6), suggesting that miR-7 may have a relatively smaller effect on regulating the
expression of molecules associated with invasion. In contrast, migratory capacity was significantly down-regulated in QGY-miR-7 cells (120 ± 3 per field for five fields; P < 0.01) versus QGY-null (180 ± 8 per field for five fields) or QGY-miR-NC cells (170 ± 6 per field for five fields) (Fig. 3). Similar results were observed in both invasion and migration assays when cells were transiently transfected with PIK3CD siRNA#3 (Supporting Fig. S7). These results indicate that Small molecule library cell assay miR-7 participates in the regulation of cell proliferation and migration by directly regulating PIK3CD expression. We further evaluated the effects of p110δ-expression inhibition by miR-7 in the PI3K/Akt-signaling pathway. We found that the transcription of AKT, mTOR, and P70S6K, which are major components of the PI3K/Akt pathway, was down-regulated to 0.4-, 0.25-, and 0.3-fold, respectively, in QGY-miR-7 cells (Fig.
4A). The transcription of eIF4E binding protein 1 (4EBP1), which is usually inhibited by mTOR, was up-regulated by 2.7-fold, as assessed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Total and phosphorylated protein levels of all four molecules described above showed the same results (Fig. 4B), indicating Akt inhibitor that miR-7 may be an important regulator of this signaling pathway. These findings were also validated
by using PIK3CD siRNA#3 (Supporting Fig. 8). Based on these results and previous studies that miRNA could regulate multiple and functionally related targets in one pathway,17 we wondered whether these four genes could be regulated by miR-7. As a result, no miR-7 target sites were in the AKT or 4EBP1 3′UTR, but one was found in the mTOR 3′UTR (Fig. 5A). Using the luciferase reporter assay, we found that relative luciferase activity was reduced to 38% ± 5% (25 ± 3.5 versus 66 ± 5.3) for the reporter plasmid that contained the putative miR-7 target site, but not the corresponding mutant counterpart that was cotransfected with miR-7 (Fig. 5B). Two putative miR-7 target sites were also found in the MCE公司 P70S6K 3′UTR (Fig. 5C). These two target sites repressed luciferase activity by approximately 50%, when combined with ectopic miR-7 expression (Fig. 5D). These data indicate that miR-7 can regulate the expression of mTOR, p70S6K, and PIK3CD by directly binding to target sites within the 3′UTR, supporting our conclusion that miR-7 can inhibit HCC cell proliferation and movement by regulating the PI3K/Akt/mTOR-signaling pathway. To further identify the function of miR-7 on the inhibition of tumor growth and metastasis in vivo, QGY-null and QGY-miR-7 cells were inoculated SC into the right and the left scapula of each mouse, respectively (n = 5).