Figure 1.
The expression levels of miR-339-5p in CRC tissues and colon cancer cell lines.
(A) Expression levels of miR-339-5p were examined by qRT-PCR in 30 colon cancer tissues and their pair-matched adjacent normal colonic tissues. Each sample was analyzed in triplicate and normalized to U6 (*P<0.05). T: tumor tissues; N: adjacent normal tissues. (B) Expression levels of mature miR-339-5p were detected by qRT-PCR in six human colonic carcinoma cell lines. The relative expression of miR-339-5p was normalized to the endogenous control U6. Each sample was analyzed in triplicate. The relative miR-339-5p expression in colon cancer cell lines was much lower than that of the three non-cancerous colonic tissues (N1, N2, and N3). (*P<0.05).
Figure 2.
Lentivirus-mediated expression of miR-339-5p inhibited cell proliferation, migration and invasion of SW620 cells.
(A) The expression of miR-339-5p was analysis in SW620 cells infected with PLVTHM-NC or pLVTHM-pre-miR-339 by qRT-PCR (*P<0.05). (B) The vitality of cells infected with PLVTHM-NC or pLVTHM-pre-miR-339 was detected using the CCK-8 assay. Values at the indicated time points were provided as the mean absorbance with an SD of five wells (*P<0.05). (C) Impact of miR-339-5p on cell cycle of SW620 cells. The percentage of cells in G1, S, and G2 phases is shown in the left panel. And the statistic analysis is also shown in the right panel. (D) Colonies formed by PLVTHM-NC or pLVTHM-pre-miR-339 infected SW620 cells were shown 2 weeks after plating. Right panel showed the quantification of the relative colony formation in PLVTHM-NC versus pLVTHM-pre-miR-339-infected cells. Values are the means ± SD of triplicate experiments (*P<0.05). (E) Transwell assay was employed to evaluate migration and invasion of SW620 cells infected with PLVTHM-NC or pLVTHM-pre-miR-339. Representative fields of migration (top) or invasive (bottom) cells on membrane (left) (Original magnification: ×200). Average number of invasive or migration cells number per field from three independent experiments ± standard (*P<0.05).
Figure 3.
Inhibition of miR-339-5p promoted the growth, migration and invasion of HCT116 cells.
(A) The expresson of miR-339-5p was analysis in HCT116 cells transfected with miR-339-5p inhibitor or negative control by qRT-PCR. (*P<0.05). (B) The vitality of cells transfected with the miR-339-5p inhibitor or negative control was detected using the CCK-8 assay. Values at the indicated time points were provided as the mean absorbance with an SD of five wells (*P<0.05). (C) Impact of miR-339-5p on cell cycle of HCT116 cells. The percentage of cells in G1, S, and G2 phases is shown in the left panel. And the statistic analysis is also shown in the right panel. (D) Transwell assay was employed to evaluate migration and invasion of HCT116 cells transfected with miR-339-5p inhibitor or negative control. Representative fields of migration (top) or invasive (bottom) cells on membrane (Original magnification: ×200). Average number of invasive or migration cells number per field from three independent experiments ± standard (*P<0.05).
Figure 4.
PRL-1 is a direct target of miR-339-5p in colon cancer cells.
(A) Expression levels of PRL-1 were detected by qRT-PCR in six human colonic carcinoma cell lines. The relative expression of PRL-1 was normalized to the endogenous control GAPDH. Each sample was analyzed in triplicate (*P<0.05). (B) The expression of PRL-1 was analysis in SW620 cells infected with PLVTHM-NC or pLVTHM-pre-miR-339 by qRT-PCR (*P<0.05) (left). The expression of PRL-1 was analysis in HCT116 cells transfected with miR-339-5p inhibitor or negative control by qRT-PCR (*P<0.05) (right). (C) The expression levels of PRL-1 were detected using Western blot. (D) A schematic illustration of base paring between miR-339-5p and the 3′UTR of PRL-1. Substitution of seven consecutive bases (GTCAAAG to ACAGGGA) at the 3′UTR of PRL-1 for the mutant reporter construct is also shown. (E) Analysis of luciferase activity. 293 cells and HCT116 cells were co-transfected with psiCHECK™-2 luciferase reporter plasmid containing either wildtype or mutant PRL-1 3′-UTR (indicated as WT or MUT on the X-axis), and either the miR-339-5p mimics or NC mimics. Luciferase activity was assayed 48 h after transfection. Renilla luciferase activity of each sample was normalized by Firefly luciferase activity. The Y-axis represents the relative luciferase activity. Data were shown as mean ± SD from three independent experiments (*P<0.05).
Figure 5.
MiR-339-5p inhibited the expression of phosphorylation of Erk1/2.
(A) The protein levels of Erk1/2 and phosphor-Erk1/2 were detected by Western Blot after transfection for 48 h. β-tublin was used as the loading control.
Figure 6.
miR-339-5p inhibited colon tumor growth in vivo.
(A) In all, 2×106 SW620/pLVTHM-NC and SW620/pLVTHM-pre-miR339 cells were independently injected into the back of 2 nude mice. Tumor sizes were measured at different time points until day 20 when mice were killed. Mean size of the tumors per animal was plotted (*P<0.05). (B) External whole-body images of SW620/pLVTHM -NC and SW620/pLVTHM-pre-miR-339 cells mice and cancers were obtained 20 days after injection. (C) Subcutaneous tumor regeneration from SW620/pLVTHM-NC and SW620/pLVTHM-pre-miR-339 cells. (D) Representative photographs of H&E staining of primary cancer tissues are shown (magnification, ×200).