Figure 1

HOXA5 as a target of miR-26a-2 in LPS cells.

(a) Schematic diagram of HOXA5 3′UTR showing seed sequence (UACUUGAA) of miR-26a-2 binding site. In mutant construct, the seed sequence was mutated to UCAUUUAG by site-directed mutagenesis. WT = wild type. (b) Summary of dual luciferase assays. pGL3-Promoter 3′UTR luciferase reporter vector was cotransfected with either miR-26a-2 expression vector or empty vector control (EV) in 293T cells. After 48 h, cells were harvested and luciferase activity was quantified. Assays were repeated three times to ensure accuracy. Fluc/Rluc = Firefly luciferase/Renilla luciferase. Data represent average Fluc/Rluc ratio ± standard deviation (SD, error bars). Asterisk (*) indicate p-value less than 0.05 by t-test. NS = not significant. (c) Endogenous mRNA expression level of miR-26a-2, HOXA5 and TP53 in LPS cell lines. Data represent average relative mRNA expression (Rel. Exp.) ± SD. (d) Endogenous mRNA expression level of HOXA5 in normal adipose tissue (N) and LPS cell lines (LPS) shown in panel c. Each dot represents HOXA5 expression level of each sample. Horizontal bars represent average HOXA5 expression level within the group. (e) Effect of forced expression of miR-26a-2 on the mRNA expression level of HOXA5 and TP53 in LPS cell lines. Cells were transfected with either miR-26a-2 expression vector or EV. After 48 h, cells were harvested and subjected to qRT-PCR. Dashed lines indicate the expression level of each gene in cells with EV. (f) Effect of inhibition of miR-26a-2 using anti-miR-26a-2 oligos on the mRNA expression level of HOXA5 and p53 in LPS cells. Cells were transfected with either scrambled oligos (SCR) or anti-miR-26a-2. After 48 h, cells were harvested and subjected to qRT-PCR. Dashed lines indicate the expression level of each gene in cells with SCR.