Figure 4.

Tmod3 Is a Target Gene of miR-145

Candidate target genes of miR-145—Krüppel-like factor 4(Klf4), semaphorin 3A (Sema3a), TMEM9 domain family member B (Tmem9b), tropomodulin 3 (Tmod3), and trio Rho guanine nucleotide exchange factor (Trio)—were identified by analyzing the seed sequence of miR-145, conserved in both human and murine, for sequence complementarity to determine predicted target mRNAs. (A) Expression levels of Klf4, Tmem9b, Tmod3, and Trio were significantly downregulated in P17 OIR retinas compared with normoxic control retinas (n = 6 per group). c-Myc, a known miR-145 target gene, served as the positive control. Expression levels were normalized to 18S and then to the levels in normoxic retinas. (B) The sequence alignment between mouse miR-145 and the putative binding sites in the 3′ UTR of target genes, with sequences recognized by the miR-145 seed sequence shown in red. (C) Luciferase reporter assays of putative miR-145 target genes were performed in HEK293T cells. Cells were transfected with 25 or 50 nM of miR-145 mimics or the negative control, and each was co-transfected with constructs of luciferase reporter containing miR-145 target sequences of Klf4, Sema3a, Tmem9b, Tmod3, and Trio. Cells transfected with the Tmod3 target sequence resulted in significant repression of luciferase activity in a miR-145 dose-dependent pattern (n = 3–6 per group). Firefly luciferase activities were normalized to the Renilla luciferase and to the levels in the negative control. (D and E) Western blotting shows the expression of TMOD3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in P17 OIR and normoxic control retinas. (E) Densitometric quantification shows that TMOD3 protein levels were substantially decreased in P17 OIR retinas compared with normoxic controls. GAPDH served as the internal loading control (n = 3 per group). (F) miR-145 expression levels were analyzed by real-time qPCR using total RNA obtained from HRMECs exposed to hypoxia for the indicated period of time. miR-145 expression was elevated over time in hypoxic HRMECs. miR-145 expressing levels were normalized to U6 snRNA and normoxia controls (n = 3 per group). (G) TMOD3 protein levels in HRMECs exposed to hypoxia for the indicated period of time were detected by western blot assay. GAPDH expression was used as a loading control. M, molecular weight markers; N, normoxia. Data are presented as means ± SEM. *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.001.