Male athymic nude mice were housed and manipulated according to the protocols approved by the Shanghai Medical Experimental Animal Care Commission. For each mouse, 5 × 106 “QGY-null” and “QGY-miR-7” subclone cells were injected subcutaneously (SC) into the right and left scapulas, respectively, in 100 μL
of serum-free medium. Tumor growth in the nude mice was measured every 5 PD0325901 in vivo days for 30 days. After the mice were sacrificed, total RNA and protein were extracted from tumor tissues to detect miR-7, PIK3CD, and PI3K/Akt-pathway components. Male athymic nude mice were randomly divided into two groups (QGY-null and QGY-miR-7; 5 mice per group), and 5 × 105 cells were injected intravenously (IV) via the tail vein. Tumor growth
and metastasis were analyzed in situ at week 7-8 after injection by green fluorescent protein (GFP) fluorescence imaging (LB981NC10D; Berthold Technologies, Oak Ridge, TN). All mice were euthanized at 9 weeks after the initial injection, and the livers and lungs were excised to examine extrahepatic metastasis from the liver to the lungs.15 All of the organs that were excised were embedded in paraffin for sectioning (5 μm) and staining with hematoxylin and eosin (H&E). The metastatic nodules on the lung and partial Decitabine in vitro liver tissues were snap-frozen for RNA and protein extraction. All treatments were performed according to the protocols described above. Ten surgical specimens (both tumor and adjacent normal tissue) were obtained from patients in Shanghai First Peoples’ Hospital and were snap-frozen in liquid nitrogen and stored at −80°C for later RNA and protein extraction. All HCC patients gave written informed consent find more for the use of clinical specimens in medical research. The studies using human tissues were reviewed and approved by the Committee for Ethical Review of Research Involving Human Subjects at Fudan University. The clinical and pathological features of these patients are described in the Supporting Table 2. Independent Student’s t tests and analysis of variance were used to compare differences between the two groups. The correlation between miR-7
and PIK3CD messenger RNA (mRNA) expression was measured using Pearson’s chi-square test. Statistical significance was determined by the log-rank test. A P value of less than 0.01 was considered to be statistically significant. Error bars represent standard error (SE), unless otherwise indicated. Given the observation that EGFR and its downstream components, Pak1 and IRS-2, are targets of miR-7,7, 9, 10 we wondered whether other miR-7 targets existed in the EGFR downstream pathways. Using TargetScan (www.targetscan.org),16 we identified PIK3CD as being a likely target of miR-7 because it contains four putative miR-7 target sites in its 3′UTR (untranslated region) (Fig. 1A). We generated a series of luciferase reporter vectors (Fig.