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• Research Paper Volume 6, Issue 3 pp 160-175

  MicroRNA-29 induces cellular senescence in aging muscle through multiple signaling pathways

  Relevance score: 7.026169

  Zhaoyong Hu, Janet D. Klein, William E. Mitch, Liping Zhang, Ivan Martinez, Xiaonan H. Wang

  Keywords: p50, p16Ink4A, RB, B-myb, sarcopenia, p85, IGF-1

  Published in Aging on March 12, 2014

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  The mechanisms underlying the development of aging-induced muscle atrophy are unclear. By microRNA array and individual qPCR analyses, we found significant up-regulation of miR-29 in muscles of aged rodents vs. results in young. With aging, p85α, IGF-1 and B-myb muscle levels were lower while the expression of certain cell arrest proteins (p53, p16 and pRB) increased. When miR-29 was expressed in muscle progenitor cells (MPC), their proliferation was impaired while SA-βgal expression increased signifying the development of senescence. Impaired MPC proliferation resulted from interactions between miR-29 and the 3'-UTR of p85a, IGF-1 and B-myb, suppressing the translation of these mediators of myoblast proliferation. In vivo, electroporation of miR-29 into muscles of young mice suppressed the proliferation and increased levels of cellular arrest proteins, recapitulating aging-induced responses in muscle. A potential stimulus of miR-29 expression is Wnt-3a since we found that exogenous Wnt-3a stimulated miR-29 expression 2.7-fold in primary cultures of MPCs. Thus, aging-induced muscle senescence results from activation of miR-29 by Wnt-3a leading to suppressed expression of several signaling proteins (p85α, IGF-1 and B-myb) that act coordinately to impair the proliferation of MPCs contributing to muscle atrophy. The increase in miR-29 provides a potential mechanism for aging-induced sarcopenia.

  

  (A) Total RNA obtained from hind-limb muscles of young and aged rats and mice were assayed for miR-29a, b and c by real time qPCR. The bar graph shows miR-29a, b and c in aged rodents muscles expressed as a fold change above the control (young rodent) which is represented by a line at 1-fold. Results are normalized to U6 RNA (Bars: mean ± s.e.; n=6 pairs; *p<0.05 vs. young). (B) Total RNA from hind-limb muscles of young and aged rats and mice were assayed for IGF-1, p85 and B-myb expression by real time qPCR. The bar graph shows IGF-1, p85 and B-myb in aged rodents muscles expressed as a percentage of control (young rodent) which is represented by a line at 100%. Results are normalized to 18S RNA (Bars: mean ± s.e.; n=6 pairs; *p<0.05 vs. young). (C) IGF-1 protein level was measured by ELISA in muscle lysates from young and old rodents. Results in the bar graph compare the amount of IGF-1 in muscle from old (O) vs. young (Y) rats and mice. All data were normalized to the muscle total protein concentration (Bars: mean ± s.e.; n=6; *p<0.05 vs. young). (D) p85α B-myb, p53, p16^INK4A, RB and GAPDH proteins were measured by western blotting of muscle lysates from young and old mice. Two bands of the RB protein were detected: the lower band is hypophosphoryated RB (pRB; MW 107 kDa) while the upper band is more highly phosphorylated RB protein (ppRB; MW 112kDa). Results in the bar graph compare the densities of protein bands in aged muscle expressed as a fold-change from levels in young mice which is represented by a line at 1-fold. All band densities were normalized to the density of GAPDH (Bars: mean ± s.e.; n=6; *p<0.05 vs. young).

  
  
  

  

  (A) MPCs were transduced with Ad-miR-29 or the control adenovirus (Ad-empty). Proliferation was assessed using a chromogenic substrate and a commercial kit (Millipore) monitoring absorbance at 420 nm (Methods). The bars show the mean ± s.e. (n=6; *p<0.05 vs. Ad-empty). (B) MPCs were transduced with Ad-miR-29 or Ad-empty. The Ki67 expression, a marker of proliferation, was assessed by immunohistology. A positive Ki67 was determined as a bright red spot localized in the nuclei. The bar graph shows cells that were positively stained for Ki67 in miR-29 treated cells and are expressed as a percent of the positive cells in the Ad-empty treated cells. Counts were made in 5 pre-defined, randomly chosen fields; each field had >500 nuclei. Red staining in nuclei was assessed using the Micro-suite Five Biological Software (Bars: mean ± s.e.; n=6/condition; *p<0.05 vs. ad-empty). (C) MPCs were transduced with Ad-miR-29 or with the Ad-empty adenovirus. To block miR-29 expression in MPCs, vectors that express an antisense of Zip-miR29 (pmiRZip29a plus pmiRZip29c) were transfected into MPCs 4 hours before the Ad-miR-29 or Ad-empty viruses were added. The blue color identifies SA-βgal is present in cells with the “fried egg morphology” that signifies cellular senescence. The bar graph shows the percentage of cells with positive staining of SA-βgal in 5 pre-defined, randomly chosen fields (Bars: mean ± s.e.; n=6/condition; *p<0.05 vs. controls; #p<0.05 vs. Ad-miR-29). (D) MPCs were transduced with Ad-miR-29 or with Ad-empty adenovirus. To block miR-29 expression in MPCs, vectors that express an antisense of miR-29a+c (pmiRZip29a plus pmiRZip29c) were transfected into MPCs 4 hours before the Ad-miR29 or Ad-empty (Ctrl) viruses were added. miR-29a expression was 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. Ad-miR29 only).

