[MiR-124 suppresses the proliferation of human prostate cancer PC3 cells by targeting PKM2].

OBJECTIVE: To explore the mechanism of miR-124 inhibiting the proliferative activity of prostate cancer PC3 cells. METHODS: Luciferase reporter gene assay was used to examine the specific binding ability of miR-124 to PKM2 mRNA 3'-UTR. After miR-124 was transfected mimic to PC3 cells, the expression levels of PKM2 mRNA and protein were detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot, respectively. The effects of miR-124 mimic and PKM2 siRNA on the proliferative activity of the PC3 cells were determined by MTT assay. RESULTS: The expressions of PKM2 mRNA and protein were upregulated (5.12 +/- 0.35) times and (4.05 +/- 0.20) times respectively in the PC3 cells as compared with those in the RWPE-1 cells (P < 0.05). Luciferase reporter gene assay demonstrated that miR-124 targeted PKM2 3'-UTR. At 24 hours after transfection with miR-124 mimic, the PKM2 protein expression in the PC3 cells was downregulated (0.16 +/- 0.04) times (P < 0.05), while the PKM2 mRNA level was not changed significantly (P > 0.05), as compared with the control group. MTT assay showed that both miRNA-124 mimic and PKM2 siRNA could inhibit the proliferation of the PC3 cells, but the former exhibited a greater inhibitory effect than the latter. After transfection with miR-124 mimic and PKM2 siRNA, the cell growth rates were (66.20 +/- 5.10)% vs (82.10 +/- 6.35)% at 24 hours (P < 0.05) and (49.34 +/- 2.37)% vs (70.10 +/- 5.80)% at 48 hours (P < 0.05). CONCLUSION: miR-124 can suppress the proliferation of PC3 cells by regulating the PKM2 gene.

Integrative analysis of the lncRNA-associated ceRNA network reveals lncRNAs as potential prognostic biomarkers in human muscle-invasive bladder cancer.

BACKGROUND: Long noncoding RNAs (lncRNAs) play important roles in competing endogenous RNA (ceRNA) networks involved in the development and progression of various cancers, including muscle-invasive bladder cancer (MIBC). PURPOSE: This study aims to construct the lncRNA-associated ceRNA network and identify lncRNA signatures correlated with the clinical features of MIBC tissue samples from The Cancer Genome Atlas (TGCA) database. METHODS: The differential expression profiles of MIBC associated lncRNAs, miRNAs and mRNAs were obtained from TCGA. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to determine the principal functions of significantly dysregulated mRNAs. The dysregulated lncRNA-associated ceRNA network of MIBC was constructed based on the bioinformatics data, and the correlations between lncRNA expression and clinical features were analyzed using a weighted gene coexpression network analysis (WGCNA). Six cancer specific lncRNAs from the ceRNA network were randomly selected to detect their expression in 32 paired MIBC tissue samples and 5 bladder cancer cell lines using quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The ceRNA network was constructed with 30 lncRNAs, 13 miRNAs and 32 mRNAs. Seventeen lncRNAs in the ceRNA network correlated with certain clinical features, and only 1 lncRNA (MIR137HG) correlated with the overall survival (OS) of patients with MIBC (log-rank test P<0.05). GO and KEGG analyses revealed roles for the potential mRNA targets of MIR137HG in epithelial cell differentiation and the peroxisome proliferator-activated receptor (PPAR) and tumor necrosis factor (TNF) signaling pathways. The expression data from TCGA were highly consistent with the verification results of the MIBC tissue samples and bladder cancer cell lines. CONCLUSION: These findings improve our understanding of the regulatory mechanism of the lncRNA-miRNA-mRNA ceRNA network and reveal potential lncRNAs as prognostic biomarkers of MIBC.

Cell-penetrating peptide conjugates of gambogic acid enhance the antitumor effect on human bladder cancer EJ cells through ROS-mediated apoptosis.

BACKGROUND: Gambogic acid (GA) is the main active ingredient of resin gamboges and possesses anti-cancer activity toward various human cancer cells. However, clinical application of GA has been limited by its poor aqueous solubility and dose-limiting toxicities. Cell-penetrating peptides (CPPs) are widely used to deliver anti-cancer drugs into cancer cells and to enhance the water solubility of drugs. PURPOSE: The object of this study was to synthesize peptide-drug conjugates in which the cell-penetrating peptide TAT (trans-activator of transcription) was conjugated to GA and evaluated the anti-cancer activity of this GA-CPP conjugate (GA-TAT) in EJ bladder cancer cells. METHODS: GA is built onto the TAT, and the GA-TAT conjugates are cleaved from the solid support and purified via HPLC. The equilibrium solubility of GA-TAT was measured using the shake-flask method. The effects of GA-TAT on EJ cell viability and proliferation were determined by MTT assay, Edu assay and colony formation assay, respectively. After treated with 1.0 µM GA-TAT for 24 h, the apoptosis rate of EJ cells were detected by Acridine orange/ethidium bromide (AO/EB) assay and flow cytometry assay. The proteins of caspase-3 (processing), caspase-9 (processing), Bcl-2 and Bax were analyzed by Western blotting, and the intracellular reactive oxygen species (ROS) production was evaluated by a reactive oxygen species assay. RESULTS: In contrast to free GA, the solubility of GA-TAT in water was significantly improved. Meanwhile, GA-TAT significantly increased EJ cellular uptake, toxicity and apoptosis. Mechanistic analysis revealed that GA-TAT enhanced the anti-cancer effect of GA against EJ cells through ROS-mediated apoptosis. The results were demonstrated that GA-TAT increased the ROS level in EJ cells, and N-acetyl-L-cysteine (NAC; a well-known ROS scavenger) inhibited GA-TAT-induced ROS generation and apoptosis. Additionally, GA-TAT activated caspase-3 and caspase-9 and down-regulated the Bcl-2/Bax ratio, but these effects were largely rescued by NAC. CONCLUSION: GA-TAT has outstanding potential for promoting tumor apoptosis and exhibits promise for use in bladder cancer therapy.

[Construction of a tissue engineering skin with epidermal stem cells].

OBJECTIVE: To constitute a composite skin substitute that can proliferate well with epidermal stem cells and fibroblasts on collagen sponge. METHODS: Epidermal stem cells were selected by rapid attachment to collagen IV for 10-15 min and cultured on 3T3 feeder layers. Collagen was extracted from rat tail. The matrix lattice was fabricated by freeze-dryer and cross-linked with glutaraldehyde. Fibroblasts were inoculated on collagen sponge and cultured for 4 days prior to inoculation of epidermal stem cells to construct composite skin substitute. The composite skin substitute were examined by means of histology, immunohistochemistry and electron microcopy, the histologic appearance was similar to that of normal epidermis. RESULTS: The epidermal stem cells formed large colonies at 7-8 days, expressed K19 antigen. The percentages of cells at G0/G1 phase of cell cycle and the percentage of alpha6 briCD71dim cells in ESC groups were higher than those in the control group. The skin substitute had epidermis and dermis, the histologic appearance was similar to that of normal skin. The artificial skin expressed keratin antigen by immunocytochemical methods. CONCLUSIONS: Epidermal stem cells proliferated well and differentiated properly on this artificial skin dermis which contained fibroblasts. It seemed that the composite skin to be a good equivalent.