Upregulated microRNA-29a by hepatitis B virus X protein enhances hepatoma cell migration by targeting PTEN in cell culture model - PubMed (original) (raw)

Upregulated microRNA-29a by hepatitis B virus X protein enhances hepatoma cell migration by targeting PTEN in cell culture model

Guangyao Kong et al. PLoS One. 2011.

Abstract

Hepatitis B virus X protein (HBx) plays important roles in the development of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) contribute to cancer development by acting as oncogenes or tumor suppressors. Previously, we reported that HBx was able to promote the migration of hepatoma HepG2 cells. However, the regulation of miRNAs in the development of HBV-related HCC is poorly understood. In the present study, we reported that miR-29a was a novel regulator of migration of hepatoma cells mediated by HBx. Our data showed that the expression of miR-29a was dramatically increased in p21-HBx transgenic mice, HBx-transfected hepatoma HepG2-X (or H7402-X) cells and HepG2.2.15 cells that constitutively replicate HBV. However, our data showed that miR-29a was upregulated in 4 of the 11 clinical HCC samples. We found that the overexpression of miR-29a promoted the migration of HepG2 cells, while a specific miR-29a inhibitor could partially abolish the enhanced migration of HepG2-X cells. Moreover, we identified PTEN was one of the target genes of miR-29a in HepG2 cells. The deletion of the miR-29a-binding site was able to abolish the role of miR-29a in suppression of luciferase activity of the PTEN 3'UTR reporter. Meanwhile, the overexpression of PTEN was able to reverse the promoted migration of HepG2 cells mediated by miR-29a. Moreover, our data showed that the modulation of Akt phosphorylation, a downstream factor of PTEN, was involved in the cell migration enhanced by miR-29a, suggesting that miR-29a is responsible for the cell migration through its target gene PTEN. Thus, we conclude that miR-29a is involved in the regulation of migration of hepatoma cells mediated by HBx through PTEN in cell culture model.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. HBx upregulates miR-29a.

(A) The relative expression of miR-29a for 6-month-old p21-HBx transgenic mice versus WT mice was detected by qRT-PCR. (B) The expression of miR-29a in stable HBx-transfected hepatoma cell lines was examined by qRT-PCR. (C) The expression levels of miR-29a in HepG2-X (or H7402-X) were examined by qRT-PCR after treatment with HBx RNAi in a dose dependent manner. (D) The relative expression level of miR-29a in HepG2.2.15 cells was examined by qRT-PCR. Plotted are the means ± SD of three samples normalized to U6. Statistically significant differences, arbitrarily set to 1.0, are indicated: *P<.05, **P<.01, Student's t test.

Figure 2. MiR-29a increases the migration of hepatoma cells.

(A) A wound healing assay was performed on HepG2 cells (1×106, 6-cm plate) transfected with either an empty vector (control) or a miR-29a expression vector (3 µg). One representative experiment is shown. Black arrows indicate the wound edge. The residual gap between the migrating cells from the opposing wound edge is expressed as a percentage of the initial scraped area. (B) Modified Boyden's chamber assays showed that the cell migration was promoted by the miR-29a in HepG2 cells. A histogram shows the relative cell number of five randomly selected fields. (C) The expression levels of miR-29a and miR-29c were examined by qRT-PCR after overexpression of miR-29a in HepG2 cells. (D) A wound healing assay was performed on HepG2, HepG2-P and HepG2-X cells transfected with a miR-29a inhibitor (100 nM) or inhibitor negative control (inhNC). One representative experiment is shown. Black arrows indicate the wound edge. The residual gap between the migrating cells from the opposing wound edge is expressed as a percentage of the initial scraped area. (E) Modified Boyden's chamber assays showed that the cell migration was blocked by miR-29a inhibitor in HepG2-X cells. A histogram shows the relative cell number of five randomly selected fields. (F) Western blot analysis showed that the expression of HBx protein was not affected by the treatment with miR-29a inhibitor in HepG2-X cells. Statistically significant differences are indicated: *P<.05, **P<.01, Student's t test.

Figure 3. MiR-29a targets PTEN.

(A) Sequence alignment between miR-29a and the 3′UTR of human PTEN mRNA. Solid line, seed match region; dashed line, seed-deleted region. (B) Luciferase reporter gene assay showed the effect of miR-29a on the activity of PTEN 3′UTR reporter. Co-transfection was performed using the plasmids, such as human PTEN 3′UTR (PTEN 3′UTR-1 or PTEN 3′UTR-2) or the miR-29a-binding site-deleted (del) PTEN 3′UTR (PTEN 3′UTR-1-del or PTEN 3′UTR-2-del), a miR-29a expression plasmid (50, 100, and 200 ng), in HepG2 cells. Empty pcDNA3.0 plasmid (200 ng) was used as a negative control. (C) Luciferase reporter gene assay showed that the decreased luciferase activities of PTEN 3′UTR reporter (PTEN 3′UTR-1 or PTEN 3′UTR-2) in HepG2 cells mediated by miR-29a overexpression (200 ng) was rescued by a miR-29a inhibitor in a dose dependent manner (30, 50, and 100 nM). (D) Immunoblot and qRT-PCR showed that miR-29a (3 µg, 6-cm plate) induced a decrease in endogenous human PTEN protein and mRNA in HepG2 and MHCC-97L cells, which was increased by transfection with a miR-29a inhibitor. GAPDH and β-actin were used as internal controls. Protein bands were quantified using Quantity One software (Bio-Rad). The value under each lane indicates the relative expression level of the PTEN, which is represented by the intensity ratio between PTEN and β-actin bands in each lane. Statistically significant differences are indicated: *P<.05, **P<.01, Student's t test.

Figure 4. PTEN inhibits migration of tumor cells.

(A) A wound healing assay was performed on HepG2 cells (1×106, 6-cm plate) transfected with random siRNA, PTEN siRNA, pcDNA3-29a, and both pcDNA3-PTEN/pcDNA3-29a, respectively. One representative experiment is shown. Black arrows indicate the wound edge. (B) Modified Boyden's chamber assays showed that enhanced cell migration induced by miR-29a was reversed by the overexpression of PTEN in HepG2 cells. A histogram shows the relative cell number of five randomly selected fields. (C) The expression of PTEN was detected by western blot analysis in above transfected HepG2 cells. Statistically significant differences are indicated: *P<.05, **P<.01, Student's t test.

Figure 5. Akt phosphorylation is regulated by miR-29a through PTEN.

(A, B) The phosphorylation level of Akt (P-Akt) and the expression level of total Akt (Akt-tot) were detected by Western blot analysis in HepG2 and H7402 cell lines after transfection of miR-29a (or a specific inhibitor). (C) Western blot analysis showed the phosphorylation of Akt (P-Akt) and the expression of total Akt (Akt-tot) in the cells treated by PTEN siRNA when the hepatoma cells were transfected with miR-29a inhibitor. Protein bands were quantified using Quantity One software (Bio-Rad). The value under each lane indicates the relative expression level of PTEN or phosphorylated Akt, which is represented by the intensity ratio between PTEN or phosphorylated Akt and β-actin or total Akt bands in each lane. (D) A wound healing assay showed that enhanced cell migration by miR-29a was abolished by Akt siRNA. One representative experiment is shown. Black arrows indicate the wound edge.

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Upregulated microRNA-29a by hepatitis B virus X protein enhances hepatoma cell migration by targeting PTEN in cell culture model - PubMed (original) (raw)