Opposing activities of oncogenic MIR17HG and tumor suppressive MIR100HG clusters and their gene targets regulate replicative senescence in human adult stem cells

Abstract

Growing evidence suggests that many diseases of aging, including diseases associated with robust changes and adipose deports, may be caused by resident adult stem cell exhaustion due to the process called cellular senescence. Understanding how microRNA pathways can regulate cellular senescence is crucial for the development of novel diagnostic and therapeutic strategies to combat these pathologies. Herein, using integrated transcriptomic and semi-quantitative proteomic analysis, we provide a system level view of the regulation of human adipose-derived stem cell senescence by a subset of mature microRNAs (termed senescence-associated-microRNAs) produced by biogenesis of oncogenic MIR17HG and tumor-suppressive MIR100HG clusters. We demonstrate functional significance of these mature senescence-associated-microRNAs in the process of replicative senescence of human adipose-derived stem cells ex-vivo and define a set of senescence-associated-microRNA gene targets that are able to elicit, modulate and, most importantly, balance intimate connections between oncogenic and senescent events.

Acute Genotoxic Stress-Induced Senescence in Human Mesenchymal Cells Drives a Unique Composition of Senescence Messaging Secretome (SMS)

“…The pH-dependent senescence-associated β-galactosidase activity (SA-β-Gal) assay was performed as described in the manufacturer's kit (BioVision) and previously published [10,27,29]. Briefly, the cultured hADSCs were fixed in fixative solution for 15 min at room temperature, washed twice with PBS and stained with X-Gal containing supplement overnight at 37°C.…”

“…Immunostaining of the indicators of senescence p21 WAF1/Cip1 , p16 and p53BP1, was performed as described previously [10,29]. Additionally, γH2AX staining was performed to assess DNA damage and BrdU staining was used to quantitate the proliferating cells [10,29].…”

“…Additionally, γH2AX staining was performed to assess DNA damage and BrdU staining was used to quantitate the proliferating cells [10,29]. The antibodies used were p21 WAF1/Cip1 (12D1), a rabbit monoclonal Alexa Fluor 647 conjugated antibody and BrdU (Bu20a), a mouse monoclonal antibody (Cell Signaling Technology, Danvers, MA), anti-phospho histone γH2AX (Ser139) mouse monoclonal (Millipore) and 53BP1, a rabbit polyclonal antibody (Bethyl Laboratories, Inc. Montgomery, TX).…”

“…The nodes are color coded to show the proteins secreted at 48 hrs by SR (blue) or SEN (red) or both (blue and red) whereas the Steiner nodes are represented in gray (for Ref. [29]). …”

“…These limitations encompass signaling, metabolic and cytoskeletal changes, which ultimately result in the diminished ability of MSC to cope with DNA damage and other stressors. Reportedly these changes result in an inability to maintain the structure and function of chromatin-a process indispensable for the controlled execution of gene transcription program [10,28,29].…”