M2 macrophage-derived exosomes promote migration and invasion of AsPC-1 cells.
To explore the role of macrophages in PC metastasis, the function of macrophages was first verified by an in vitro cell assay. AsPC-1 cells were co-cultured with M0 macrophages and induced M2 macrophages, respectively, and a transwell assay was conducted. Based on the results, M2 macrophages enhanced the migration and invasion of AsPC-1 cells compared with M0 macrophages obviously (Figs. 1A and B). When the exosome release inhibitor GW4869 is added to M2 macrophages, the ability to promote the migration of AsPC-1 cells can be reversed (Figs. 1C and D). These results suggested that the invasion and migration of AsPC-1 cells may be mediated by exosomes secreted by M2 macrophages.
Identification of M2 macrophage-derived exosomes and their role in promoting AsPC-1 migration.
Exosomes were isolated from macrophage culture supernatant by ultracentrifugation. Under transmission electron microscopy revealed a large number of exosomes of fixed shape and size in macrophage culture supernatants with a typical bilayer structure and an average diameter of about 100 nm (Figs. 2A and B). In exosome extracts, marker proteins TSG101, CD63, and CD81 were significantly increased by western blot analysis (Figs. 2C and D). The above results indicate that the extraction from macrophage exosomes was successful. Moreover, laser confocal microscopy revealed that AsPC-1 cells can take up exosomes produced by M2 macrophages when exosomes are labeled and cultured with AsPC-1 cells (Fig. 2E). To further verify the direct effects of macrophage exosomes derived from M0 and M2 macrophages on PC metastasis, exosomes derived from both macrophages were added to the AsPC-1 culture medium. After co-culture, we found that M2 macrophage-derived exosomes enhanced the migratory capacity of AsPC-1 cells (Figs. 2F and G). Finally, the protein expression of the epithelial cell marker E-cadherin was significantly downregulated, while the mesenchymal cell marker vimentin was significantly upregulated, suggesting that M2 macrophage-derived exosomes promote EMT in AsPC-1 cells (Fig. 2H).
Exosomal lncRNA sequencing and Myt1l discovery.
After high-throughput sequencing of lncRNA samples from M0 and M2 macrophage-derived exosomes, we discovered NONMMUG008725.2 (LncRNA Myt1l) expression was significantly increased. This suggests the M2 macrophage-derived exosomes are promoting AsPC-1 metastasis by an undiscovered molecular mechanism (Fig. 3A and B). Myt1l was predominantly localized in the cytoplasm as determined by fluorescence in situ hybridization (Fig. 3C).
Myt1l targets MiR-135.
In recent years, evidence has accumulated that lncRNAs modulate miRNA expression by acting as ceRNAs. To further investigate the role of Myt1l in AsPC-1 metastasis, we labeled Myt1l with biotin, and its corresponding pattern is shown in Fig. 4A. qRT-PCR analysis of miR-95, miR-92, miR-135, miR-1-3p, and miR-25-3p enrichment levels was performed to determine which of the target miRNAs was highly enriched with Myt1l in AsPC-1 cells. We found that among the five potential miRNA targets, Myt1l pulled down higher levels of miR-135 (Fig. 4B). Therefore, miR-135 was shown to may be a target gene for Myt1l. According to bioinformatics prediction, the specific binding site of Myt1l and miR-135 is shown in Fig. 4C. The wild-type (WT) and mutant (Mut) sequences of Myt1l were transfected into the luciferase vector for further confirmation that Myt1l binds to miR-135. Based on the results, the activity of the luciferase in the Myt1l-WT group decreased when miR-135 mimic was applied, and accordingly, when miR-135 is inhibited, the Myt1l-WT group exhibits an increase in luciferase activity. However, in the Myt1l-MUT group (which had a mutated binding site between Myt1l-WT and miR-135), the luciferase signal did not change significantly regardless of overexpression or inhibition of miR-135. This indicated that miR-135 can interact with Myt1l and bind to miR-135 through the sites in the figure to play a targeting role. In addition, compared to the M0-exo group, the M2-exo group expressed significantly more Myt1l. To further observe the interaction between Myt1l and miR-135, M2-exo-lncRNA Myt1l OE and M2-exo-lncRNA Myt1l KD groups were set up separately by regulating the expression profile of Myt1l (Fig. 4D). After that, we detected miR-135 levels in M0-exo, M2-exo, M2-exo-lncRNA Myt1l OE, and M2-exo-lncRNA Myt1l KD groups. In contrast to M0-exo group, a significant reduction in miR-135 expression was found in the M2-exo group. In M2 macrophage exosomes, miR-135 expression was significantly decreased following overexpression of Myt1l, while miR-135 expression was strongly increased following knockdown of Myt1l (Fig. 4E). Based on these results, Myt1l reduces the expression of miR-135 in M2-derived exosomes by sponging miR-135.
MiR-135 targets Slug in AsPC-1 cells.
