2). These results indicate that miR-7 may arrest cell-cycle progression by repressing p110δ expression. To verify our observations, we established relevant stable
subclones in QGY-7703, which were named QGY-null (mock), QGY-miR-NC (noneffective control), and QGY-miR-7, respectively. Ectopic expression of miR-7 was elevated by https://www.selleckchem.com/products/AC-220.html approximately 7-fold (Supporting Fig. 3A), which resulted in a 0.24-fold reduction of PIK3CD mRNA (Fig. 2B). Western blotting analysis showed that miR-7 specifically repressed p110δ protein expression (Fig. 2B), but did not affect the expression of the other two p110 catalytic subunits (p110α and p110β) or their corresponding regulatory subunit, p85 (Supporting Fig. 3B). We further investigated the effect of the stable expression of miR-7
on HCC cell growth in vitro. Using the cell-proliferation assay, we observed a significant decrease in cell number in QGY-miR-7 cells (538.8 ± 39.0 × 103, n = 3; P < 0.01) versus QGY-null cells (1,164 ± 34.1 × 103, n = 3; P < 0.01) or QGY-miR-NC cells (949 ± 48.1 × 103, n = 3; P < 0.01) on day 7 (Fig. 2C). No apoptosis was observed on day 4 (Supporting Fig. 3C) when miR-7 was stably expressed, indicating that the decrease in cell numbers might be caused by the arrest of cell-cycle progression (Fig. 2C). A similar inhibition in cell proliferation was observed in the PIK3CD siRNA#3 group, but not in the control siRNAs (Supporting see more Fig. 4). To further validate our results, we assayed for alterations in cell-cycle progression every 2 hours for 24 hours after 30 hours of serum starvation (Fig. 2D). A G0/G1 cell-cycle arrest that was detected in QGY-miR-7 cells was associated with miR-7 overexpression. It took QGY-miR-7 cells 8-9 hours to recover after serum starvation (G0/G1 ≤60%), whereas the controls recovered in approximately 5 hours, and the percentage Pyruvate dehydrogenase of cells in the G0/G1 phase remained over 50% and had no significantly periodic change
when miR-7 was stably expressed, which was obviously higher than those in S or G2/M phase (Fig. 2D, top). By analyzing changes in the cell proportion in S or G2/M phase, we found that QGY-miR-7 required 14 hours to complete a cell cycle after serum recovery, compared to approximately 12 hours for control cells (Fig. 2D, middle and bottom). All the results were consistent with those observed in transient transfection experiments. These data strongly suggest that miR-7 inhibits HCC cell growth by G0/G1 arrest, but not by triggering apoptosis. We further investigated whether overexpression of miR-7 could weaken the invasiveness and migratory capabilities in HCC. Using the wound-healing assay (Supporting Materials and Methods), we found that ectopic expression of miR-7 decreased cell motility in QGY-miR-7 cells, compared to QGY-null and QGY-miR-NC cells (Supporting Fig. 5).