Figure 1.
MiRNA expression profiles in CRC with or without lymph node metastasis.
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. * P<0.05, ** P<0.01.
Table 1.
The clinicopathologic characteristics of 202 cases of primary CRC patients used in this study.
Figure 2.
The effect of miR-145 overexpression on the HCT-8 cells.
(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.
Figure 3.
MiR-145 promoted invasion and metastasis of CRC cells in vitro and in vivo.
(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. ** P<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). ** P<0.01.
Figure 4.
The expression profiles of Hsp-27 were detected in CRC cells or CRC tissues.
(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.
Figure 5.
Knockdown of Hsp-27 by siRNA attenuated the prometastatic effect of miR-145.
(A) HCT-8-mir-145 (mir-145) or HCT-8-NC (NC) cells were transfected with Hsp-27 siRNA (mir-145+si-hsp27) or a negative control siRNA (mir-145+mock). The expression of Hsp-27 protein was detected by western blot assay. (B) Wound-healing assay to evaluate the effect of Hsp-27 siRNA in HCT-8-miR-145 cells. (C) Knockdown of Hsp-27 by siRNA in HCT-8-miR-145 cells significantly inhibited cell invasion. The images were representatives of at least three independent experiments. Average number of invasion cell number per field from at least three independent experiments ± SD is shown by column figure. ** P<0.01.