Reversible HuR-microRNA Binding Controls Extracellular Export of miR-122 & Augments Stress Response (2016)
Figure 2

Figure 2. Human ELAV protein HuR binds miRNA in starved cells and is necessary for extracellular export of miRNA

(A) Relative amount of miRNAs bound to HuR that are immunoprecipitated either from Fed or Starved Huh7 cells. Immunoprecipitated materials were separated into two equal halves to do western blot analysis of HuR and relative quantification of associated miR-122 and miR-21 were done by quantitative RT-PCR (mean +/- s.e.m., n=3). GFP used as negative control to confer the specificity of the anti-HuR 3A2 antibody in immunoprecipitating HuR. The miR-122 content in the GFP-antibody immunoprecipitated materials was too low to get detected reliably using the qRT-PCR.

(B-C) Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells. Schemes of HuR depletion experiments in Huh7 cells (B). Relative levels of miRNAs were measured in EVs released from siCon or siHuR treated cells by qRT-PCR. Cellular miRNA contents were normalized against U6snRNA. Levels of each miRNA in control siRNA (siCon) treated set were taken as unit. In siHuR treated cells, depletion of HuR was monitored by western blotting (mean+/- s.e.m., n=4) (C). CAT-1 and Aldolase mRNA levels of siHuR and siCon treated Fed Huh7 cells were quantified by qRT-PCR and normalized against GAPDH mRNA.

(D) Effect of HuR knockdown on cellular level of different miRNAs in starved Huh7 cells. Relative levels were estimated by real time qPCR, normalized with respect to U6 snRNA and plotted in the left panel. Levels of miR-122 in EVs of starved siHuR and siCon treated Huh7 cells were also measured by RT-PCR and plotted in the right panel (mean+/- s.e.m., n=3).

(E) Level of eIF-2 α and its phosphorylated form, estimated by western blot using specific antibodies, in siCon or siHuR treated and starved Huh7 cell extracts. Western blot data of HuR confirm reduction of this protein upon siRNA treatment. CAT-1 and Aldolase mRNA levels were estimated by real time quantification and normalized against GAPDH level. Values of siCon treated samples were taken as unit (mean+/- s.e.m., n=3). In the right panel the relative quantity of phosphorylated eIF-2 α measured by densitometry were plotted. Levels of p38 and phosphor-p38 levels in siCon and siHuR treated Starved Huh7 cells were detected by western blot.

(F) Western blot analysis to detect the level of phosphorylated eIF-2 α and eIF-4E-BP1 in lysates of Starved and HuR depleted Huh7 cells either transfected with ant-let-7a or anti-miR-122 oligos. miR-122 inactivation was monitored by measuring cat-1 level by qRT-PCR using GAPDH as control (mean+/- s.e.m., n=3). * denotes the phosphorylated eIF-4E-BP1. β-Actin was used as loading control. Relative levels of phosphorylated eIF2-α were measured by densitometric estimation of three independent measurements.

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.