Mitochondrial DNA Hypomethylation Is a Biomarker Associated with Induced Senescence in Human Fetal Heart Mesenchymal Stem Cells

Figure 5

Knockdown of DNMTs induces senescence in MSCs. (a) Downregulation of DNMTs in senescent MSCs. After induction of cellular senescence, cells were harvested and the expression of DNMTs was determined by semiquantitative PCR. (b) Knockdown of DNMTs by shRNAs. Lentiviruses containing DNMT shRNAs (sh-DNMT1, sh-SNMT3a, and sh-DNMT3b) or scramble control (sh-CT) were transduced into HMSCs. After 72 h, transduction efficiency was assessed by the observation of GFP positive cells. Cells were harvested and RNA were extracted, and the expression of DNMTs was determined by RT-PCR. (c) Induction of cellular senescence by DNMT-shRNA knockdown. Left panel: HMSCs morphology taken 7 days after DNMT-shRNA transduction. Sh-CT: shRNA scramble control; sh-DNMTs: HMSCs were transducted by lentiviruses containing DNMT1, DNMT3a, and DNMT3b shRNAs. Middle panel: lentiviral transduction efficiency as shown by copGFP fluorescence of the shRNA vector. Right panel: senescent-associated β-Gal staining. After DNMT-shRNA knockdown, MSCs were stained for β-Gal activity. Note the occurrence of senescence in DNMT-knockdown MSCs in the absence of peroxide treatment. (d) Expression of COX and ND2 in DNMT-shRNA-treated MSCs. β-Actin was used as the internal control for PCR reaction. Cox1 was upregulated in DNMT-knockdown MSCs in parallel with cellular senescence.

(a) Methylation enzymes

(b) shRNA knockdown

(c) β-Gal staining

(d) Mitochondrial gene expression