Figure 4

Calreticulin is a miR-27a target and affects MHC class I cell surface exposure. (a) Schematic representation of human calreticulin (CRT) mRNA: the highly conserved miR-27a-binding site located in the 3′UTR is illustrated in red. (b) Immunoblot analysis of CRT levels in HCT116 CTRL and derivative clones miR27a_KD or miR27a_OE cells after transfection with the specific target protector (CRT-TP) or negative control (NC-TP) for 72 h. β-Actin was used as a loading control. *P≤0.05, ns=non significant. (c) CRT mRNA qRT-PCR analysis of HCT116 CTRL and derivative clones miR27a_KD or miR27a_OE cells transfected with the specific CRT-TP or negative control (NC-TP) for 72 h. *P≤0.05 (Student's t-test). (d) CRT immunoblot analysis of HCT116 CTRL and derivative clones miR27a_KD or miR27a_OE cells transfected with three specific CRT siRNAs or Control siRNA for 72 h. Densitometric analysis is reported below the bands. **P≤0.01 (Student's t-test). (e) Flow cytometry assessment of cell surface MHC class I molecules in the same cells as in (b) after transfection with CRT-TP or CRT-siRNAs or relative negative controls for 72 h. (f) Immunoblot analysis of MHC class I molecules present in the plasma membrane fraction of HCT116 CTRL cells and their derivative clones miR27a_KD or miR27a_OE transfected with the CRT-siRNAs or scrambled siRNA control (Ctrl-siRNA). Data normalized to E-cadherin are shown in the histograms. *P≤0.05, **P≤0.01 versus HCT116 CTRL cells; ^#P≤0.05 versus Ctrl-siRNAs transfected cells