microRNA-18b modulates insulin-like growth factor-1 expression in deer antler cell proliferation by directly targeting its 3' untranslated region

Abstract

Insulin-like growth factor-1 (IGF-1) is a multipromoter gene that has complex biological functions and plays an important role in Chinese sika deer antler cell differentiation and proliferation. microRNAs and their roles in deer antler growth have attracted much attention. In the present study, to investigate the effect of microRNAs on the regulation of IGF-1 during the rapid growth of antlers, miRNA GeneChip analysis and TargetScan Human software were used to screen microRNAs that bind to the 3' untranslated region (3'UTR) of IGF-1. The results indicated that a significantly differential expression of miR-18b was observed in cartilage and mesenchymal of antler tip tissue and the presence of miR-18b-binding sites within the IGF-1 3'UTR. A miR-18b mimic was then transfected into antler cartilage cells to overexpress miR-18b and the expression levels were quantified by real-time PCR. Real-time PCR showed that the expression level of miR-18b in transfected cells was significantly increased compared with the control group (p<0.01). Dual luciferase assays revealed that miR-18b decreased the fluorescence value of the luciferase reporter gene in the group transfected with the wild-type vector of IGF-1 3'UTR. In contrast, the relative luciferase activity in the group transfected with the mutant vector of IGF-1 3'UTR did not change obviously. MTT assays and cell cycle analyses confirmed that overexpression of the miR-18b mimic inhibited the proliferation of cartilage cells. In contrast, transfection of a miR-18b inhibitor increased the cell proliferation rate. Furthermore, Western blot analyses revealed that overexpression of miR-18b mimics downregulated the protein levels of IGF-1, while IGF-1 expression increased after transfection of miR-18b inhibitors. Taken together, our findings show that miR-18b is a potentially novel target in deer antler cell proliferation. miR-18b may modulate IGF-1 expression of sika deer antler.

FIG. 1.

Conservative analysis of the microRNA-binding sites in the 3′UTR of IGF-1. (A) microRNAs targeting the 3′UTR of IGF-1 were screened using a microRNA GeneChip to determine the microRNA content in cartilage and mesenchymal cells. Ratios of microRNA content between the two cell types that were more than double or less than half were considered to indicate differential expression. This figure shows the seed region sequence of miR-18b and its conserved target site in the 3′UTR of IGF-1, which was downloaded from the TargetScan website. Predicted miR-18b target sequences in the 3′UTR of IGF-1 are presented. (B) Comparison of microRNA fold changes. The relative quantitative results for endogenous microRNAs screened in cartilage and mesenchymal cells are shown. The U6 was used as an internal control. The results showed that the level of endogenous microR-18b was differentially expressed in cartilage and mesenchymal cells. Data are presented as the mean±SD (n=3). 3′UTR, 3′ untranslated region; IGF-1, insulin-like growth factor-1.

FIG. 2.

IGF-1 is a direct target of microRNA-18b. (A) Schematic diagram of the IGF-1 3′UTR luciferase reporters with wild type or one target site mutated. Constructs were generated using PCR. The seed sequence is underlined and mutant site is in red font. (B) Relative luciferase activity analysis in cartilage cells. Sika deer cartilage cells were cotransfected with the recombination firefly luciferase reporter plasmid pmiR-RB-Report™-IGF-1-3′UTR (wild type or mutant), the scramble group, and miR-18b mimic as indicated. Following 24, 48, and 72 h, the activity of firefly luciferase was measured. Luciferase activity decreased compared with the control group in cells that were transfected with the wild type of IGF-1 3′UTR. Luciferase activity did not change obviously compared with the control group, in cells that were transfected with the mutant of IGF-1 3′UTR. Data are presented as the mean±SD (n=3). Similar results were found after three independent experiments. *p<0.05; **p<0.01 compared with the control.

FIG. 3.

miR-18b content increased markedly following transfection. The relative quantitative results for the content of miR-18b in cartilage cells following transfection for 24, 48, and 72 h are shown. The U₆ gene was used as a loading control. The content of miR-18b in the cartilage cells increased markedly compared with that of the control cells and untransfected cells. Data are presented as the mean±SD (n=3). Similar results were found in three independent experiments. **p<0.01 compared with the control. NG, normal group.

FIG. 4.

miR-18b inhibits cartilage cell growth. Cartilage cells were transfected with the negative scramble control, microRNA-18b mimic, or inhibitor as indicated. The rate of cell inhibition was measured at the indicated times post-transfection using an MTT assay. Compared with the control, cell inhibition decreased after 24, 48, and 72 h, in cells that were transfected with miR-18b mimic. Also, cell inhibition increased after 24, 48, and 72 h in cells that were transfected with the miR-18b inhibitors compared with the control. Data are presented as the mean±SD (n=3). Similar results were found in three independent experiments. *p<0.05; **p<0.01, compared with the control.

FIG. 5.

miR-18b affects cell cycle distribution. After 24, 48, and 72 h, the cultured cell medium was replaced with a fresh serum-free medium. Cell cycle analysis was conducted at the indicated times postserum deprivation. Cell cycle analysis revealed that a miR-18b mimic prevented cartilage cells from entering S phase. The percentage of cells in G1 was higher compared with the control, and the percentage of cells in S phase was lower compared with the control. Meanwhile, the percentage of cells in S phase that were transfected with a miR-18b inhibitor was higher compared with the control. Data are presented as the mean±SD (n=3). Similar results were found in three independent experiments. *p<0.05, compared with the control.

FIG. 6.

Western blotting analysis of the effect of the microRNA mimic or inhibitor on the expression of IGF-1 protein. miR-18b inhibited the expression of IGF-1 protein. The expression of IGF-1 protein and GAPDH in sika deer cartilage cells was determined using Western blot analysis. Protein levels of IGF-1 decreased compared with the control group following 24, 48, and 72 h in cells that were transfected with the miR-18b mimic. Protein levels of IGF-1 increased compared with the control group transfected with the miR-18b inhibitor. Each panel includes three lanes and indicates the change of IGF-1 protein expression level after transfection for 24, 48, and 72 h. Primary antibodies against IGF-1 for the scrambled group, miR-18b mimics, and miR-18b inhibitor are rabbit polyclonal anti-IGF-1 antibodies. The primary antibody against GAPDH is the rabbit polyclonal anti-GAPDH antibody. Secondary antibodies are all goat anti-rabbit IgG-HRP conjugated.