Hypoxia-inducible MicroRNA-224 Promotes the Cell Growth, Migration and Invasion by Directly Targeting RASSF8 in Gastric Cancer

Background: Hypoxia plays an important role in the development of various cancers. MicroRNAs (miRNAs) act as post-transcriptional regulators of gene expression and modulate the tumorigenesis, including gastric cancer. However, the roles and molecular mechanism of miR-224 in gastric cancer under hypoxia remain poorly understood.

Method: Real-time PCR and Northern blot assay were used to examine the effects of hypoxia and HIF-1α on miR-224 expression. Luciferase and ChIP assays were performed to determine whether miR-224 was a transcriptional target of HIF-1α. Then MTT, colony formation, in vitro scratch and invasion assays were used to detect the effects of miR-224 on cell growth, migration and invasion under hypoxia, as well as the in vivo animal study. Luciferase assay and Western blot were performed to validate the targets of miR-224. Functional studies were performed to determine the roles of RASSF8 as that of miR-224 under hypoxia. The effects of RASSF8 knockdown on the transcriptional activity and translocation of NF-κB were investigated using Luciferase assay and Western blot, respectively. Finally, the expression levels of miR-224 and RASSF8 were detected using real-time PCR in gastric cancer tissues as well as lymph node metastasis tissues.

Results: We demonstrated that miR-224 was upregulated by hypoxia and HIF-1α. HIF-1α affected miR-224 expression at the transcriptional level. MiR-224 inhibition suppressed cell growth, migration and invasion induced by hypoxia, while miR-224 overexpression resulted in opposite effects. MiR-224 inhibition also suppressed tumor growth in vivo. We then validated that RASSF8 was a direct target of miR-224. RASSF8 overexpression inhibited cell growth and invasion, while RASSF8 knockdown ameliorated the inhibitory effects of miR-224 inhibition on cell growth and invasion. Furthermore, we found that RASSF8 knockdown enhanced the transcriptional activity of NF-κB and p65 translocation, while RASSF8 overexpression resulted in opposite effects. Inhibition of NF-κB activity by PDTC attenuated the effects of RASSF8 knockdown on cell proliferation and invasion. Finally, miR-224 was upregulated in both gastric cancer tissues and lymph node metastasis positive tissues, while RASSF8 expression was opposite to that of miR-224.

Conclusion: These results indicate that hypoxia-inducible miR-224 promotes gastric cancer cell growth, migration and invasion by downregulating RASSF8 and acts as an oncogene, implying that inhibition of miR-224 may have potential as a therapeutic target for patients with hypoxic gastric tumors.