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

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 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

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

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

Microscopy images of H&E stained normal liver tissue slides.

<p>The normal liver tissues from groups treated with three drug formulations via spleen injection and PBS groups were collected and fixed using 10% formalin for 24 hrs at room, and then hematoxylin and eosin staining (H&E staining) was carried out. Magnification = 400×.</p

The anti-tumor effect of Dox-loaded galactosylated liposomes in animal model via spleen injection.

<p>Three formulations (Dox alone, CL-Dox and Gal-Dox) were introduced into tumor-bearing mice on day 7 post cell inoculation via two administration routes, tail vein injection and spleen injection. The drug dose administrated was 6 mg/kg. A) Tumor progression in liver was assessed by the mean value of hepatic tumor weight. B) Mesenteric lymph node metastasis was assessed by the mean weight of metastatic carcinoma from mesenteric lymph node. The results represent the mean ± SE. “*” indicate significant difference (p<0.05).</p

The effect of temperature on cell uptake by ASGPR+/− cells.

<p>The HCT-8 cells and HepG2 cells were incubated with either conventional liposomes (CL) or galactosylated liposomes (Gal-lipo) at 4°C for 1 hr, and then warmed to 37°C with continued incubation for an additional 1 hr. Cellular uptake of liposomes labeled by 25-NBD-cholesterol was visualized under fluorescence microscopy. The mean fluorescence intensity was quantitatively determined using Image-Pro Plus Imaging software. A) Fluorescence imaging of cellular uptake of liposomes. B) Quantitative analysis of the mean fluorescence intensity. “*” indicates significant difference (p<0.001). CL: conventional liposomes; Gal: galactosylated liposomes.</p

The effect of incubation time on cell uptake by ASGPR+/− cells.

<p>The HCT-8 cells (ASGPR−) and HepG2 cells (ASGPR+) were incubated with either conventional liposomes (CL) or galactosylated liposomes (Gal-lipo) at 37°C for different time duration. Cellular uptake of liposomes labeled by 25-NBD-cholesterol was visualized under fluorescence microscopy. The mean fluorescence intensity representing durg uptake was determined using Image-Pro Plus Imaging software. A) Fluorescence imaging of cellular uptake of liposomes. B) Quantitative analysis of the mean fluorescence intensity for the treatment of 1 hr incubation. “*” indicates significant difference (p<0.001). CL: conventional liposomes; Gal: galactosylated liposomes.</p