At low ASO concentrations (5 to 25 nmol/L), we observed an average decrease Pazopanib purchase in cell proliferation of 55% at 72 hours post-transfection with either miR-17�C92 polycistron or miR-21 ASOs (Figure 6A). We also observed a markedly smaller, albeit significant, reduction in cell proliferation (~20%) following control transfections with an ASO against miR-122, a major liver miRNA that is not expressed in HepG2 cells (Figure 6A). A t-test demonstrated that as compared with control, the reduction seen in all test groups was significant at P < 0.001, including treatment with control ASO (miR-122). However, reduction caused by treatment with ASO to either miR-21 or the miR-17�C92 polycistron was significant as compared with control ASO (miR-122) P < 0.001 at 5 nmol/L and 10 nmol/L respectively and maintains this significance for all other doses.

Thus, although control ASO (miR-122) treatment had a minor, yet statistically significant effect on proliferation, it could not account for the significant decrease in proliferation seen with ASO treatment to miR-17�C92 or miR-21. In addition, treatment with ASOs that selectively repress individual members of the miR-17�C92 polycistron revealed that loss of a single miRNA could not account for the proliferative decrease that resulted from knockdown of the complete polycistron (Figure 6B). Figure 6 Knockdown the miR-17�C92 polycistron or miR-21 reduces HepG2 cell proliferation. Cell proliferation was measured by MTS, dose-response data from 0 to 100 nmol/L total ASO transfected, 72 hours after transfection. Data expressed as % of control .

.. Fluorescence-activated cell sorter analysis demonstrated that the reduction in proliferation following ASO miRNA treatments of HepG2 cells is attributed to a retardation of the cell cycle (Figure 7). At 48 hours following transfection with the ASO mixture directed against the miR-17�C92 polycistron, we observed a statistically significant increase in the percentage of cells in G1 as compared to non-treated cells or cells treated with a sequence-scrambled ASO (Figure 7A). Furthermore, ASO treatment against individual members of the miR-17�C92 polycistron demonstrated that loss of miR-17.5p expression alone resulted in the retention of cells in G1 (Figure 7B). Therefore, miR-17.5p may be the most effective miRNA in the polycistron that affects the G1-S phase progression of HepG2 cells.

Additionally, in WHK44 cells, a woodchuck hepatoma cell line expressing N-myc 1,24 cells accumulated in G1 on treatment with ASOs against miRNAs of the miR-17�C92 polycistron (supplemental Figure 3, see Cilengitide http://ajp.amjpathol.org), demonstrating the conserved role of the miR-17�C92 polycistron in in vitro models for HCC. Figure 7 Knockdown of the miR-17�C92 polycistron causes a retardation of the cell cycle.