He-Chan Pian inhibits the metastasis of non-small cell lung cancer via the miR-205-5p-mediated regulation of the GREM1/Rap1 signaling pathway

Background: He-Chan Pian (HCP), a traditional Chinese medicinal formula, shows promising efficacy for the treatment of lung Cancer.

Purpose: Gremlin (GREM1) plays an important role in gastrointestinal tumor metastasis; however, little is known about its role in lung Cancer. We determined the mechanism underlying the protective effect of HCP against metastasis in a mouse model of non-small cell lung Cancer (NSCLC) and demonstrated the role of GREM1.

Methods: Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) was used to analyze the herbal components and metabolites from the serum of HCP-treated mice. The tumor, liver, and kidney were examined histologically, and the antitumor effects and toxicity of HCP were evaluated. Levels of epithelial-mesenchymal transition (EMT)-associated transcription factors were measured using western blotting in tumors from five groups (i.e., model, HCP [L], HCP [M], HCP [H], and positive control [cisplatin, DDP]). Differentially expressed proteins and genes were identified using protein chip and Sequencing analyzes, respectively. Short hairpin RNAs and overexpression plasmids were introduced into cells to evaluate the effects of GREM1. To evaluate proliferation, migration, and invasion, the expression levels of proteins involved in the Rap1 pathway and EMT were measured in vitro. Xenograft tumors with overexpression-GREM1 (OE-GREM1) in A549 cells were examined for cell proliferation. A dual-luciferase assay was performed to verify the direct interaction of GREM1 with miR-205-5p in lung Cancer.

Results: Thirty-six ingredients and bioactive constituents detected in the serum of HCP-treated mice were identified as the key compounds involved in the inhibition of tumor growth. Animal experiments revealed that HCP significantly decreased tumor volumes and had no adverse effects on the liver or kidney or side effects. GREM1 upregulation was closely related to tumor metastasis and was regulated by miR-205-5p, as confirmed using a dual-luciferase reporter assay. OE-GREM1 promoted A549 cell migration and invasion, promoted EMT, and increased the expression of Rap1 pathway intermediaries, whereas shGREM1 had the opposite effects. Furthermore, the effects of OE-GREM1 on proliferation in the A549 xenograft mouse model were attenuated, although HCP has an inhibitory effect on tumors.

Conclusion: Our results suggest that HCP contributes to the inhibition of NSCLC metastasis via the Gremlin/Rap1 signaling pathway regulated by miR-205-5p.

Gremlin/Rap1; He-Chan Pian, NSCLC; Metastasis; miR-205-5p.