Reversible HuR-microRNA Binding Controls Extracellular Export of miR-122 & Augments Stress Response (2016)
Figure 1

Figure 1. Starvation induces extracellular export of miR-122 in mammalian hepatic cells.

(A-B) Levels of miR-122 in Huh7 cells and released EVs either untreated (Fed) or subjected to starvation for metabolites including amino acids for 16h (Starved). miR-122 signals were detected by Northern blotting and position of the 32P-labelled Oligos that served as size markers are shown in the lane (A). U6 snRNA was used as loading control. Cellular CAT-1 levels were measured by qRT-PCR using GAPDH mRNA as control (B). A scheme of experiment is shown in the top panel in B.

(C-D) Cellular and extracellular levels of different miRNAs and EV associated proteins in Fed and Starved Huh7 cells. Relative changes in cellular and extracellular levels (EV associated) of miR-122 and miR-24 in Fed or 8h Starved Huh7 cells were quantified by qRT-PCR and plotted (mean+/- s.e.m., n=3). Levels of individual miRNAs in Fed condition were taken as unit. Fold change (with SD) in the cellular level of all miRNAs and pre-miR-122 measured were shown in the bottom panel (C). Cellular miRNA levels were normalized against U6 snRNA. Western blot analysis of different exosomal proteins and Ago2 in EVs isolated under Fed or Starved conditions. CD63 is an exosomal marker protein and levels of HuR in EVs were quantified against respective CD63 levels. Mean of three independent measurements are plotted (middle panel in D). Calnexin and GAPDH are not detected in EVs but visible in the cellular extract. EV associated HA-HuR levels for Fed and Starved Huh7 cells expressing HA-HuR were also detected in the bottom panel in D.

(E) Effect of GW4869 treatment on cellular miRNA content in Fed and Starved cells. Levels of miRNAs were measured by real-time quantification and normalized against U6 snRNA. Mean data are from three independent experiments. DMSO treatment was used as control for GW4869 treated cells.

(F) CAT-1 and Aldolase mRNA expression in DMSO or GW4869 treated starved Huh7 cells. qRT-PCR techniques was adopted for quantification using 18S rRNA values for normalization (mean +/- s.e.m., n=3).

(G) Effect of siSMPD2 treatment on miR-122 content of EVs from Fed and Starved Huh7 cells. EVs from Fed and Starved Huh7 cells, depleted for SMPD2, were isolated and miR-122 content were measured by qRT-PCR and normalized against protein content of EVs. Fold change in EV associated miR-122 levels upon starvation both for control and siSMPD2 treated cells were shown above the respective bars.

(H) Effect of siSMPD2 treatment on CAT-1 mRNA content (left panel) and miR-122 levels (right panel) in Huh7 cells. RNAs, from Fed and Starved Huh7 cells, depleted for SMPD2, were isolated and miR-122 and CAT-1 mRNA contents were measured by qRT-PCR. miR-122 contents were normalized against U6 snRNA. GAPDH mRNA was used for normalization of CAT-1 mRNA levels (mean +/- s.e.m., n=3). miR-122 levels for siCon treated cells (Fed and Starved) were considered as units.

(I-J) A schematic representation of the experiment to separate Extracellular Vesicles (EVs) on OptiPrep@ density gradient (left panel). Densities of fractions 1-10 are plotted and a best-fit curve is drawn (right upper panel). CD63 levels in individual fractions of Fed and Starved cells were detected by western blots to confirm the presence of exosomes of Fed and Straved cells (right lower panel). Mean Ct values of miR-122 in RNAs isolated from Exosome enriched (8-9) vs Non-exosomal (1-3) fractions were analyzed and plotted (J) (mean +/- s.e.m., n=3).

(K) Effect of Starvation on apoptic cell numbers in Huh7 cells. Western Blot detection of apoptosis marker Cytochrome C in cellular and in EV associated fractions of Fed and Starved Huh 7 cells. β-Actin and CD63 are used as loading control for cellular and EV associated fraction respectively (left panel). TUNEL positive cells in Fed and Starved Huh7 cells. TUNEL assays of Fed and Starved Huh 7 cells were performed and TUNEL positive cells were quantified. Fed, but DNase treated, cells were used as positive control (+ve control) (right panel).

(L) Effect of thapsigargain (TG) treatment of Huh7cells on cellular and EV associated miR-122 level. A schematic representation of the experiment (upper panel). miR-122 levels were measured by qRT-PCR in total cellular RNA and in EVs released by the treated cells. Values were normalized either against U6 snRNA or protein content of EVs (mean+/- s.e.m., n=3) (lower panels) for cellular and EV associated RNA respectively.

(M) Effect of TG on cellular level of eIF2-α and its phosphorylated form, and Ago2 and CD63 in EVs. β-Actin was used as loading control for cellular samples.

For statistical analysis all experiments were done minimum three times and P-values were calculated. 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. For estimations of relative level of either miRNAs or mRNAs Ct values only within the range of 20-32 were considered for analysis.