MiR-143-3p Inhibits Osteogenic Differentiation of Human Periodontal Ligament Cells by Targeting KLF5 and Inactivating the Wnt/β-Catenin Pathway

Abstract

Human periodontal ligament cells (hPDLCs) play a vital role in cell regeneration and tissue repair with multi-directional differentiation potential. microRNAs (miRs) are implicated in the osteogenesis of hPDLCs. This study explored the mechanism of miR-143-3p in osteogenesis of hPDLCs. Osteogenic differentiation of isolated hPDLCs was induced. KLF5 expression during osteogenic differentiation of hPDLCs was detected and then silenced in hPDLCs. Binding relationship between KLF5 and miR-143-3p was predicted and verified. hPDLCs were treated with miR-143-3p mimic or overexpressing KLF5, and then osteogenic specific markers and mineralized nodules were measured. The key factors of the Wnt/β-catenin pathway during osteogenesis of hPDLCs were measured. KLF5 expression was upregulated during osteogenesis of hPDLCs. KLF5 silencing or miR-143-3p mimic reduced osteogenic specific markers and mineralized nodules. Overexpression of KLF5 could reverse the inhibitory effect of miR-143-3p on osteogenic differentiation. miR-143-3p mimic and KLF5 silencing inactivated the Wnt/β-catenin pathway. Activation of the Wnt/β-catenin pathway reversed the repression effect of miR-143-3p mimic on osteogenesis of hPDLCs. In conclusion, miR-143-3p inhibited osteogenic differentiation of hPDLCs by targeting KLF5 and inactivating the Wnt/β-catenin pathway.

Keywords: KLF5; Wnt/β-catenin pathway; human periodontal ligament cells; microRNA-143-3p; osteogenic differentiation.

Figure 1

KLF5 expression was upregulated during osteogenic differentiation of hPDLCs. (A) Morphology of hPDLCs at passage 3. (B) Vimentin was measured using immunohistochemistry staining (400×, bar = 40 μm). (C) Expressions of surface markers of hPDLCs were identified using flow cytometry. (D,E) Osteogenic differentiation of hPDLCs was analyzed using ARS staining and ARS semi-quantitative analysis. (F) ALP activity was measured using ALP staining. (G,H) Levels of osteogenic specific markers (OCN, OPN, ALP, and Runx2) were detected using Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting. (I,J) KLF5 expression during the osteogenic differentiation of hPDLCs was detected using RT-qPCR and Western blotting. The experiment was repeated three times. Data are expressed as mean ± standard deviation. One-way ANOVA was applied to assess data in panels (E,F,I,J), and two-way ANOVA was applied to assess data in panels (G,H), followed by Dunnett’s multiple comparisons test or Tukey’s multiple comparisons test, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 2

KLF5 silencing inhibited the osteogenic differentiation of hPDLCs. (A,B) Silencing effect of siRNA on KLF5 was measured using RT-qPCR and Western blotting. (C,D) Effect of KLF5 on osteogenic differentiation of hPDLCs was measured using ARS staining and ARS semi-quantitative analysis. (E) ALP activity was measured using ALP staining. (F,G) Changes of OCN, OPN, ALP, and Runx2 in hPDLCs were measured using RT-qPCR and Western blotting. Data are expressed as mean ± standard deviation. One-way ANOVA was applied to assess data in panels (A,B,D,E), and two-way ANOVA was applied to assess data in panels (F,G), followed by Dunnett’s multiple comparisons test or Tukey’s multiple comparisons test, ^**p < 0.01, ^***p < 0.001.

Figure 3

miR-143-3p targeted KLF5 in hPDLCs. (A) Binding site of miR-145-3p and KLF5 was predicted by Starbase. (B) Binding relationship between miR-145-3p and KLF5 was verified using dual-luciferase reporter gene assay. (C) Effect of miR-145-3p mimic on miR-145-3p was measured using RT-qPCR. (D) Effect of miR-145-3p mimic on mRNA level of KLF5 was measured using RT-qPCR. (E) Effect of miR-145-3p mimic on protein levels of KLF5 was measured using Western blotting. The experiment was repeated three times. Data are expressed as mean ± standard deviation. One-way ANOVA was applied to assess data in panel (B), followed by Tukey’s multiple comparisons test, and two-way ANOVA was applied to assess data in panels (C–E), followed by Sidak’s multiple comparisons test or Tukey’s multiple comparisons test, ^***p < 0.001.

Figure 4

miR-143-3p targeted KLF5 to inhibit osteogenic differentiation of hPDLCs. (A) Effect of pcDNA-KLF5 on KLF5 expression was measured using RT-qPCR. (B,C) Osteogenic differentiation of hPDLCs was measured using ARS staining and ARS semi-quantitative analysis. (D) ALP activity was measured using ALP staining. (E,F) Levels of osteogenic specific markers (OCN, OPN, ALP, and Runx2) were detected using RT-qPCR and Western blotting. The experiment was repeated three times. Data are expressed as mean ± standard deviation. One-way ANOVA was applied to assess data in panels (A,C,D), and two-way ANOVA was applied to assess data in panels (E,F), followed by Tukey’s multiple comparisons test, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 5

miR-143-3p targeted KLF5 and inhibited the Wnt/β-catenin pathway in hPDLCs. (A,B) After intervention of miR-143-3p and KLF5, protein levels of Wnt7b and β-catenin during osteogenic differentiation of hPDLCs were detected using RT-qPCR and Western blotting. The experiment was repeated three times. Data are expressed as mean ± standard deviation. Two-way ANOVA was applied to assess data in panels (A,B), followed by Tukey’s multiple comparisons test, ^**p < 0.01, ^***p < 0.001.

Figure 6

Activation of the Wnt pathway reversed the inhibitory effect of miR-143-3p mimic on osteogenic differentiation of hPDLCs. (A,B) Osteogenic differentiation of hPDLCs was measured using ARS staining and ARS semi-quantitative analysis. (C) ALP activity was measured using ALP staining. (D,E) Levels of osteogenic specific markers (OCN, OPN, ALP, and Runx2) were detected using RT-qPCR and Western blotting. The experiment was repeated three times. Data are expressed as mean ± standard deviation. One-way ANOVA was applied to assess data in panels (B,C), and two-way ANOVA was applied to assess data in panels (D,E), followed by Dunnett’s multiple comparisons test or Tukey’s multiple comparisons test, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.