Sasa veitchii Extract Mitigates Mycophenolate Mofetil-Induced Human Palatal Cell Proliferation Inhibition by Downregulating microRNA-4680-3p

Abstract

Cleft palate is a common birth defect worldwide and is caused by both genetic and environmental factors. Intrauterine drug exposure is one of the environmental factors that can induce cleft palate. Mycophenolate mofetil (MPM) is an immunosuppressant drug with teratogenic effects, including cleft palate. However, the research on MPM-induced cleft palate remains limited. Sasa veitchii extract (SE), a medical plant extract, is commercially available in Asia and has been reported to show effectiveness against oral diseases. The purpose of the present study is to evaluate whether SE protects against MPM-induced immunosuppression in human embryonic palatal mesenchymal (HEPM) cells. Cell viability and G1 phase-related cell cycle markers were assessed by co-treatment with MPM and SE. Furthermore, we quantified cleft palate-associated miRNA levels and the expression of its downstream genes. MPM treatment reduced cell viability in a concentration-dependent manner. Co-treatment with SE alleviated MPM-induced inhibition of HEPM cell proliferation. Additionally, SE reduced MPM-induced miR-4680-3p upregulation and the downregulation of its downstream genes (ERBB2 and JADE1). These results suggest that SE alleviated MPM-induced cell proliferation inhibition through modulating miR-4680-3p expression.

Keywords: Sasa veitchii; cell cycle; cleft palate; microRNA; mycophenolate mofetil.

Figure 1

Proliferation of HEPM cells treated with MPM (0.03–10 µM) for 48 h. ** p < 0.01, and *** p < 0.001 versus control (n = 6).

Figure 2

Protective effect of SE against MPM-induced inhibition of HEPM cell proliferation. 1 µM MPM and 100 µg/mL SE were used. * p < 0.05, and *** p < 0.001 versus control (n = 6).

Figure 3

The absorption spectra of SCC and SE. (a) indicated SCC (0.02 mg/mL) and (b) indicated SE (0.04 mg/mL). The arrow shows the main peak of SCC.

Figure 4

Sodium copper chlorophyllin failed to alleviate MPM-induced cell proliferation inhibition in HEPM cells. 1 µM MPM and 1 µg/mL SCC were used. *** p < 0.001 (n = 6). N.S.; Not significant.

Figure 5

SE attenuated MPM-induced cell cycle arrest in HEPM cells. (a) BrdU staining (green) of HEPM cells after treatment with 1 µM MPM and/or 100 µg/mL SE for 48 h. BrdU-positive cells were stained green, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). Arrow indicated BrdU-positive cells. Scale bar, 50 µm. The graph shows the quantification of BrdU-positive cells. ** p < 0.01 and *** p < 0.001 (n = 8–10). (b) Western blotting of HEPM cells treated with 1 µM MPM and/or 100 µg/mL SE for 48 h. β-ACTIN served as an internal control.

Figure 6

SE downregulated of miR-4680-3p levels and upregulated of ERBB2 and JADE1 in HEPM cells. (a) Quantitative RT-PCR analysis of Let-7c-5p and miR-4680-3p expression after treatment with 1 µM MPM and/or 100 µg/mL SE in HEPM cells. * p < 0.05, ** p < 0.01, and *** p < 0.001. N.S.; Not Significant. (b) Quantitative RT-PCR analysis of BACH1, PAX3, ERBB2, and JADE1 expression after treatment with 1 µM MPM and/or 100 µg/mL SE in HEPM cells. ** p < 0.01, and *** p < 0.001. N.S.; Not Significant.

Figure 7

Proposed mechanism of SE against MPM-induced cell proliferation inhibition.