ABSTRACT
Macrophage migration inhibitory factor (MIF), a proinflammatory and immunoregulatory chemokine, plays important roles in cancer-related biological processes. However, few studies have focused on the clinical relevance of MIF and cyclin D1 expression in hepatocellular carcinoma cells (HCCs). In this study, MIF and cyclin D1 expression levels in HCC tissues and cell lines were significantly upregulated compared with adjacent normal tissues or a normal liver cell line. In HCC specimens, MIF expression positively correlated with cyclin D1 expression. Additionally, MIF and cyclin D1 expression positively correlated with tumor size. MIF knockdown inhibited the proliferation of PLC and HepG2 cells and promoted apoptosis. However, small interfering RNA (siRNA) against MIF did not influence the cell cycle in these cells. In an in vivo xenograft model, MIF knockdown reduced the tumor growth rate. The expression levels of Bcl-2, p-caspase-3, BIM and Bax were upregulated, while the expression levels of cyclin D1, p-Akt and p-ERK were downregulated in MIF-knockdown cells. These findings indicate that MIF siRNA reduces proliferation and increases apoptosis in HCC cells. MIF knockdown inhibits the expression of growth-related proteins and induces the expression of apoptosis-related proteins, supporting a role for MIF as a novel therapeutic target for HCC.
Subject(s)
Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/therapy , Cyclin D1/metabolism , Liver Neoplasms/genetics , Liver Neoplasms/therapy , Macrophage Migration-Inhibitory Factors/deficiency , RNA, Small Interfering/genetics , Animals , Apoptosis/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Cycle/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Female , Hep G2 Cells , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Macrophage Migration-Inhibitory Factors/genetics , Male , Mice, Inbred BALB C , Mice, Nude , Middle Aged , RNA, Small Interfering/administration & dosage , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: According to cancer-related microRNA (miRNA) expression microarray research available in public databases, miR-362 expression is elevated in gastric cancer. However, the expression and biological role of miR-362 in gastric progression remain unclear. METHODS: miR-362 expression levels in gastric cancer tissues and cell lines were determined using real-time PCR. The roles of miR-362, in promoting gastric cancer cell proliferation and apoptosis resistance, were assessed by different biological assays, such as colony assay, flow cytometry and TUNEL assay. The effect of miR-362 on NF-κB activation was investigated using the luciferase reporter assay, fluorescent immunostaining. RESULTS: MiR-362 overexpression induced cell proliferation, colony formation, and resistance to cisplatin-induced apoptosis in BGC-823 and SGC-7901 gastric cancer cells. MiR-362 increased NF-κB activity and relative mRNA expression of NF-κB-regulated genes, and induced nuclear translocation of p65. Expression of the tumor suppressor CYLD was inhibited by miR-362 in gastric cancer cells; miR-362 levels were inversely correlated with CYLD expression in gastric cancer tissue. MiR-362 downregulated CYLD expression by binding its 3' untranslated region. NF-κB activation was mechanistically associated with siRNA-mediated downregulation of CYLD. MiR-362 inhibitor reversed all the effects of miR-362. CONCLUSION: The results suggest that miR-362 plays an important role in repressing the tumor suppressor CYLD and present a novel mechanism of miRNA-mediated NF-κB activation in gastric cancer.
Subject(s)
Apoptosis/genetics , MicroRNAs/metabolism , NF-kappa B/metabolism , Signal Transduction/genetics , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , 3' Untranslated Regions/genetics , Base Sequence , Cell Line, Tumor , Cell Proliferation , Deubiquitinating Enzyme CYLD , Down-Regulation/genetics , Gene Expression Regulation, Neoplastic , Humans , MicroRNAs/genetics , Molecular Sequence Data , Tumor Suppressor Proteins/metabolism , Up-Regulation/geneticsABSTRACT
SPHK1 expression is elevated in gastric cancer and is associated with shorter survival times for patients. However, the molecular mechanism of SPHK1 up-regulation in gastric cancer remains unclear. In the present study, we report that miR-124 down-regulated SPHK1 expression by directly targeting its 3'-untranslated region (3'-UTR) and that miR-124 expression was inversely correlated with SPHK1 expression in gastric cancer samples. Furthermore, we demonstrated that, similar to the effect of silencing SPHK1, up-regulation of miR-124 markedly inhibited proliferation and tumourigenicity of gastric cancer cells both in vitro and in vivo. This was found to be mechanistically associated with induction of cyclin-dependent kinase inhibitors p21$^{{\rm Cip1}}$ and p27$^{{\rm Kip1}}$, enhancement of the transcriptional activity of FOXO1 and suppression of AKT activity. Moreover, we showed that the re-introduction of SPHK1 (without the 3'-UTR), but not with the 3'-UTR, could abrogate the miR-124-mediated induction of p21$^{{\rm Cip1}}$ and p27$^{{\rm Kip1}}$, as well as rescue the miR-124-induced proliferation inhibition. Together, these results suggest that miR-124 has an important role in the suppression of gastric cancer and presents a novel mechanism of miRNA-mediated SPHK1 expression in cancer cells.
