Ranunculi Ternati Radix regulates miR-125/STAT3 signaling to reprogram tumor microenvironment and inhibit melanoma growth

Background: Melanoma is a malignancy with a high mortality rate. STAT3, a transcription factor that plays an important role in melanoma cell proliferation and immune evasion, is a well-established target for treating melanoma. Ranunculi Ternati Radix (RTR), the dried root of Ranunculus ternatus Thunb., is commonly prescribed to treat cancer in China. However, pharmacological effects of RTR on melanoma, and mechanisms of its actions are unclear. Some constituents of RTR, such as ursolic acid, have anti-melanoma effects, and have been shown to
inhibit STAT3 signaling.

Purposes: To investigate the anti-melanoma effects of the ethyl acetate fraction of the 70% ethanol extract of RTR (EA-RTR) and explore the involvement of STAT3 signaling in the effects.

Methods: Human melanoma cells A375 and A2058 were used to examined the effects of EA-RTR on cell viability. A B16 melanoma allograft mouse model was used to evaluate the effects of EA-RTR on tumor growth and tumor microenvironment. Immune cells and immunosuppressive factors in the tumors were detected using a flow cytometry-based assay and ELISA assay, respectively. The mRNA levels of STAT3 and STAT3-targeted genes were detected by RT-qPCR, and their protein levels were examined by immunoblotting. A375STAT3C (overexpressing a constitutive activation variant of STAT3) and A375NC (harbouring the empty vector) stable cell lines were used to determine the role of STAT3 in the anti-melanoma effects of EA-RTR. A microRNA array analysis of tumor tissues from the B16 melanoma-bearing mice was performed to investigate the mechanisms of action of EA-RTR on STAT3 signaling.

Results: EA-RTR dose-dependently reduced cell viabilities in A375 and A2058 cells and inhibited tumor growth in B16 melanoma-bearing mice without obvious toxicity. EA-RTR increased dendritic (DC) cells and decreased the production of STAT3-regulated immunosuppressive factors MIP-1α, TNF-α, and IFN-γ in the B16 tumors. Mechanistic studies demonstrated that EA-RTR lowered the protein and mRNA levels of STAT3 and STAT3-targeted genes MCL-1 and BCL-XL that are responsible for maintaining melanoma cell survival. Overactivation of STAT3 in melanoma cells significantly diminished the effects of EA-RTR in reducing cell viability, suggesting that inhibiting STAT3 contributes to the anti-melanoma effects of EA-RTR. Results of microRNA array analysis revealed that the levels of miR-125 family members, miR-125a, miR-125b and miR-351, were upregulated by EA-RTR in the B16 tumor tissues. miR-125 family microRNAs have been reported to act as tumor suppressors by targeting STAT3.

Conclusion: Our data demonstrate that EA-RTR inhibits melanoma growth and reprograms melanoma microenvironment; and upregulating miR-125 family microRNAs to inhibit STAT3 signaling is associated with the effects of EA-RTR. This study lays the groundwork for developing EA-RTR as an effective and safe modality for treating melanoma.