FIGURE 5.

Tpm1 is a potential miR-29c target during renal fibrosis. (A) Schematic diagram summarizing target genes identified using four microRNA target prediction programs (miRNADA, microCosm, miRDB, and miRNAMAP) with the potential targets identified by all four of the programs. (B) Luciferase reporter activity assay performed in HEK 293T cells infected with a WT reporter plasmid (WT) + miR-29c or a mutant 3’UTR TPM1 reporter plasmid (MUT) + miR-29c. Representative western blots show protein levels of TPM1 in kidneys transfected with pre-miR-29c (C) or miR-29c inhibitor (D) 10 days after the sham procedure or UUO surgery. Protein expression was normalized with β-actin. (E) Quantitative analysis of the protein levels in (C). (F) Quantitative analysis of the protein levels in (D). RT-PCR results of TPM1 expression in kidneys transfected with pre-miR-29c (G) or miR-29c inhibitor (H) 10 days after the sham procedure or UUO surgery. Representative western blots show levels of α-SMA, HGF, fibronectin, and Col3a1 proteins in kidneys transfected with pre-miR-29c + AAV-TPM1 OE (I) or miR-29c inhibitor + AAV-shTPM1 construct (K) 10 days after the sham procedure or UUO surgery. Protein expression was normalized with β-actin. (J) Quantitative analysis of the protein levels in (I). (L) Quantitative analysis of the protein levels in (K). RT-PCR results for the expression of several fibrosis-related genes (α-SMA, HGF, fibronectin, and Col3a1) in kidneys transfected with pre-miR-29c + AAV-TPM1 OE (M) or miR-29c inhibitor + AAV-shTPM1 construct (N) 10 days after the sham procedure or UUO surgery. Representative photomicrographs of the kidney sections transfected with pre-miR-29c + AAV-TPM1 OE (O) or miR-29c inhibitor + AAV-shTPM1 construct (P) 10 days after the sham procedure or UUO surgery, stained for α-SMA, and counterstained with DAPI (blue). The data are presented as mean ± SEM values. Symbols (“^∗”, “#”, “&” and “^∧”) represent statistical significance.