  
  
  

  

  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).

  
  
  

  

  (A) MPCs were transfected with pLuc-ctrl, pLuc-p85α-3'-UTR or pLuc-mutant-p85α (p85α with a mutated 3'-UTR binding site for miR-29). Then they were treated with the empty virus (Ctrl), the miR-29 adenovirus to over-express miR-29 (miR-29) or miR-29 inhibitor (Zip29a or Zip29c). Luciferase activity in cells that received the control virus was designated as the 100% activity level (designated by a horizontal line in the graph). The other bars all show the response of the cells to miR-29 or zip-miR-29 (indicated below each bar) expressed as a percent of the control level for each experiment. Left (white bars): the effect of miR-29 on p85α 3'-UTR; middle (black bar): the effect of miR-29 on mutated p85α 3'-UTR; right (gray bars): the effect of inhibited endogenous miR-29 on p85α 3'-UTR. Triplicate determinations were made in each condition and each experiment was repeated a total of three times; the results were combined to calculate differences in firefly luciferase activity normalized by renilla luciferase activity. The data represent mean ± s.e.; (n=9; *p<0.05 vs. Ad-ctrl).(B) MPCs were transfected with constructs containing the two miR-29 binding sites on IGF-1 3'-UTR, pMIR-IGF/321-3217 (IGF/a) and pMIR-IGF/3275-5574 (IGF/b), before being treated with the empty adenovirus (Ctrl), Ad-miR-29 (miR29) or miR-29 inhibitor (Zip29a or Zip29c). Luciferase activity in cells that received the control virus was designated as the 100% activity level (designated by a horizontal line in the graph). The other bars all show the response of the cells to miR-29 or zip-miR-29 (indicated below each bar) expressed as a percent of the control level for each experiment. Left (white bars): the effect of miR-29 on the two IGF 3'-UTR binding sites (IGF/a or IGF/b); middle (black bars): the effect of miR-29 on mutated (m-) IGF/a or IGF/b; right (gray bars): inhibited endogenous miR-29 effect on IGF/a or IGF/b. The results were combined to calculate differences in firefly luciferase activity normalized by renilla luciferase activity. The data represent mean ± s.e.; (n=9; *p<0.05 vs. Ad-ctrl). (C) MPCs were transfected with pLuc-ctrl, pLuc-3'UTR-B-myb (pLuc-myb) or pLuc-mutant-B-myb (pLuc-mut-myb). Then they were treated with the empty virus (ctrl), the miR-29 adenovirus (miR29) to over-express miR-29 or miR-29 inhibitor (Zip29a or Zip29c). Luciferase activity in cells that received the control virus was designated as the 100% activity level (designated by a horizontal line in the graph). The other bars all show the response of the cells to miR-29 or miR-29 inhibitor (Zip29a or Zip29c) expressed as a percent of the control level for each experiment. Left (white bars): the effect of miR-29 on B-myb 3'-UTR; middle (black bar): the effect of miR-29 on mutated B-myb 3'-UTR; right (gray bars): inhibited miR-29 effect on B-myb 3'-UTR. The results were combined to calculate differences in firefly luciferase activity normalized by renilla luciferase activity. The data represent mean ± s.e.; (n=9; *p<0.05 vs. Ad-ctrl).

  
  
  

  

  (A) MPCs were transfected with pGL3-miR-29a/b1 (gray bar) or pGL3-miR-29C/b2 (black bar) to assay miR-29 promoter activities. Transfected cells were treated as follows: left to right, treated with TGF-β; co-transfected with NFκB plasmid (pCMV1.p65); transduced with Ad-Wnt-3a; or treated with Wnt-3a-conditioned media. The bar graph represents firefly luciferase activity corrected for renilla luciferase activity (FFL/RL) and compared to control (luciferase activity of pGL3 untreated, set to 1 and designated by a horizontal line in the graph). The data represent the means ± s.e.; (n=9, *p<0.05 vs. pGL3 (Ctrl). (B) MPCs were cultured in Wnt-3a conditioned media and cells harvested at the indicated times. Total RNA was isolated and miR-29a expression was measured by qPCR. The bar graph shows miR-29a from Wnt-3a conditional medium expressed as a fold-change vs. miR-29a levels in cells cultured with control media (set to 1). Results are normalized to U6 RNA as an internal control. The data represent the means ± s.e.; (n=3 pairs; *p<0.05 vs. control).