Hepatitis B virus induces cell proliferation via HBx-induced microRNA-21 in hepatocellular carcinoma by targeting programmed cell death protein4 (PDCD4) and phosphatase and tensin homologue (PTEN).

Hepatitis B viral infection-induced hepatocellular carcinoma is one of the major problems in the developing countries. One of the HBV proteins, HBx, modulates the host cell machinery via several mechanisms. In this study we hypothesized that HBV enhances cell proliferation via HBx-induced microRNA-21 in hepatocellular carcinoma. HBx gene was over-expressed, and miRNA-21 expression and cell proliferation were measured in Huh 7 and Hep G2 cells. miRNA-21 was over-expressed in these cells, cell proliferation and the target proteins were analyzed. To confirm the role of miRNA-21 in HBx-induced proliferation, Hep G 2.2.1.5 cells (a cell line that expresses HBV stably) were used for miRNA-21 inhibition studies. HBx over-expression enhanced proliferation (3.7- and 4.5-fold increase; n = 3; p<0.01) and miRNA-21 expression (24- and 36-fold increase, normalized with 5S rRNA; p<0.001) in Huh 7 and Hep G2 cells respectively. HBx also resulted in the inhibition of miRNA-21 target proteins, PDCD4 and PTEN. miRNA-21 resulted in a significant increase in proliferation (2- and 2.3-fold increase over control cells; p<0.05 in Huh 7 and Hep G2 cells respectively) and decreased target proteins, PDCD4 and PTEN expression. Anti-miR-21 resulted in a significant decrease in proliferation (p<0.05) and increased miRNA-21 target protein expression. We conclude that HBV infection enhances cell proliferation, at least in part, via HBx-induced miRNA-21 expression during hepatocellular carcinoma progression

Effect of miRNA-21 on phospho-akt levels.

<p>A, shows the Western blot results of phsopho-akt and akt proteins in the miRNA-21-transfected cells. This is a representative picture from three experiments. Lane 1, Control; lane 2, NS-miRNA transfected cells; and lane 3, miRNA-21 transfected cells. B, Western blots were quantified and the data are presented in the graph (n = 3; *p<0.05).</p

Proposed Model of HBx-miRNA-21 relationship.

<p>Schematic diagram showing the relationship between HBx, miRNA-21, akt and cellular proliferation in hepatic cells.</p

Role of miRNA-21 on proliferation of hepatoma cells.

<p>miRNA-21 was over-expressed in Hep G2 (A & C) and Huh 7 (B & D) cells. Relative expression of miRNA-21 was measured in the transfected cells (A & B) to determine the miRNA-21 levels (n = 3; *p<0.05 and **p<0.01). As an internal control, 5S RNA was quantified in all real time PCR experiments. Both C & D show the cell proliferation assays in miRNA-21 over-expressing cells. Cells were plated at a density of 8000/well in a 96-well plate for the proliferation assays. As a control in all these experiments NS-miRNA was used in parallel experiments (n = 3; *p<0.05).</p

Transfection efficiency in Huh 7 and Hep G2 cells.

<p>Both Huh 7 (A and B) and Hep G2 (C and D) cells were transfected with eGFP-N1 plasmid using the similar transfection conditions as HBx. After 48 hours of transfection, the cells were observed (10× magnification). Both A and C are fluorescent pictures and B and D are corresponding phase-contrast pictures.</p

Effect of miRNA-21 on PDCD4 and PTEN proteins.

<p>A, shows the Western blot results of miRNA-21 target proteins, PDCD4 and PTEN in miRNA-21 over-expressing cells. As an internal control β-actin was used in all the Western blot experiments. This is a representative picture from three experiments. Lane 1, Control; lane 2, NS-miRNA transfected cells; and lane 3, miRNA-21 transfected cells. The Western blots were quantified and the data are presented for Huh 7 (B) and Hep G2 (C) cells (n = 3; *p<0.05).</p

Effect of miRNA-21 on PDCD4 and PTEN proteins in LX2 and Hela Cells.

<p>A, shows a representative picture of Western blot results of miRNA-21 target proteins, PDCD4 and PTEN in miRNA-21 over-expressing LX2 and Hela cells. As an internal control β-actin was used in all the Western blot experiments. Lane 1, Control; lane 2, NS-miRNA transfected cells; and lane 3, miRNA-21 transfected cells. The Western blots were quantified and the data are presented for LX2 (B) and Hela (C) cells (n = 3; *p<0.05).</p

Effect of HBx on PDCD4 and PTEN.

<p>HBx protein was over-expressed in both Huh 7 (A) and Hep G2 (B) cells and the expression of miRNA-21 was estimated using real time PCR (n = 3; *p<0.001). HBx was over-expressed in both Huh 7 and Hep G2 cells and the target proteins for miRNA-21 were determined using Western blots. This picture is a representative of the three experiments (C). Lane 1, Control; lane 2, Empty vector; and lane 3, HBx transfected cells. β-actin was used as loading control. These Western blots were quantified using Li-COR's image studio lite software and the data are presented for Huh 7 (D) and Hep G2 (E) cells (n = 3; *p<0.05).</p

Effect of anti-miR21 on proliferation and miRNA-21 target proteins.

<p>Intracellular miRNA-21 was inhibited using anti-miR-21 oligos in Hep G 2.2.1.5 cells. A, Relative expression of miRNA-21 was measured in the anti-miR-21 transfected cells and NS-anti-miR was used as control in all the experiments (n = 3; *p<0.01). B, The proliferation assay was also performed in anti-miR-21 transfected cells (n = 3; *p<0.05). C, Western blot was performed to measure the protein levels of miRNA-21 target proteins, PDCD4 and PTEN in anti-miR-21 transfected cells. As an internal control β-actin was used in all the Western blot experiments. Lane 1, Control; lane 2, NS-anti-miR transfected cells; and lane 3, anti-miR-21 transfected cells. D, The Western blots were quantified and the data are presented from three experiments (*p<0.05).</p