IGF-1/IGF-1R/hsa-let-7c axis regulates the committed differentiation of stem cells from apical papilla

Abstract

Insulin-like growth factor-1 (IGF-1) and its receptor IGF-1R play a paramount role in tooth/bone formation while hsa-let-7c actively participates in the osteogenic differentiation of mesenchymal stem cells. However, the interaction between IGF-1/IGF-1R and hsa-let-7c on the committed differentiation of stem cells from apical papilla (SCAPs) remains unclear. In this study, human SCAPs were isolated and treated with IGF-1 and hsa-let-7c over/low-expression viruses. The odonto/osteogenic differentiation of these stem cells and the involvement of mitogen-activated protein kinase (MAPK) pathway were subsequently investigated. Alizarin red staining showed that hsa-let-7c low-expression can significantly promote the mineralization of IGF-1 treated SCAPs, while hsa-let-7c over-expression can decrease the calcium deposition of IGF-1 treated SCAPs. Western blot assay and real-time reverse transcription polymerase chain reaction further demonstrated that the expression of odonto/osteogenic markers (ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN, COL-I/COL-I, DSPP/DSP, and DMP-1/DMP-1) in IGF-1 treated SCAPs were significantly upregulated in Let-7c-low group. On the contrary, hsa-let-7c over-expression could downregulate the expression of these odonto/osteogenic markers. Moreover, western blot assay showed that the JNK and p38 MAPK signaling pathways were activated in Let-7c-low SCAPs but inhibited in Let-7c-over SCAPs. Together, the IGF-1/IGF-1R/hsa-let-7c axis can control the odonto/osteogenic differentiation of IGF-1-treated SCAPs via the regulation of JNK and p38 MAPK signaling pathways.

Figure 1. Identification of SCAPs and screening of MOI values for lentiviral infection.

(a) Isolated SCAPs with typical fibroblast- or spindle-like morphology. (b) Isolated SCAPs were immunopositive against STRO-1 by flow cytometry. (c) Isolated SCAPs were positive for CD73, CD90 and CD105, respectively, but negative for CD45 and CD34 by flow cytometry. (d) Cell morphology under microscope in α-MEM and α-MEM+POL (Polybrene) groups with different virus titers. (e) Cell fluorescence expression under an inverted microscope in α-MEM and α-MEM+POL groups with different virus titers.

Figure 2. Negative correlation between hsa-let-7c and IGF-1R.

(a) Hsa-let-7c detection in IGF-1-treated SCAPs by miRNA microarray. (b) The predicted consequential pairing of target region (top) and miRNA (bottom) between IGF-1R and hsa-let-7c on the TargetScanHuman website. (c) Immunofluorescent staining of IGF-1R in SCAPs. Nuclei are stained in blue and IGF-1 receptor was in green. (d) Real-time RT-PCR analysis for the expression of hsa-let-7c in Con-over group, Let-7c-over group, Con-low group and Let-7c-low group, respectively. Values were described as the means ± SD, n = 3. **2^−∆∆Ct > 2, P < 0.01; *1 < 2^−∆∆Ct < 2, P < 0.01. (e) Western blot assay for the expression of IGF-1R in Con-over group, Let-7c-over group, Con-low group and Let-7c-low group, respectively. (f) Quantitative analysis for western blot results. Values were described as the means ± SD, n = 3. **P < 0.01.

Figure 3. IGF-1/IGF-1R/hsa-let-7c axis had no significant influence on the proliferation of SCAPs.

(a) CCK8 assay of 13 consecutive days presented no significant difference (P > 0.05) between Con-over group and Let-7c-over group. (b) CCK8 assay of 13 consecutive days displayed no significant difference (P > 0.05) between Con-low group and Let-7c-low group. (c) Flow cytometry (FCM) analysis of Con-over group. (d) Flow cytometry analysis of Let-7c-over group. (e) Flow cytometry analysis of Con-low group. (f) Flow cytometry analysis of Let-7c-low group.

Figure 4. IGF-1/IGF-1R/hsa-let-7c axis can regulate the odonto/osteogenic differentiation of SCAPs.

(a) Alizarin red staining for control group, insulin-like growth factor-1 (IGF-1) group, mineralization-inducing media (MM) group and MM+IGF-1 group at day 14, respectively. (b) Calcium nodules in different groups under the inverted microscope. Scale bars = 100 μm. (c) Quantitative analysis for calcium contents in the Let-7c-over group. Values were the means ± SD, n = 3. **P < 0.01. (d) Quantitative analysis for calcium contents in the Let-7c-low group. Values were the means ± SD, n = 3. **P < 0.01. (e) Real-time RT-PCR analysis for the expression of DMP-1, COL-I, RUNX2, OSX, DSPP and OCN in Con-over group and Let-7c-over group respectively. (f) Real-time RT-PCR analysis for the expression of DMP-1, COL-I, RUNX2, OSX, DSPP and OCN in Con-low group and Let-7c-low group respectively. Values were described as the means ± SD, n = 3. **2^−∆∆Ct > 2, P < 0.01; *1 < 2^−∆∆Ct < 2, P < 0.01. (g) Western blot assay for the odonto/osteogenic proteins (OCN, DSP, OSX, RUNX2, ALP, COL-I and DMP1) in Con-over group and Let-7c-over group respectively. (h) Western blot assay for the odonto/osteogenic proteins (OCN, DSP, OSX, RUNX2, ALP, COL-I and DMP1) in Con-low group and Let-7c-low group respectively. (i) Quantitative analysis for western blot results in Con-over group and Let-7c-over group respectively. Values were described as the means ± SD, n = 3. *P < 0.05, **P < 0.01. (j) Quantitative analysis for western blot results in Con-low group and Let-7c-low group respectively. Values were described as the means ± SD, n = 3. *P < 0.05, **P < 0.01.

Figure 5. IGF-1/IGF-1R/hsa-let-7c axis can regulate MAPK signaling pathway in SCAPs.

(a) Western blot assay for the expression of MAPK related proteins at 0.5 hour (ERK, phosphorylated ERK, JNK, phosphorylated JNK, p38 and phosphorylated p38, respectively). (b) The ratio changes of p-ERK/ERK, p-JNK/JNK and p-p38/p38 at 0.5 hour in different groups. Values were described as the means ± SD, n = 3. *P < 0.05, **P < 0.01. (c) Western blot assay for the expression of MAPK related proteins at 1 hour. (d) The ratio changes of p-ERK/ERK, p-JNK/JNK and p-p38/p38 at 1 hour in different groups. Values were described as the means ± SD, n = 3. *P < 0.05, **P < 0.01. (e) Western blot assay for the expression of MAPK related proteins at 6 hour. (f) The ratio changes of p-ERK/ERK, p-JNK/JNK and p-p38/p38 at 6 hour in different groups. Values were described as the means ± SD, n = 3. *P < 0.05, **P < 0.01.

Figure 6. Schematic diagram for IGF-1/IGF-1R/hsa-let-7c MAPK axis.

ERK, JNK and p38 MAPKs are members of MAPK family which can be activated by a variety of environmental factors. JNK and p38, which are activated by hsa-let-7c low-expression and IGF-1R over-expression, can translocate to the nucleus where they phosphorylate the transcription factors and subsequently regulate the downstream gene expression.