Journal: Life Science Alliance

Article Title: Mutual feedback regulation between Poly(A)-specific ribonuclease (PARN) and cognate microRNAs

doi: 10.26508/lsa.202503341

Figure Lengend Snippet: (A, B) miR-29a-3p and miR-1207-5p overexpression down-regulate PARN mRNA and PARN protein. (A) qRT-PCR Relative Quantitive Chart representing PARN mRNA levels upon miR-29a-3p (blue columns) or miR-1207-5p (pink columns) overexpression. (C) Green bar indicates PARN mRNA levels from WT (not transfected) cells used for normalization. Numbers below the columns (24, 48) indicate hours post transfection. (B) Western blot showing PARN protein levels upon miR-29a-3p and miR-1207-5p overexpression 48 and 72 h post transfection. Extracts from WT (lane 1) and empty-vectortransfected H520 cells (lane 2) served as controls. β-actin was used as a loading reference. (C, D) miR-29a-3p and miR-1207-5p inhibition up-regulates PARN. (C) PARN mRNA levels in the presence of miR-29a-3p or miR-1207-5p LNAs in NCI-H520 cells. Cells were transfected with LNA against miR-29a-3p (blue columns) or LNA against miR-1207-5p (pink bars) and PARN mRNA levels were measured with qRT-PCR. A scramble LNA was used as a negative control (gray bars). (C) Green bar indicates PARN mRNA levels from WT (not transfected) cells used for normalization. Numbers below the columns indicate hours post transfection. (D) Levels of PARN protein upon transfection with LNAs against miR-29a-3p or miR-1207-5p. Numbers (48 and 72) above the lanes indicate hours post transfection. The type of LNAs is indicated above numbers: scramble, lanes 1 and 2; miR-29a-3p, lanes 3 and 4; miR-1207-5p, lanes 5 and 6. The red arrowhead indicates the position of 74-kD PARN (B, D).

Article Snippet: The extracts were precleared with 50 μl of protein A-Sepharose CL-4B beads (Acris Antibodies) at 4°C for 1 h with rotation followed by centrifugation at 10,000 g in 4°C for 10 min.

Techniques: Over Expression, Quantitative RT-PCR, Transfection, Western Blot, Inhibition, Negative Control