ABSTRACT
To investigate the molecular mechanism of resveratrol inhibiting the metastasis of liver cancer . HepG2 and Huh7 cells were treated with different concentrations of resveratrol, and the cell viability was determined by CCK-8 assay to determine the optimal concentration of resveratrol for subsequent experiments. The expressions of miR-186-5p in liver cancer tissues and liver cancer cells were determined by quantitative real-time RT-PCR. The migration and invasion of HepG2 and Huh7 cells were detected by wound healing assay and Transwell assay, and the expression levels of epithelial-mesenchymal transition (EMT) related proteins were determined by Western blotting. Resveratrol with concentration of had no effect on the viability of HepG2 and Huh7 cells, so the concentration of resveratrol in subsequent experiments was 6.25 μmol/L. Resveratrol inhibited the wound healing and invasion of liver cancer cells; increased the expression of E-cadherin, and decreased the expression of vimentin and Twist1. The expression of miR-186-5p was significantly down-regulated in liver cancer tissues and cells compared with the adjacent tissues and normal liver cells (both <0.05). Furthermore, resveratrol induced the expression of miR-186-5p in liver cancer cells (both <0.01). Overexpression of miR-186-5p suppressed the migration, invasion and EMT of liver cancer cells. Knockdown of miR-186-5p blocked the inhibition effects of resveratrol on the migration, invasion and EMT of liver cancer cells. Resveratrol could inhibit the metastasis of liver cancer , which might be associated with up-regulating miR-186-5p.
Subject(s)
Humans , Cell Line, Tumor , Cell Movement , Cell Proliferation , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Hep G2 Cells , Liver Neoplasms/genetics , MicroRNAs/genetics , Neoplasm Invasiveness/genetics , Resveratrol/pharmacologyABSTRACT
AIM: To investigate the expression of miR-186-5p in colon cancer and the effect on colon cancer, and provide a new therapeutic target for the treatment and prognosis of colon cancer. METHODS: Serum from eligible patients were collected and divided into five groups (n=20) including the healthy group and clinical stages I, II, III and IV. The miRNAs in patients' serum were extracted by miRVana microRNA isolation kit. The differences of miR-186-5p expression in patients' serum of colon cancer at different stages were detected with real-time quantitative PCR. The full-length gene of miR-186-5p was cloned into pENTR/D-Topo vector to construct cell lines with high expression of miRNA. 5×103 cells were seeded in 96-well plates. The expression of mir-186-5p was decreased by using miR-186-5p inhibitor. The proliferation of cells was detected with cell proliferation ELISA kit after 24 hours of incubation with BrdU and absorbance was measured at 370 and 492 nm. The effect of miR-186-5p on the sensitivity of colon cancer cell lines to chemotherapeutic drugs was detected by MTT assay. RESULTS: The expression of miR-186-5p was down-regulated in the serum of patients with colon cancer compared to normal group. Expression of miR-186-5p in serum of patients with stage I, II, III and IV was significantly decreased (P<0.01) compared with the normal group. Colon cells were transfected with a plasmid integrating the miR-186-5p gene, and miR-186-5p was highly expressed in the HCT116 and SW480 cell lines (P<0.01). The expression of mir-186-5p was decreased by mir-186-5p inhibitor (hsa-miR-186-5p inhibitor) in HCT116 and SW480 cells. The proliferation rate of HCT116 and SW480 cells with high expression of miR-186-5p was lower than control group (P<0.01). After treating with mir-186-5p inhibitor, the cell proliferation rate was higher than that of the control group (P<0.01). After treatment with different concentrations cisplatin for 24 hours, the mortality of HCT116 and SW480 cells with high expression of mir-186-5p was higher than that of the control group (P<0.01). The mortality of the cells with low expression of mir-186-5p was lower than that of the control group (P<0.01). The protein level of PLK1 was decreased after transfecting with miR-186-5p plasmid and increased after stimulating with miR-186-5p inhibitor in HCT116 and SW480 cells. CONCLUSION: The expression of miR-186-5p is decreased in colon cancer patients. Overexpression of miR-186-5p can inhibit the proliferation of colon cancer cells and reduce the drug resistance of colon cancer cells; inhibition of miR-186-5p expression can increase the proliferation and drug resistance of colon cancer cells.
ABSTRACT
PURPOSE: Long intergenic non-protein coding RNA 665 (LINC00665) plays a vital role in the development of cancer. Its function in hepatocellular carcinoma (HCC), however, remains largely unknown. MATERIALS AND METHODS: The expressions of LINC00665, miR-186-5p, and MAP4K3 were determined by qRT-PCR. Cell viability and apoptosis were evaluated by MTT and flow cytometry, respectively. Autophagic puncta formation was observed by fluorescence microscopy. Bioinformatics analysis, luciferase reporter assay, RNA immunoprecipitation, and RNA pulldown were performed to identify associations among LINC00665, miR-186-5p, and MAP4K3. Western blot was utilized to examine the expressions of MAP4K3, Beclin-1, and LC3. Tumor growth was evaluated in a xenograft model. RESULTS: Elevations in LINC00665 were observed in HCC tissues and cells. The overall survival of HCC patients with high levels of LINC00665 was shorter than those with low levels. In vitro, LINC00665 depletion inhibited viability and induced apoptosis and autophagy. miR-186-5p interacted with LINC00665 and was downregulated in HCC tissues and cells. Upregulation of miR-186-5p inhibited viability and induced apoptosis and autophagy, which were attenuated by upregulation of LINC00665. MAP4K3 was found to possess binding sites with miR-186-5p and was upregulated in HCC tissues and cells. MAP4K3 depletion inhibited viability and induced apoptosis and autophagy, which were attenuated by miR-186-5p inhibitor. In vivo, miR-186-5p expression was negatively correlated with LINC00665 or MAP4K3 in HCC tissues, while LINC00665 was positively correlated with MAP4K3. LINC00665 knockdown suppressed tumor growth. CONCLUSION: LINC00665 was involved in cell viability, apoptosis, and autophagy in HCC via miR-186-5p/MAP4K3 axis, which may provide a new approach for HCC treatment.