Baohuoside-I suppresses cell proliferation and migration by up-regulating miR-144 in melanoma

Abstract

Context: Baohuoside-I was reported to induce apoptosis in non-small-cell lung cancer and inhibit the growth of multiple myeloma cells. The antitumour potential of baohuoside-I has not been demonstrated in melanoma yet.

Objective: To investigate the potential antitumour activity of baohuoside-I against melanoma and elucidate its underlying molecular mechanism.

Materials and methods: Cell viability was evaluated by MTT assay. The malignant invasion capacity was measured with trans-well assay. The relative expression change of microRNAs was profiled with microarray. TargetScan was utilized for prediction of target gene of miR-144. Regulatory effect of miR-144 on SMAD1 was determined by dual luciferase reporter assay. Endogenous SMAD1 protein in response to ectopic expression of miR-144 was determined by immunoblotting. Xenograft mice were employed to evaluate antitumour potential of baohuoside-I (25 mg/kg by tail intravenous injection every two days) in vivo.

Results: Baohuoside-I significantly inhibited proliferation (45 ± 4% reduction in M14 and 35 ± 3% reduction in MV3 at 24 h) and migration (70 ± 4% reduction in M14 and 72 ± 3% reduction in MV3) in melanoma cells. Mechanistically, baohuoside-I up-regulated miR-144 expression levels (3 ± 0.2-fold). Silence of miR-144 reversed the inhibition of baohuoside-I in melanoma. We have identified that SMAD1 was the novel target of miR-144. Moreover, baohuoside-I suppressed melanoma in vivo (52 ± 8% reduction in xenograft tumour size at day 20).

Conclusions: Our data suggested significant antitumour potential of baohuoside-I against melanoma both in vitro and in vivo, which warrants further laboratory investigation and clinical trial.

Keywords: Antitumour effects; SMAD1; invasion.

Figure 1.

Baohuoside-I inhibits cell proliferation and migration in melanoma cells. M14 and MV3 cells were treated with 20 μg/mL baohuoside-I or DMSO. (A–B) The cell proliferation abilities were analyzed by MTT assay. Data were presented as mean ± SD from three independent experiments with triple replicates per experiment. **p < 0.01 compared to DMSO group. (C–D) Transwell migration assay was employed to analyze the migration abilities of the cells. Data were presented as mean ± SD from three independent experiments with triple replicates per experiment. **p < 0.01 compared to DMSO group.

Figure 2.

Baohuoside-I upregulates miR-144 expression levels. M14 was treated with 20 μg/mL baohuoside-I or DMSO. (A) After 24 h, the RNAs of the cells were isolated and were analyzed by microarray, and the different expression genes (DEGs) were displayed as volcano plot. (B) Cells were treated as above, the expression levels of differential miRNAs (miR-144, miR-124) were detected by qRT-PCR. Data were presented as mean ± SD from three independent experiments with triple replicates per experiment. **p < 0.01 compared to DMSO group. **p < 0.01 compared to DMSO group.

Figure 3.

Silence of miR-144 reverses the inhibition of baohuoside-I in melanoma. M14 and MV3 cells were transfected with Anti-miR-144 or control (Anti-miR-NC), and then were treated with 20 μg/mL baohuoside-I or DMSO. (A–B) The cell proliferation abilities were analyzed by MTT assay. Data were presented as mean ± SD from three independent experiments with triple replicates per experiment. **p < 0.01 compared to DMSO + Anti-miR-NC group. ##p < 0.01 compared to baohuoside-I + Anti-miR-NC group. (C–D) Transwell migration assay was employed to analyze the migration abilities of the cells. Data were presented as mean ± SD from three independent experiments with triple replicates per experiment. **p < 0.01 compared to DMSO + Anti-miR-NC group. ##p < 0.01 compared to baohuoside-I + Anti-miR-NC group.

Figure 4.

SMAD1 is the new target of miR-144. (A) TargetScan program was used to predict the targets of miR-144, and the putative seed-matching sites or mutant sites (the letter “G” in the 16th, 18th and 20th position) between miR-144 and 3′-UTR of SMAD1 according the program. (B) Luciferase reporter assay was employed to the luciferase activities of the WT and mut reporters. Data were presented as mean + SD from three independent experiments with triple replicates per experiment. **p < 0.01. (C) miR-144 and SMAD1 expression levels of melanoma specimens (n = 49) were analyzed by qRT-PCR and the relationship between which was tested by Pearson’s method. (D) M14 and MV3 cells were transfected with miR-144 or control mimics (miR-NC). After 48 h, the expressions of SMAD1 were detected by western blotting.

Figure 5.

Baohuoside-I suppresses melanoma in vivo. M14 cells were resuspended in serum-free RPMI-1640 medium and were injected into each side of posterior flank of the nude mice, and the mice were treated with baohuoside-I (25 mg/kg) or DMSO by tail vein injection every two days. (A) The tumours were measured every two days. Data were presented as mean ± SD from eight tumours. **p < 0.01 compared to DMSO group. (B–C) In day 20 the tumours were stripped, photographed and weighed. Data were presented as mean ± SD from eight tumours. **p < 0.01 compared to DMSO group. (D) The RNA expression levels of miR-144 and SMAD1 were analyzed by qRT-PCR in the tumours. Data were presented as mean ± SD from eight tumours. **p < 0.01 compared to DMSO group. (E) The protein expression levels of SMAD1 of the tumours were tested by western blotting.