BackgroundHypoxia 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.MethodReal-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.ResultsWe 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.ConclusionThese 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.
Osteosarcoma (OS) is the most commonly diagnosed malignant cancer of bone that occurs in adolescents and children. Mounting number of studies have indicated that miRNAs are increasingly playing fundamental roles in OS development. Thus, the biological function of miR-429 in OS progression was explored. The results of RT-qPCR revealed that miR-429 was downregulated in OS tissues and OS cell lines (MG-63, U2OS, Saos-2) while homeobox A9 (HOXA9) was markedly increased. Moreover, HOXA9 was confirmed as a direct target of miR-429 by using luciferase reporter assay. It was identified that miR-429 exhibited a suppressive effect on OS progression while HOXA9 showed the oncogenic function in OS progression by using MTT and Transwell assays. More importantly, rescue assays manifested that HOXA9 can partially overturn the suppressive effect of miR-429 on OS. Overexpression of miR-429 inhibited the activation of Wnt/β-catenin signaling pathway. In conclusion, miR-429 suppressed OS progression by targeting HOXA9 through Wnt/β-catenin pathway.
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