Research Paper Volume 13, Issue 8 pp 11470—11490

Excessive expression of miR-1a by statin causes skeletal injury through targeting mitogen-activated protein kinase kinase kinase 1

Mitogen-activated protein kinase kinase kinase 1 (MAP3K1) is a target gene of miR-1a. (A) Computational target-scan analysis showed that miR-1a is able to bind the highly conserved target site (1589-1595, 5′-ACAUUCC-3′) in the 3′-UTR of MAP3K1 mRNA. (B) Plasmid of luciferase reporter construction containing 3′-UTR of MAP3K1 mRNA (WT-MAP3K1-UTR) or mutant of MAP3K1-UTR (MT-MAP3K1-UTR) was co-transfected with miR-1a negative control (NC) or mimics in HEK293 cells. The luciferase activities in total cell lysates were assayed. N is 5 in each group. **PC, D) Cultured skeletal muscle cells were transfected with miR-1a NC and mimics for 48 hours. The mRNA level of MAP3K1 in (C) and protein level in (D) were analyzed using real-time PCR and western blot, respectively. N is 5 in each group. *P **P post-hoc tests was used to determine P value in (B–D).

Figure 3. Mitogen-activated protein kinase kinase kinase 1 (MAP3K1) is a target gene of miR-1a. (A) Computational target-scan analysis showed that miR-1a is able to bind the highly conserved target site (1589-1595, 5′-ACAUUCC-3′) in the 3′-UTR of MAP3K1 mRNA. (B) Plasmid of luciferase reporter construction containing 3′-UTR of MAP3K1 mRNA (WT-MAP3K1-UTR) or mutant of MAP3K1-UTR (MT-MAP3K1-UTR) was co-transfected with miR-1a negative control (NC) or mimics in HEK293 cells. The luciferase activities in total cell lysates were assayed. N is 5 in each group. **P<0.01 vs. NC. (C, D) Cultured skeletal muscle cells were transfected with miR-1a NC and mimics for 48 hours. The mRNA level of MAP3K1 in (C) and protein level in (D) were analyzed using real-time PCR and western blot, respectively. N is 5 in each group. *P < 0.05 or **P < 0.01 vs. NC. A one-way ANOVA followed by Tukey post-hoc tests was used to determine P value in (BD).