Hypoxia-inducible microRNA-224 promotes the cell growth, migration and invasion by directly targeting RASSF8 in gastric cancer

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.

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.

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.

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.