Adipo-differentiation of A-MSCs from GC Patients Is inhibited and Negatively Correlated with miR-155
We initially analysed GC patients survival through a tumor database (http://kmplot.com) revealed that high miR-155 expression level is a poor prognostic factor in GC patients (Figure 1A). Then, the level of miR-155 in serum of GC patients and healthy donors was measured. A high level of miR-155 expression was present in serum of GC patients (Figure 1B). Then we also detected the capacity of adipo-differentiation in A-MSC isolated from peritoneum adipose tissue of GC patients (GC group), and A-MSC from subcutaneous adipose tissue of healthy donors (HD group) as normol control. Investigated by Oil Red O staining, the accumulation of lipid droplets in A-MSCs isolating from peritoneum of GC patients was decreased (Figure 1C). As a crucial transcription factor of early adipo-differentiation stage, C/EBPβ was significantly decreased in A-MSCs isolating from GC patients (Figure 1D-E). We performed RT-PCR to detect the expression level of miR-155 and the results indicated that miR-155 in A-MSC was obviously increased in the GC group compared to HD group (Figure 1F). In conclusion, the results above suggested that miR-155 can promote GC progression and negatively correlated with adipogenesis capacity of A-MSCs.
Identification And Characterization Of A-mscs And Gc Exosomes
The morphology of A-MSCs present like fibroblasts (Fig. 2A) and the lipid drops formation was observed after culturing in adipogenic differentiation medium (Fig. 2B-C). A-MSCs were positive for CD44, CD73, CD90, CD105, CD166 and HLA-ABC, but negative for CD31, CD133, CD14, CD34, CD45 and HLA-DR (Fig. 1D). In our preliminary experiment, TEM showed that SGC7901-exosomes exhibited a round-shaped morphology (Fig. 1E) with a size ranging from 50 to 100 nm (Fig. 1G). In addition, Tgs101 and CD63 were highly expressed by SGC7901 exosomes and MGC803 exosomes (Fig. 1F). Exosomes stained with PKH26 were added into the medium of A-MSCs, the phagocytosis was recorded at 2 h, 4 h and 6 h after co-culture through confocal microscopy. It showed that the uptaken of SGC7901 exosomes increases with the time of co-culture and over 80% of the A-MSCs exhibited PKH67 staining although the intensity of the red fluorescence of the positive MSCs (Fig. 1H). Taking together, our results proved that A-MSCs and exosomes with high purity were successfully isolated, as well as SGC7901-exosomes can be taken up by A-MSCs.
Gc Cell Derived Exosomes Inhibited Adipo-differentiation Of A-mscs
Since SGC7901 exosomes and MGC803-exosomes could be taken up by A-MSCs, then we determined whether the adipogenic differentiation of A-MSCs was changed by GC exosomes. A-MSCs were treated with different doses of SGC7901 exosomes and MGC803 exosomes (50ug/ml and 70ug/ml) in adipogenic induction medium for 5 days. GC-exosomes treatment significantly decreased lipid droplets in A-MSCs as the concentration exosomes increased, compared with GES-1-exosomes (50ug/ml) treatment groups (Fig. 3A). GC exosomes treatment, both SGC7901 exosomes and MGC803 exosomes, also decreased mRNA expression levels of adipogenic transcription factors C/EBPα, and PPARγ, while GC-exosomes had no effect on the expression of C/EBPβ mRNA (Fig. 3B, D). Moreover, the protain expression of C/EBPβ, C/EBPα, and PPARγ in A-MSCs cultured in adipogenic induction medium were inhibited by GC-exosomes (Fig. 3C, E). The results above suggested that GC exosomes acted as a negative regulator in adipo-differentiation of A-MSCs.
