Research Paper Volume 6, Issue 3 pp 160—175

Figure 3. miR-29 expression in muscles of mice decreases IGF-1, p85α, B-myb but increases cell cycle arrest proteins.

The mouse microRNA mimic, miR-29a (mmu-miR-29a), was injected into the right tibialis anterior (TA) muscle of 4 month old normal mice; permeation increased by muscle electroporation. The left TA muscle was used as a control and injected with mouse mimic miR-ctrl (mmu-miR-ctrl) and electroporated. Muscles were harvested at 7 and 30 days after electroporation. (A) RNA isolated from TA muscles and miR-29a expressions were measured using real-time qPCR; U6 was the internal control. The bar graph shows the amount of miR-29a expressed as a fold change from the level in controls (Bars: mean ± s.e.; ctrl defined as 1 fold; n=3 determinations per condition; *p<0.05 and **p<0.01 vs. control). (B) IGF-1 protein level was measured by ELISA in muscle lysates. Results in the bar graph compare the protein amount of IGF-1 in control muscle (ctrl) vs. miR-29 overexpressing muscle. All data were normalized to the muscle total protein concentration (Bars: mean ± s.e.; n=6; *p<0.05 vs. control). (C) Protein levels of P85α, B-myb, P53, P16^INK4A, RB and GAPDH in lysates from muscles at 7 days after electroporation were measured by Western blotting. There were two RB protein bands: the lower is hypophosphoryated RB (pRB; MW 107 kDa) and the upper band is more highly phosphorylated RB protein (ppRB; MW 112kDa). The bar graph shows the density of each protein band expressed as a fold-change from control levels (mimic miR-ctrl was set to 1, indicated by a horizontal line in the graph). All band densities were normalized to the density of GAPDH (Bars: mean ± s.e.; n=6; *p<0.05 vs. ctrl). (D) The proliferation marker (Ki67) was assessed by immuno-histochemistry in TA muscles at 30 days after electroporation of mmu-miR-29 or mmu-miR-ctrl into muscles. The bar graph shows the percentage of positive staining nuclei of Ki67 in 10 predetermined, random fields for each condition(Bars: mean ± s.e.; n=6/condition; *p<0.05 vs. controls). (E) The level of the senescence marker, SA-βgal, was assessed in cross sections of TA muscles at 30 days after electroporation of the mmu-miR-29 or mu-miR-ctrl. The blue color shows the presence of SA-βgal indicating the presence of cellular senescence. The bar graph shows the percentage of positive staining in 10 predetermined, random fields for each condition(Bars: mean ± s.e.; n=6/condition; *p<0.05 vs. controls).