MicroRNA-195a-5p inhibits mouse medullary thymic epithelial cells proliferation by directly targeting Smad7

Abstract

MiR-195 has been implicated in inhibiting cell proliferation in different types of tumors. Whether it contributes to the process of thymic epithelial cells (TECs) proliferation remains unclear. In this study, we found that miR-195a-5p was highly up-regulated in the TECs isolated from the aging mice. Further experiments showed that miR-195a-5p mimic transfection inhibited the proliferation of mouse medullary thymic epithelial cell line 1 (MTEC1), whereas the transfection of miR-195a-5p inhibitor in MTEC1 had the opposite effect. In addition, miR-195a-5p had no obvious effect on MTEC1 apoptosis. Furthermore, Smad7, a negative regulator of transforming growth factor β pathway, was confirmed as a direct target of miR-195a-5p by luciferase assays. Taken together, our results indicate that miR-195a-5p inhibits MTEC1 proliferation, at least in part, via down-regulation of Smad7.

Keywords: cell proliferation, Smad7; miR-195a-5p; mouse thymic epithelial cell.

Figure 1.

The expression of miR-195a-5p in isolated TECs and the effects of miR-195a-5p supplementation on cell viability and apoptosis (A) miR-195a-5p was detected by qRT-PCR in TECs from 1-, 10- and 19-month-old mice, U6 was used as a reference. (B, C) Cell viability analysis was performed by CCK-8 at 24, 48, and 72 h after transfection. (D) At 48 h after transfection, cell apoptosis was analyzed by flow cytometer. Data were presented as the mean ± SD of three experiments. *P < 0.05, **P < 0.01.

Figure 2.

The cell cycle distribution of MTEC1 cells at 48 h after treatment with miR-195a-5p mimic or inhibitor Cell cycle was performed by flow cytometry after transfection with miR-195a-5p mimic/mimic-NC (A, B) and miR-195a-5p inhibitor/inhibitor-NC (C, D). Data were presented as the mean ± SD of three experiments. *P < 0.05, **P < 0.01.

Figure 3.

Expressions of cell cycle-related genes analyzed by qPCR and western blot analysis (A) The mRNA expressions of Cyclin D1, Cyclin E1, Cyclin D3, Cdk1, Cdk2, Cdk4, E2F1, and C-myc in miR-195a-5p mimic group. (B) The expressions of Cyclin D1, Cyclin E1, Cyclin D3, Cdk4, Cdk6, E2F1, and C-myc in miR-195a-5p inhibitor group. (C, D) The protein expressions of Cdk4, Cyclin D1, Cyclin E1, and C-myc in miR-195a-5p mimic and inhibitor-transfected MTEC1 cells, respectively. The data were normalized to the level of β-actin for mRNA and to the level of tubulin for protein in each sample. *P < 0.05, **P < 0.01.

Figure 4.

Confirmation of Smad7 as a direct target gene of miR-195a-5p (A) The putative binding sites in Smad7 3′UTRs for miR-195a were predicted by Targetscan and the three muted nucleotides in mutant Smad7 3′UTR were shown. (B) The luciferase activity assay in HEK-293T cells were calculated as ratio of firefly to renilla after the cells were co-transfected with miR-195a-5p mimic or mimic-NC with pmiRGLO-Smad7-mut and pmiRGLO-Smad7-wt. Data were presented as the mean ± SD of three experiments. **P < 0.01. (C) The mRNA and (D) protein expression levels of Smad7 in MTEC1 cells 48 h after transfected with miR-195a-5p mimic, mimic-NC, miR-195a-5p inhibitor or inhibitor-NC. Data were normalized to the level of β-actin for mRNA and to the level of tubulin for protein in each sample. *P < 0.05.