Up-Regulation of MicroRNA-145 Associates with Lymph Node Metastasis in Colorectal Cancer

<div><p>Metastasis is the main cause of mortality in patients with solid tumours. Identifying the exact molecules associated with CRC metastasis may be crucial to understand the process, which might also be translated to the diagnosis and treatment of CRC. In this study, we investigate the association of microRNA expression patterns with the lymph node metastasis of colorectal cancer. Among these candidate miRNAs, the expression of miRNA-145 was significantly related to lymph node metastasis of CRC. Both <i>in vitro</i> and <i>in vivo</i> study demonstrated that up-regulation of miR-145 could improve the ability of migration and invasion of colorectal cancer cell, while no effect on proliferation was observed. The mechanism of this promotion is associated with the stabilization of Hsp-27, a protein which plays an important role in the promotion of metastasis. These results may be crucial to understanding CRC metastasis and may be translated to the diagnosis and treatment of CRC.</p></div

The clinicopathologic characteristics of 202 cases of primary CRC patients used in this study.

<p>The clinicopathologic characteristics of 202 cases of primary CRC patients used in this study.</p

MiR-145 promoted invasion and metastasis of CRC cells <i>in vitro</i> and <i>in vivo.</i>

<p>(A) Migration and invasion (B) assay of HCT-8-miR-145 or HCT-8-NC cells. The images were representatives of at least three independent experiments. Average number of migration cell number per field from at least three independent experiments ± SD is shown by column figure. ** <i>P</i><0.01. (C) Photo images of mesenteric lymph node metastasis from nude mice which was injected into the liver with HCT-8-miR-145 or HCT-8-NC cells followed by surgical suture. Animals were killed 2 weeks post intra-hepatic cell inoculation. (D) Incidence of mesenteric lymph node metastasis in mice (table) and mean number of visible metastatic nodules in mesentery (column figure). ** <i>P</i><0.01.</p

The effect of miR-145 overexpression on the HCT-8 cells.

<p>(A) qRT-PCR analysis of miR-145 expression in HCT-8 cells transfected with the lenti-miR-145 expression vector or the miRNA negative control vector using a lentivirus system. (B) Proliferation rates of HCT-8-miR-145 or HCT-8-NC cells detected by CCK-8 assay. (C) Cell cycle analysis of HCT-8-miR-145 or HCT-8-NC cells by Flow cytometry. Data represents average +SD of three independent experiments.</p

Prediction of Solubility Properties from Transfer Energies for Acidic Phosphorus-Containing Rare-Earth Extractants Using Implicit Solvation Model

<p>The differences of thermodynamics energies from the pure phase to a solution were used to predict the solubility properties of acidic phosphorus–containing rare-earth extractants. Four solvents, namely tributylphosphate, <i>n</i>-dodecane, toluene, and <i>n</i>-octanol were used. The thermodynamic cycle of the implicit solvation model and the structure model with short carbon chains were used. The relationship obtained by simulation of the solubility properties and extractant structures agreed qualitatively with reported experimental results. These results provide guidance for the design of new efficient extractants.</p

The expression profiles of Hsp-27 were detected in CRC cells or CRC tissues.

<p>(A) The expression levels of Hsp-27 were examined in HCT-8-miR-145 or HCT-8-NC cells by western blotting analysis. (B) The expression of Hsp-27 protein was detected in CRC and adjacent normal tissues by western blot assay. The relative Hsp27/actin ratios of individual bands are shown as the mean + SD of values derived from all patient samples (Non-tumor tissue, n = 47; LNM-P tumor tissue, n = 41; LNM-N tumor tissue, n = 43). (C–D) MiR-145 Enhanced Hsp-27 Stability in CRC Cells.HCT-8-miR-145 or HCT-8-NC cells were incubated with the protein synthesis inhibitor cycloheximide (CHX, 0.5 µg/µL) (C) or proteasome inhibitor MG-132 (5 µM) (D) for 24 hours. The level of total Hsp-27 was detected by western blotting analysis. The relative Hsp27/actin ratios of individual bands are shown as the mean ± SD of values normalized to beta-actin.</p

MiRNA expression profiles in CRC with or without lymph node metastasis.

<p>A. The certified result of microarray analysis. Hierarchical clustering of 32 significantly dysregulated miRNAs expression profiles in human primary colorectal cancer tissues derived from colorectal cancer patients with (LNM-P, n = 4) or without (LNM-N, n = 4) lymph node metastasis. B.Validation of selected miRNAs predicted to be dysregulated in CRC with or without lymph node metastasis using qRT-PCR in the same tissues used for microarray analysis. Data shown in B is representative of three independent experiments, and presented as fold expression normalized to U6 ± SD (standard deviation).C. QRT-PCR analysis of the relative expression of miR-145 in additional 202 (LNM-N = 99; LNM-P = 103) cases of human CRC tissues, including tumor sample (T) and matching non-tumor tissue sample (NT) from the same patient. Each sample was analyzed in triplicate and normalized to U6. * <i>P</i><0.05, ** <i>P</i><0.01.</p

Lanthanide Complex-Based Luminescent Probes for Highly Sensitive Time-Gated Luminescence Detection of Hypochlorous Acid

Two novel lanthanide complex-based luminescent probes, ANMTTA-Eu³⁺ and ANMTTA-Tb³⁺ {ANMTTA, [4′-(4-amino-3-nitrophenoxy)­methylene-2,2′:6′,2″-terpyridine-6,6″-diyl] bis­(methylenenitrilo) tetrakis­(acetic acid)}, have been designed and synthesized for the highly sensitive and selective time-gated luminescence detection of hypochlorous acid (HOCl) in aqueous media. The probes are almost nonluminescent due to the photoinduced electron transfer (PET) process from the 4-amino-3-nitrophenyl moiety to the terpyridine-Ln³⁺ moiety, which quenches the lanthanide luminescence effectively. Upon reaction with HOCl, the 4-amino-3-nitrophenyl moiety is rapidly cleaved from the probe complexes, which affords strongly luminescent lanthanide complexes HTTA-Eu³⁺ and HTTA-Tb³⁺ {HTTA, (4′-hydroxymethyl-2,2′:6′,2″-terpyridine-6,6″-diyl) bis­(methylenenitrilo) tetrakis­(acetic acid)}, accompanied by the remarkable luminescence enhancements. The dose-dependent luminescence enhancements show good linearity with detection limits of 1.3 nM and 0.64 nM for HOCl with ANMTTA-Eu³⁺ and ANMTTA-Tb³⁺, respectively. In addition, the luminescence responses of ANMTTA-Eu³⁺ and ANMTTA-Tb³⁺ to HOCl are pH-independent with excellent selectivity to distinguish HOCl from other reactive oxygen/nitrogen species (ROS/RNS). The ANMTTA-Ln³⁺-loaded HeLa and RAW 264.7 macrophage cells were prepared, and then the exogenous HOCl in HeLa cells and endogenous HOCl in macrophage cells were successfully imaged with time-gated luminescence mode. The results demonstrated the practical applicability of the probes for the cell imaging application