AsPC-1 cells metastasis is regulated by miR-135, but the molecular mechanisms behind this are unknown. We performed bioinformatics analysis using Targetscan bioinformatics and found that the Slug-3′UTR region is a conservative miR-135 binding site (Fig. 5A). Based on this binding site, toward elucidating Slug and miR-155 target binding behavior, we constructed wild-type (Slug-WT) and mutant (Slug-MUT) reporter vectors. As shown in Fig. 5A, a significant amount of miR-135 overexpression inhibits the luciferase activity of the Slug-WT group, whereas miR-135 inhibition produced the opposite result. However, regardless of overexpression or inhibition of miR-135, luciferase activity in the Slug-Mut group showed no significant change, indicating that miR-135 and Slug can target binding, the results were as expected. Furthermore, compared to M0-exo, M2-exo showed significantly higher levels of Slug protein expression (Fig. 5B). Exosomes of M2 macrophages expressed significantly more Slug when Myt1l was overexpressed whereas it was reduced significantly when Myt1l was inhibited, suggesting overexpression of Myt1l could significantly promote Slug expression (Fig. 5C). Next, we measured E-cadherin and vimentin levels in the groups M0-exo, M2-exo, M2-exo-lncRNA Myt1l OE, and M2-exo-lncRNA Myt1l KD. According to the results, Myt1l overexpression significantly lowered the expression of E-cadherin, whereas vimentin was significantly upregulated. When the expression of Myt1l was inhibited, the opposite effect was observed (Fig. 5D). This suggests that the overexpression of Myt1l in M2-exosomes can accelerate the EMT in AsPC-1 cells. This conclusion was reinforced by cell migration experiments (Fig. 5E). M2 macrophage exosomes co-incubated with AsPC-1 cells significantly promoted their migration when Myt1l was overexpressed, whereas Myt1l inhibition negatively influenced AsPC-1 migration (Fig. 5F).
For further research on the migration of AsPC-1 cells in response to Myt1l and miR-135, our study examined the effects of lncRNA Myt1l OE and lncRNA Myt1l OE + miR-135 OE on E-cadherin and vimentin levels as well as the number of cells migrating into AsPC-1 cells using western blots (Fig. 5G—I). According to the study, Myt1l overexpression increased vimentin expression in AsPC-1 cells and promoted their migration, but E-cadherin expression was inhibited. In contrast, overexpression of miR-135 can antagonize these processes. Accordingly, vimentin protein levels and migration of AsPC-1 cells significantly decreased when Myt1l expression was inhibited, whereas E-cadherin protein levels increased. When knocking down Myt1l and inhibiting miR-135, the protein expression of E-cadherin, vimentin, and the migration of AsPC-1 cells could be restored to the control level. The above indicated that miR-135 negatively regulates the metastasis of AsPC-1 cells and weakens the function of Myt1l. In our study, we detected that Slug positively correlated with the Myt1l/miR-135 axis, which promotes AsPC-1 cells' metastasis. Based on these results, we identified that Slug promotes the metastasis of AsPC-1 cells by targeting the Myt1l/miR-135 axis.
M2 macrophage-derived exosomal Myt1l promotes AsPC-1 cell metastasis to the lung.
To further demonstrate that Myt1l promotes metastasis of AsPC-1 cells, we used a mouse model for validation. Firstly, 4—6 weeks old female mice were injected with control, M2-exo, M2-exo-lncRNA Myt1l OE and M2-exo-lncRNA Myt1l KD through the tail vein, followed four weeks later by a shot of AsPC-1 cells via the same vein. The fluorescence expression of the different groups was observed by an in vivo fluorescence imaging system every 2 weeks thereafter. A significant increase in fluorescence intensity in mouse lungs when Myt1l was overexpressed was observed compared to the M2-exo group, however, a significant decrease was observed when Myt1l expression was inhibited, indicating that AsPC-1 cells can be effectively metastasized into the lungs by exosomes derived from M2 macrophages (Fig. 6A). After 8 weeks, the mice were executed and the levels of TNF-α, IL-1β, IL-10, and IL-4 in the lung tissues were assessed by ELISA kits. Compared to the control group, the levels of inflammatory cytokines IL-10 and IL-4 were increased and the levels of TNF-α and IL-1β were decreased in the M2-exo group. When Myt1l overexpression group was compared to M2-exo group, the expression of TNF-α and IL-1β was further suppressed, while the expression of IL-10 and IL-4 was further enhanced. The opposite effect was seen when Myt1l expression was inhibited (Fig. 6B). We then examined the expression of Slug proteins in different groups and found that overexpression of Myt1l significantly promoted and positively correlated with the protein levels of Slug (Fig. 6C). Moreover, each group of lung tissues was examined for expression of E-cadherin and vimentin, which showed that overexpression of Myt1l increased vimentin expression while inhibiting E-cadherin expression, Inhibition of Myt1l had the opposite effect, whereas inhibiting Myt1l expression caused the opposite effect (Fig. 6D). Accordingly, M2 macrophage-derived exosomes containing Myt1l cause EMT and lung metastasis in AsPC-1 cells.