Gc-exosomal Mir-155 Tageting C/ebpβ Expression In A-mscs
The miR-155 binding sites in the 3’UTR of C/EBPβ mRNA are shown in Fig. 4A. Transfection of miR-155 mimics markedly reduced luciferase activity, whereas luciferase levels were relatively enhanced by miR-155 inhibitors. However, the inhibitory activity was lost when the binding sites were mutated (Fig. 4B). The data indicated miR-155 suppressed C/EBPβ expression by binding to C/EBPβ 3’UTR in adipocytes. Additionally, to further explore the influence of miR-155 on the adipogenic differentiation of A-MSCs, normal control mimics, miR-155 mimics, normal control inhibitors, and miR-155 inhibitors were directly transfected into A-MSCs. As shown in Figs. 4C and 4D, RT-PCR was used to analyze the level of miR-155 in above groups. Cultured in adipogenic differentiation medium for 5 days later, western blotting was used to assess the expression level of C/EBPβ. The expression of C/EBPβ in A-MSCs were much lower in miR-155 mimics group than that in normal control mimics group, while the expression of C/EBPβ was significantly enhanced in A-MSCs transfected with miR-155 inhibitors compared with normal control inhibitors. However there was no significant change in C/EBPβ mRNA (Fig. 4E-G). Moreover, we also found that miR-155 was highly expressed in exosomes derived from SGC7901 cells and MGC803 cells (Fig. S-A). To verify the function of miR-155 in GC-exosomes, SGC7901 cells were relatively transfected with miR-155 inhibitors or normal control inhibitors, then isolating exosomes of each group (SGC exosomes In.miR-155 or SGC exosomes In.NC) and co-cultured with A-MSCs (Fig. 4H). miR-155 level in SGC7901 cells exosomes and A-MSCs treated with SGC exosomes In.miR-155 or SGC exosomes In.NC (100 µg/ml) for 8 h were measured by RT-PCR, and that the level of miR-155 was markedly decreased in SGC exosomes In.miR-155 (Fig. 4I) and A-MSCs treated with that (Fig. 4J). Under adipo-differentiational condition for 5 days, the expression of C/EBPβ, C/EPBα and PPARγ in A-MSCs treated with SGC exosomes In.miR-155 (100 µg/ml) were much higher than those in SGC exosomes In.NC group (Fig. 4L,M). But no significant change of C/EBPβ mRNA in A-MSCs was obvioused in those two groups (Fig. 4K). These results suggested that C/EBPβ was a downstream target gene of miR-155, the upregulation of GC exosomal miR-155 expression may be responsible for the decreased expression level of C/EBPβ, as well as the downstream protein C/EPBα and PPARγ in A-MSCs.
GC Exosomal miR-155 Contributes to The Lose of Adipose Mass in Vivo
To identify whether GC Exosomal miR-155 suppressed adipo-differentiation of A-MSCs and promoted cancer cachexia in vivo, BALB/c mice were subcutaneously injected with SGC7901 cells transfected with lentivirus containing a miR-155 overexpressing (miR-155 OE) sequence or miR-155 shRNA (miR-155 KD) or control lentivirus(NC). Then PBS was used as a negative control (Fig. 5A). 4 weeks later, all of the tumors, plasma and inguinal adipose tissues were harvested. The sizes of tumors and weight of mice were measured during the experiment. Compared with control group, the diameter of tumors was remarkably enhanced in the miR-155-OE group, while in the control group, the growth of tumors was visibly inhibited (Fig. 5B). Similarly, weight of mice was obviously reduced in miR-155 OE group, while which was increased in the miR-155 KO group (Fig. 5C). The tumors obtained were photographed and analyzed (Fig. 5D-E). Images of inguinal adipose tissues from the four groups are shown in Fig. 5F, and the lengths and weights of these adipose tissues in miR-155 OE group were decreased. As expected, the miR-155 KO rescued that trend (Fig. 5G-H). Additionally we detected the expression of fatty acid binding protein-4 (FABP4) in inguinal adipose tissues. A down-regulation of FABP4 was detected in tumor-implanted groups, which shown a negative correlation with miR-155 level (Fig. 5I). The data above demonstrated that GC-exosomes carried miR-155 can be delivered into adipose tissue and lead to decrease of adipose mass which is caused by suppressing C/EBPβ.
GC exosomal miR-155 suppresses A-MSCs adipo-differentiation via targeting C/EBPβ/C/EBPα/PPARγ in vivo
To further investigated the biological role of miR-155 in CAC, initially we detected lipid-formed capacity in mA-MSCs after culturing in adipogenic differentiation medium (Fig. 6A), miR-155 in mA-MSCs and serum esosomes were distinctly upregulated in the miR-155 OE group (Fig. 6B, Figure S-B). The expression of C/EBPβ, C/EBPα and PPARγ in mA-MSCs of each group indicated a negtive relationship with miR-155 level of adipose tissue (Fig. 6C, D). We further investigated the expression of C/EBPβ, C/EBPα and PPARγ in mA-MSCs adipo-differentiated with adipogenic medium for 5 days. The results a significant decrease of C/EBPβ, C/EBPα, PPARγ expression due to implanted-tumor, especially in miR-155 OE group, but this decrease was rescued when miR-155 was knocked down in implanted-tumor (Fig. 6E). These findings indicated that GC-exosomes carried miR-155 acted as a promotive role on CAC via suppressing of C/EBPβ in A-MSCs..