Figure 1.
miR-203 modulates Hakai levels in HeLa cells.
A, schematic representation of Hakai mRNA depicting two predicted miR-203 target sites within the Hakai 3′UTR. Alignment of consensus sequences of Hakai mRNA with miR-203; top strand, Hakai sequence; bottom strand, miR-203. B, miR-203 levels measured by RT-qPCR after 48 h of untransfected HeLa cells, transfection with scrambled control miRNA (Ctrl) or the precursor miR-203 (Pre-miR-203); U6 snRNA levels was used as control to monitor loading differences. C, levels of Hakai mRNA normalized to control HPRT mRNA were measured 48 h after transfecting HeLa cells with the indicated miRNAs. D, the effect of the indicated transfected miR-203 on Hakai levels in HeLa cells were tested in HeLa whole-cell lysates by Western blotting (Top) and quantified by densitometry (Bottom), using Hakai antibody and α-tubulin as loading control for normalization. E, the levels of Hakai and loading control α-tubulin were tested in whole-cell lysates by Western blotting 48 h after transfecting HeLa cells with the anti-miR-203 or scrambled control miRNA. F, the levels of Hakai mRNA 48 h after transfection of HeLa cells with the anti-miR-203, or scrambled control miRNAs (each normalized to HPRT levels), were analyzed by RT-qPCR. G, western blotting analysis (Top) and quantification by densitometry (Bottom) of AKT2 levels in HeLa cells expressing either pre-miR-203 or anti-miR-203, processed as described in D. Western blotting signals were quantified by densitometry and shown in 1D. Values in B–D and F–G are the means ± SEM from three independent experiments. Statistical analyses indicate the significant difference in the indicated transfected miRNAs with respect to transfected scrambled control (Ctrl) miRNA (*p<0.05, **p<0.01, ***p<0.001). The western blotting data are representative of three experiments.
Figure 2.
Influence of miR-203 on Hakai levels in several cell lines.
A, the effect of modulating miR-203 levels in 293 cells was studied as described in 1D and 1E; Western blot analysis (left panel) and quantification by densitometry (right panel). B, the effect of modulating miR-203 levels in A549 cells was studied as described 1D and 1E. Western blot analysis (left panel) and quantification by densitometry (right panel). C, endogenous Hakai expression levels in human colorectal cell lines (SW480 and SW620), as assessed by western blotting (upper panel) and by RT-qPCR (lower panel) analysis. D, endogenous miR-203 levels in human colorectal cell lines (SW480 and SW620) by RT-qPCR (lower panel). Values (A–D) are the means ± SEM from three independent experiments. Significant differences in the indicated transfected miRNA with respect to transfected scrambled control (Ctrl) miRNA for A–B, and the between SW480 and SW620 cell lines for C–D are indicated (*p<0.05, **p<0.01, ***p<0.001, n = 3). The western blotting data are representative of three independent experiments.
Figure 3.
miR-203 influence on Hakai reporter construct.
A, schematic of EGFP reporter constructs bearing either no Hakai mRNA sequences (pEGFP) or miR-203 target sites on the Hakai 3′-UTR (pEGFP-3′UTR). B, 48 h after cotransfection of the plasmids with scrambled control miRNA or Pre-miR-203, the level of EGFP expressed was analyzed by Western blotting. C, quantification of panel B showing the effect of scrambled Ctrl miRNA or pre-miR-203 on pEGFP and pEGFP-3′UTR. Significant differences in EGFP expression from pEGFP-3′UTR cotransfected with scrambled control miRNA (Ctrl) and pEGFP-3′UTR cotransfected with pre-miR-203 are indicated (***p<0.001). Western blotting data are the means ± SEM from three independent experiments.
Figure 4.
Influence of miR-203 on cell proliferation.
A, 48 h after transfection of HeLa cells with the miRNAs shown, cell numbers were measured using a hemocytometer and represented as percentage of cells relative to the scrambled Ctrl miRNA group. B, measurement of BrdU incorporation by 48 h after transfection of HeLa cells with the indicated miRNAs. The data in panels A, and B are the means ± SEM from three experiments. Significant differences in cell number (A) and BrdU incorporation (B) in the indicated transfected miRNA compared to transfected scrambled (Ctrl) miRNA are indicated (*p<0.05, **p<0.01). C, forty-eight h after transfection with scrambled Ctrl miRNA, Anti-miR-203, or Pre-miR-203, HeLa cells were subjected to FACS analysis. D, the relative G1, S, and G2/M compartments shown in C were calculated and represented in left panel, and total cell numbers in every stage cell cycle compartment were included in the right panel. Data (C and D) are representative of three independent experiments.
Figure 5.
Influence of miR-203-regulated Hakai on cell proliferation.
A, levels of Hakai and loading control α-tubulin were tested in whole-cell lysates by Western blotting 48 h after transfecting HeLa cells with two different siRNAs oligos for Hakai relative to the scrambled Ctrl small RNA group. B, 48 h after transfection of HeLa cells with the indicated siRNA oligos, cell numbers were measured by BrdU incorporation assay and represented as percentage of cells. C, measurement of BrdU incorporation 48 h after transfection of HeLa cells with the indicated small RNAs. D, measurement of cell number by hemocytometer after 48 h of transfection of HeLa cells with the indicated small RNAs. E, forty-eight h after transfecting HeLa cells with the indicated small RNAs, Hakai and α-tubulin levels were measured by Western blot analysis. Western blotting data are representative of three independent experiments. The data in panels B–D are the means ± SEM from three experiments. Significant differences in BrdU incorporation (B and C) and in cell number (D), compared to the transfected scrambled (Ctrl) small RNA are indicated (*p<0.05, **p<0.01).
Figure 6.
Hakai and miR-203 expression in colon tissues.
A, immunohistochemical analysis of Hakai protein expression in normal colon versus colon cancer tissues. Differences between tumour samples compared to its paired healthy tissues are statistically significant (***p<0.001, n = 19). B, in situ hybridization (ISH) for miR-203 (upper panel) and U6 snRNA (as control probe, middle panel) expression in colon cancer tissues compared to normal colon tissue. Haematoxylin and eosin (H&E)-stained section (lower panel) was included to identify tumour area. Scale bar, 200 µm.