.93 (1.50–5.30)
T staging			0.83
T1	5	2.20 (1.50–2.60)
T2	11	2.36 (0.60–5.30)
T3	21	2.60 (1.10–4.20)
T4	3	2.30 (2.20–2.40)
N staging			0.32
N0	8	2.01 (0.60–2.60)
N1	8	2.34 (1.10–3.30)
N2	13	2.50 (1.00–4.20)
N3	11	2.85 (1.50–5.30)
M staging			0.15
M0	38	2.37 (0.60–5.30)
M1	2	4.20 (4.20, 4.20)
TNM stage			0.02
I	5	1. 82 (0.60–2.60)
II	15	2.33 (1.00–4.20)
III	18	2.56 (1.20–5.30)
IV	2	4.20 (4.20, 4.20)

Abbreviations: TNM, tumor, node, and metastasis; miR-221, microRNA-221; GC, gastric cancer; CRP, C-reactive protein; CEA, carcinoembryonic antigen.

Figure 2 Characterization of exosomes that originated from BM-MSCs.

Notes: (A) Protein expression of CD9 and CD63 according to Western blotting indicated the successful extraction of exosomes from BM-MSCs. β-actin was used as the internal reference. (B) The particle size (nm) of exosomes from BM-MSCs after transfection with miR-221 mimics was assayed by nanoparticle tracking analysis. (C) After transfection with miR-221 mimics or inhibitor for 48 hours, the miR-221 expression level was assessed by qRT-PCR and was correspondingly changed in BM-MSCs. U6 was used as the internal control. The difference was considered statistically significant at ***(P<0.001).
Abbreviations: BM-MSCs, bone marrow mesenchymal stem cells; miR-221, microRNA-221; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 3 miR-221 in exosomes that originated from BM-MSCs promotes GC cell proliferation.

Notes: (A) miR-221 expression in BGC-823 and SGC-7901 cells, which was determined by qRT-PCR, was increased in the mimics group but reduced in the inhibitor group. U6 was used as the internal reference. (B) The corresponding expression of PTEN and p27, as examined by Western blotting in GC cells cultured with exosomes, suggested the feasibility of exosome transfection. (C) Viability of BGC-823 and SGC-7901 cells, as determined by MTT, was significantly enhanced in the miR-221 mimics group compared with the miR-221 inhibitor group. The difference was considered statistically significant at ***(P<0.001).
Abbreviations: miR-221, microRNA-221; BM-MSCs, bone marrow mesenchymal stem cells; GC, gastric cancer; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 4 miR-221 in exosomes that originated from BM-MSCs promotes GC cell migration, invasion, and adhesion to the matrix.

Notes: (A–D) GC cell migration ability, which was evaluated by wound-healing and Transwell assays, was enhanced after culture with exosomes transfected with miR-221 mimics, while cell migration was restrained in cells cultured with exosomes that were transfected with miR-221 inhibitor. Histograms show the OD value of the BGC-823 and SGC-7901 cells that transmigrated in the Transwell assays and that were stained with crystal violet. (E) GC cell invasion ability, which was evaluated by Transwell assays, was upregulated after culture with exosomes transfected with miR-221 mimics, while invasion ability was downregulated in cells cultured with exosomes transfected with miR-221 inhibitor. Histograms show the OD value of the invasive BGC-823 and SGC-7901 cells that were stained with crystal violet. (F) Adhesion to the matrix of GC cells, as tested by adhesion assays, was significantly higher in the cells cultured with exosomes transfected with miR-221 mimics than in the cells cultured with exosomes transfected with the miR-221 inhibitor. Histograms show the OD value of adherent BGC-823 and SGC-7901 cells, as shown by MTT assay. The difference was considered statistically significant at *(P<0.05), **(P<0.01), and ***(P<0.001).
Abbreviations: miR-221, microRNA-221; BM-MSCs, bone marrow mesenchymal stem cells; GC, gastric cancer; OD, optical density.