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Background: The programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) pathway plays crucial role in various types of cancer. However, the underlying mechanism of PD-L1 expression dysregulation in GC has not been revealed. High expression of PD-L1 and low expression of miR-15a/16 often leads to poor prognosis of gastric cancer, therefore PD-L1 was regarded as a key target for the treatment of gastric cancer. This study aims to determine whether exosomes with miR-15a/16 could target PD-L1 and inhibit immune escape in gastric cancer (GC).
Methods: The protein expression levels of PD-L1 in tumor tissues and normal tissues of patients were evaluated by Western blot (WB) and immunohistochemistry (IHC), then confirmed that PD-L1 was a target of miR-15a/16 by luciferase reporter assay. HEK293T cells were transfected with miR-15a/16, exosomes were isolated by sequential differential centrifugation then cultured with gastric cancer cell lines, the protein and RNA levels of the PD-L1 were determined by western blotting and real-time qPCR. A mouse xenograft model was adopted to assess the association between exosome loaded miR-15a/16 and tumor growth in vivo. Flow cytometry was performed to analysis the effect of exo-miR-15a/16 on CD8+T lymphocytes and INFγ+ of mice.
Results: We found that compared to adjacent noncancerous tissues, PD-L1 protein expression was significantly increased while miR-15a/16 expression level was significantly inhibited in tumor tissues. In addition, we found that miR-15a/16 mimics could downregulate PD-L1 expression, while miR-15a/16 inhibitors enhanced PD-L1 expression in human gastric cancer cell line SGC7901. Exo-miR-15a/16 could target PD-L1 and inhibit immune escape in gastric cancer.
Conclusions: Our study confirmed that miR-15a/16 in exosomes could suppress tumor growth and inhibit immune escape by targeting PD-L1 in vivo and in vitro, which might serve as a potential biomarker in monitoring the activity of gastric cancer.
Keywords
Exosomes；PD-L1 ; miR-15a/16; gastric cancer
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Primary research
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 28 May, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: The programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) pathway plays crucial role in various types of cancer. However, the underlying mechanism of PD-L1 expression dysregulation in GC has not been revealed. High expression of PD-L1 and low expression of miR-15a/16 often leads to poor prognosis of gastric cancer, therefore PD-L1 was regarded as a key target for the treatment of gastric cancer. This study aims to determine whether exosomes with miR-15a/16 could target PD-L1 and inhibit immune escape in gastric cancer (GC).
Methods: The protein expression levels of PD-L1 in tumor tissues and normal tissues of patients were evaluated by Western blot (WB) and immunohistochemistry (IHC), then confirmed that PD-L1 was a target of miR-15a/16 by luciferase reporter assay. HEK293T cells were transfected with miR-15a/16, exosomes were isolated by sequential differential centrifugation then cultured with gastric cancer cell lines, the protein and RNA levels of the PD-L1 were determined by western blotting and real-time qPCR. A mouse xenograft model was adopted to assess the association between exosome loaded miR-15a/16 and tumor growth in vivo. Flow cytometry was performed to analysis the effect of exo-miR-15a/16 on CD8+T lymphocytes and INFγ+ of mice.
Results: We found that compared to adjacent noncancerous tissues, PD-L1 protein expression was significantly increased while miR-15a/16 expression level was significantly inhibited in tumor tissues. In addition, we found that miR-15a/16 mimics could downregulate PD-L1 expression, while miR-15a/16 inhibitors enhanced PD-L1 expression in human gastric cancer cell line SGC7901. Exo-miR-15a/16 could target PD-L1 and inhibit immune escape in gastric cancer.
Conclusions: Our study confirmed that miR-15a/16 in exosomes could suppress tumor growth and inhibit immune escape by targeting PD-L1 in vivo and in vitro, which might serve as a potential biomarker in monitoring the activity of gastric cancer.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
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