Reversible HuR-microRNA Binding Controls Extracellular Export of miR-122 & Augments Stress Response (2016)
Figure 3

Figure 3. HuR is sufficient for EV-mediated export of miRNAs

(A) Northern blot of miR-122 in Huh7 cells expressing the Control and HA-HuR expression plasmids. The U6 was used for loading control. Position of the 32P-labelled Oligos, blotted to the membrane used for Northern blotting, served as size markers are shown in the M lane.

(B) Schematic representation of experiments done in Huh7 cells expressing HA-HuR (bottom). For control, pCIneo transfected Huh7 cells were used. Relative expression of HuR in transfected cells were analysed by western blot (top). β-actin blots were used as loading controls.

(C) Cellular and EV-associated miRNA levels were measured in Huh7 cells either expressing the pCIneo or HA-HuR expressing vectors by qRT-PCR (mean+/- s.e.m., n=5). Cellular miRNA levels were normalized against U6 snRNA.

(D) Experimental scheme for EV isolation either by affinity purification technique using CD63 positive biotinylated beads or by ultracentrifugation from Huh7 cell conditioned medium (Huh7CM).

(E) Effect of HA-HuR expression on miRNA levels on EV and non-EV fractions of the culture supernatant collected from control and HA-HuR encoding plasmid transfected Huh7 cells. For isolation of non-EV fraction, supernatant obtained after removal of EVs by ultracentrifugation was used for RNA preparation (left panel) (mean+/- s.e.m., n=3). Effect of HuR expression on CD63, Alix and HuR levels in EVs. Extracts from EVs isolated from control or HA-HuR expressing Huh7 cells were western blotted for respective proteins (right panel).

(F) Relative miR-122 levels in CD63 positive affinity purified EVs isolated from HA-HuR and pCIneo transfected Huh7 cells were quantified by qRT-PCR (mean+/- s.e.m., n=3). The levels of miR-122 were normalized against CD63 content of EVs.

(G) Effect of GW4869 on EV associated content of three different miRNAs in HA-HuR expressing Huh7 cells quantified by qRT-PCR (mean+/- s.e.m., n=5). EVs from DMSO treated cells were used as control.

(H) A schematic representation of the experiment done to characterize EVs isolated from Huh7 cells expressing HA-HuR or pCIneo respectively by running OptiPrepTM density gradient (left panel) and western blotting for CD63 (right upper panel). Relative miR-122 levels in fractions 1-2 and 8-9 were compared by qRT-PCR (right lower panel).

(I) Levels of apoptic cells in HA-HuR expressing Huh7 cells. Western Blot detection of apoptosis marker Cytochrome C was done (top) for EV associated and cellular fractions of HA-HuR and pCIneo (control) expressing Huh 7 cells respectively. Western blot for HA-HuR was done to analyse the expression of HA-HuR in EVs isolated from HA-HuR and pCIneo expressing Huh7 cells. CD63 and β-actin are used as loading control for EV associated fractions and cellular fractions respectively (upper panels). TUNEL assay of HA-HuR and pCIneo expressing and non-transfected Huh 7 cells was performed and representative pictures of HA-HuR and pCIneo expressing cells along with picture of non-transfected but DNase treated (positive control ) cells were compared (lower panels). Scale bar 50 µM.

ns: non-significant, * P<0.05, **P<0.01, ***P<0.0001.P values were determined by paired t test. Position of size markers in protein gels used for respective western blot analysis are shown against each panels. HC; Heavy Chain and LC; Light Chain of IgG used for immunoprecipitation reaction. For estimations of relative level of either miRNAs or mRNAs Ct values only within the range of 20-32 were considered for analysis.