Objective      To investigate the regulation of mitochondrial microRNAs (MitomiRs) by polynucleotide phosphorylase (PNPase) inhibition

and the possible protection effects on mitochondrial DNA (mtDNA).       Methods      Human hepatocellular cell lines SK-Hep1 and Hep G2 and human bone sarcoma cell line U2OS were transfected by PNPase shRNA lentivirus with green fluorescent protein (GFP). PNPase expression was detected by Western blotting. Then the mitochondria were separated

and MitomiRs were extracted. MitomiRs were analyzed by microRNA chips before and after PNPase shRNA treatment. MtDNA damage frequency was assessed by Q-PCR

and mitochondrial 8-hydroxydeoxyguanosine (8-OHdG) was detected by ELISA.       Results      Visible green fluorescence was found in SK-Hep1

HepG2 and U2OS cells transfected with PNPase shRNA lentivirus in 12 to 24 h later. When the MOI (transfection plural) was 20

the transfection efficiencies of 3 kinds of cells were more than 80%. In the next 2 consecutive weeks

the transfection efficiencies maintained at 70% to 80%. PNPase expression in the cells transfected with PNPase shRNA lentivirus was lower than that in the normal control and negative control cells. MicroRNA chip analysis results showed the obvious changes of MitomiRs profiles in the PNPase shRNA-treated cells. MiR-30c-2-3p

miR-494

miR-1273-g-3p

and miR-4443 were upregulated

while miR-324-3p

miR-574-5p

miR-371-b-5p

miR-6068

and miR-21-5p were downregulated. MtDNA damage frequency was reduced in the cells transfected with PNPase shRNA

and the content of 8-OHdG in the mitochondria of those cells was lowered.       Conclusion      Inhibition of  PNPase expression in tumor cell mitochondria results in changes of MitomiRs expression. At the same time

mtDNA damage is reduced

implying that MitomiRs may participate in the regulation of mtDNA replication.