Fig. 3

miR-20a-5p enhances CRC cell proliferation and migration, and up-regulates Epithelial-Mesenchymal Transition (EMT) markers partly through targeting PTEN. A Western blot analysis indicated that while the expression levels of N-cadherin and Vimentin significantly increased after miR-20a-5p mimic transfection, there was a notable decrease in the protein level of E-cadherin following the transfection. β-actin was employed as an endogenous loading control. The cropped blots are presented correspondingly. Full-length blots are presented in Supplementary Fig. 4. B TargetScan 7.2 predicted the complementary binding site between miR-20a-5p and PTEN's 3'-UTR (http://www.targetscan.org). C A luciferase assay demonstrated a significant reduction in luciferase activity upon introducing the miR-20a-5p mimic, indicating the binding affinity between miR-20a-5p and PTEN's 3'-UTR. D, E The transcript expression level of PTEN was assessed following the use of both the miR-20a-5p mimic and siRNA targeting PTEN. In both cases, PTEN expression decreased at the mRNA level, and a similar pattern was achieved. F Transfection of miR-20a-5p mimic to SW480 CRC cells led to an observable increase in the cell count, compared to the negative control group. Notably, transfection of CRC cells with si-PTEN had a similar effect and enhanced CRC cell proliferation. Importantly, the suppression of miR-20a-5p with its specific inhibitor effectively decreased cell numbers. However, the inhibitory effect of the miR-20a-5p inhibitor on SW480 CRC cell proliferation was partially rescued when SW480 CRC cells were concurrently transfected with si-PTEN. G Representative photomicrographs of the migration potential of SW480 CRC cells in different conditions after 48 h of transfection, assessed using the transwell assay. H Quantitative assessment of migrated cells revealed that the transfection of CRC cells with either the miR-20a-5p mimic or si-PTEN can give rise to substantial increase in the migration rate of CRC cells. Conversely, the miR-20a-5p inhibitor resulted in a reduction in the number of migrated cells. Of significant importance, the suppressive effect of the miR-20a-5p inhibitor on SW480 CRC cell migration was partially diminished when SW480 CRC cells were concurrently transfected with si-PTEN. I, J The mean normalized ratios measured for EMT-related markers N-cadherin and Vimentin indicated that transfection of miR-20a-5p inhibitor led to reduced expression levels of each marker. Importantly, introduction of si-PTEN into SW480 CRC cells caused a significant up-regulation in the transcript expression levels of N-cadherin and Vimentin. K The mean normalized ratios measured for E-cadherin showed that transfection of the miR-20a-5p inhibitor led to increased expression levels of E-cadherin. Transfection of si-PTEN into SW480 CRC cells caused a down-regulation in the transcript expression level of E-cadherin to some extent. However, when si-PTEN was introduced concurrently with the miR-20a-5p inhibitor, it simultaneously resulted in promoting the expression of E-Cadherin. Columns represent means of three different experiments; bars represent SD. *P < 0.05, **P < 0.01