Objective:The relative expression of miR-4698 in liver cancer tissues and cell lines was detected, and its effect on the proliferation and migration of liver cancer cells and its molecular mechanism were analyzed.Methods:Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to analyze the relative expression of miR-4698 in liver cancer tissues and liver cancer cell lines. Among the lowest-expressing hepatocellular carcinoma cell lines, miR-4698 mimic and control mimic were transfected with liposome transfection method, and named as experimental group and control group. qRT-PCR was used to detect transfection efficiency. Cell counting kit-8 (CCK-8) and transwell migration experiments were used to detect the effects of overexpressing miR-4698 on the proliferation and migration ability of liver cancer cells. Bioinformatics and dual luciferase reporter gene experiments were used to predict and verify the binding of miR-4698 to target gene. qRT-PCR and Western blot were used to detect the relative expression of target gene at mRNA and protein levels, respectively.Results:Compared with the adjacent tissues, miR-4698 was significantly lower in the liver cancer ( P<0.01). Compared with normal hepatocytes, miR-4698 was significantly lower in hepatoma cell lines ( P<0.05), and the lowest in Huh7 cells ( P<0.01). After transfection, the expression of miR-4698 in Huh7 cells in experimental group was significantly increased compared with that in the control group ( P<0.01). Overexpression of the miR-4698 can inhibit the proliferation and migration of Huh7 cells ( P<0.05). Bioinformatics showed that the target gene of miR-4698 was polypeptide-n-acetylgalactosamine transferase 4 (GALNT4), and the double luciferase reporter gene confirmed that miR-4698 could bind to GALNT4 ( P<0.01). qRT-PCR showed that overexpression of miR-4698 could inhibit the expression of GALNT4 gene ( P<0.01). Western blot results showed that overexpression of miR-4698 decreased the protein expression of GALNT4, cyclin B and CDK1, and increased the protein expression of N-cadherin and ZEB-2. Conclusions:The expression of miR-4698 in liver cancer tissues and cell lines is significantly reduced. Overexpression of miR-4698 can inhibit the expression of GALNT4 gene and reduce the proliferation and migration ability of liver cancer Huh7 cells.

PURPOSE: The purpose of this study is to examine the role of the double-stranded RNA (dsRNA) activated Toll–interleukin-1 receptor domain-containing adaptor inducing interferon β (TRIF) signal pathway in triggering apoptosis in hepatocellular carcinoma (HCC) cells. MATERIALS AND METHODS: First, siRNA targeted autophagy–related gene LC3 (pU6H1-LC3 siRNA and siLC3) and a dsRNA used as a Toll-like receptor 3 (TLR3) ligand was constructed and synthesized, respectively. Then, a human HCC cell line was transfected with dsRNA, siLC3, and cotransfected with siLC3 and dsRNA (siLC3+dsRNA), respectively. Finally, quantification real-time polymerase chain reaction, western blotting, and immunofluorescence staining were used in the HCC line (SMMC7721), and MTT assay, flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling, and transmission electron microscopy were used in an HCC xenograft model of nude mice. Human umbilical vein endothelial cell tube forming assay, color Doppler ultrasonographic flow image examination, and CD34-positive microvessel density were used in vitro and in vivo. RESULTS: Compared with untreated cells, the protein and mRNA expression of TLR3 and TRIF was up-regulated, in order, siLC3+dsRNA, dsRNA, and siLC3. Expression of LC3 was obviously down-regulated and the autophagosomes were significantly decreased in siLC3+dsRNA and siLC3, whereas in dsRNA (p < 0.05). LC3 and TRIF colocation was observed in HepG2 cells. Decreased cell viability, increased apoptosis, decrease in xenograft tumor volume, and angiogenesis potential were also observed in order (p < 0.05). CONCLUSION: Suppression of intracellular autophagy resulted in decreased degradation of TRIF protein, which can promote triggering of apoptosis by the TLR3-TRIF pathway. dsRNA and siLC3 could play anticancer roles in coordination.
Animals ; Apoptosis ; Autophagy ; Blotting, Western ; Carcinoma, Hepatocellular* ; Cell Line ; Cell Survival ; Endothelial Cells ; Flow Cytometry ; Fluorescent Antibody Technique ; Hep G2 Cells ; Heterografts ; Humans ; In Vitro Techniques ; Interferons ; Mice ; Mice, Nude ; Microscopy, Electron, Transmission ; Microvessels ; Real-Time Polymerase Chain Reaction ; RNA, Double-Stranded ; RNA, Messenger ; RNA, Small Interfering ; Signal Transduction ; Toll-Like Receptor 3 ; Tumor Burden ; Umbilical Veins

Objective:To analyze the expression of hsa-microRNA-6832-5p (hsa-miR-6832-5p) in bladder tumor tissues and bladder tumor cells, and to explore its interference on the expression of proline-rich protein 11 (PRR11) gene in bladder tumor cells and its effect on cell proliferation and migration.Methods:Real-time quantitative polymerase chain reaction (qPCR) was used to detect the expression level of hsa-miR-6832-5p in bladder tumor tissues, paratumorous tissues, bladder tumor cell lines and normal bladder epithelial cells. Bioinformatics predicts and the dual luciferase reporter gene validates the downstream target gene of hsa-miR-6832-5p. hsa-miR-6832-5p or miR-NC were transfected to the bladder tumor cell lines with the lowest expression level of hsa-miR-6832-5p, respectively, and named as miR-6832-5p group and miR-NC group. qPCR was used to detect the expression levels of hsa-miR-6832-5p and target gene mRNA in the transfected cells. Western blot was used to detect the expression level of target gene protein. Methyl thiazolyl tetrazolium (MTT) assay and transwell assay were used to detect cell proliferation and migration, respectively.Results:The expression of hsa-miR-6832-5p was lower in bladder tumor tissues than in adjacent tissues ( P<0.01). The expression of hsa-miR-6832-5p in bladder tumor cells was lower than that in normal bladder epithelial cells ( P<0.05), and T24 cells had the lowest hsa-miR-6832-5p expression level ( P<0.01). Bioinformatics and dual luciferase reporter genes showed that hsa-miR-6832-5p can directly act on the 3′-untranslated region of the PRR11 gene ( P<0.01). The expression of hsa-miR-6832-5p in the miR-6832-5p group was significantly higher than that in the miR-NC group ( P<0.01). The expression of PRR11 in the miR-6832-5p group was significantly lower than that in the miR-NC group ( P<0.01). Western blot results were consistent with qPCR results. Compared with the miR-NC group, the proliferation of bladder tumor cells was significantly decreased after transfection with hsa-miR-6832-5p ( P<0.05), and the migration ability of cells was significantly decreased ( P<0.01). Conclusions:The expression of hsa-miR-6832-5p was significantly decreased in bladder tumor tissues and cell lines. hsa-miR-6832-5p inhibited the proliferation and migration of bladder tumor cells by down-regulating the expression of PRR11 gene.