Figure 3.

Inhibition of vIL6 and hIL6 expression by miRNAs. (A) Generation of a miRNA-resistant vIL6 (miR-re vIL6) by introduction of point mutations into the miR-1293 seed match. (B) Repression of vIL6 expression by ectopic miR-1293 in HEK293 cells. The cells were pretreated with 10 nM Pre-miR-1293 or a negative control (NC) Pre-miR-NC for 48 h before transfection with an indicated vIL6-FLAG expression vector. Cell lysates prepared 24 h after transfection were blotted for vIL6 expression by using an anti-FLAG antibody. (C) Two putative, overlapped miRNA seed matches in the ORF of hIL6 mRNA corresponding to the miR-1293 responsive region of vIL6 mRNA. (D) Repression of hIL6 expression by miR-608, but not by miR-1293. HeLa cells pretreated for 48 h with 10 nM Pre-miR-608, Pre-miR-1293 or Pre-miR-NC were transfected with an hIL6-FLAG expression vector for additional 24 h before Western blotting for hIL6 expression using an anti-FLAG antibody. The numbers below panels B and D indicate a relative level of vIL6 and hIL6 in each sample after being normalized to β-tubulin. (E) The IL6 sequences for swapping between hIL6 and vIl6. The vIL6 sequences indicated by the black box in (F) for swapping are bolded to replace the corresponding hIL6 sequence in Italic. The conserved nts between vIL6 and hIL6 are marked with *. (F) Diagram of the strategy for swapping the vIL6 region (black box) into hIL6. (G) The vIL6 region responsive to miR-1293 functions in hIL6 in the response to miR-1293-mediated repression. HEK293 cells transfected with Pre-miR-NC or Pre-miR-1293 for 48 h were transfected again with the indicated IL6-FLAG expression vector for additional 24 h before being immunoblotted with anti-FLAG M2 and β-tubulin antibodies. The numbers below each sample set indicate a relative level of hIL6 in each sample in a representative gel after being normalized to β-tubulin.