Protein abundance and phosphorylation convey important information about pathway activity and molecular pathophysiology in diseases including cancer, providing biological insight, informing drug and diagnostic development, and guiding therapeutic intervention. Analyzed tissues are usually collected without tight regulation or documentation of ischemic time. To evaluate the impact of ischemia, we collected human ovarian tumor and breast cancer xenograft tissue without vascular interruption and performed quantitative proteomics and phosphoproteomics after defined ischemic intervals. Although the global expressed proteome and most of the >25,000 quantified phosphosites were unchanged after 60 min, rapid phosphorylation changes were observed in up to 24% of the phosphoproteome, representing activation of critical cancer pathways related to stress response, transcriptional regulation, and cell death. Both pan-tumor and tissuespecific changes were observed. The demonstrated impact of pre-analytical tissue ischemia on tumor biology mandates caution in interpreting stress-pathway activation in such samples and motivates reexamination of collection protocols for phosphoprotein analysis. Molecular

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BackgroundMicroRNAs (miRNAs) are a new class of naturally occurring, small, non-coding RNAs that regulate protein-coding mRNAs by causing mRNA degradation or repressing translation. The roles of miRNAs in lineage determination and proliferation, as well as the localization of several miRNA genes at sites of translocation breakpoints or deletions, have led to speculation that miRNAs could be important factors in the development or maintenance of the neoplastic state.ResultsWe showed that miR-9 was downregulated in human gastric adenocarcinoma. Overexpression of miR-9 suppressed the growth of human gastric adenocarcinoma cell line MGC803 cell as well as xenograft tumors derived from them in SCID mice. Bioinformatics analysis indicated a putative miR-9 binding site in the 3'-untranslated region (3'UTR) of the tumor-related gene NF-κB1 mRNA. In an EGFP reporter system, overexpression of miR-9 downregulated EGFP intensity, and mutation of the miR-9 binding site abolished the effect of miR-9 on EGFP intensity. Furthermore, both the NF-κB1 mRNA and protein levels were affected by miR-9. Finally, knockdown of NF-κB1 inhibited MGC803 cell growth in a time-dependent manner, while ectopic expression of NF-κB1 could rescue MGC803 cell from growth inhibition caused by miR-9.ConclusionThese findings indicate that miR-9 targets NF-κB1 and regulates gastric cancer cell growth, suggesting that miR-9 shows tumor suppressive activity in human gastric cancer pathogenesis.

MicroRNAs are short regulatory RNAs that negatively modulate gene expression at the post-transcriptional level, and are deeply involved in the pathogenesis of several types of cancers. To investigate whether specific miRNAs and their target genes participate in the molecular pathogenesis of laryngeal carcinoma, oligonucleotide microarrays were used to assess the differential expression profiles of microRNAs and mRNAs in laryngeal carcinoma tissues compared with normal tissues. The oncogenic miRNA, microRNA-21 (miR-21), was found to be upregulated in laryngeal carcinoma tissues. Knockdown of miR-21 by specific antisense oligonucleotides inhibited the proliferation potential of HEp-2 cells, whereas overexpression of miR-21 elevated growth activity of the cells, as detected by the colony formation assay. The cell number reduction caused by miR-21 inhibition was due to the loss of control of the G1-S phase transition, instead of a noticeable increase in apoptosis. Subsequently, a new target gene of miR-21, BTG2, was found to be downregulated in laryngeal carcinoma tissues. BTG2 is known to act as a pan-cell cycle regulator and tumor suppressor. These findings indicate that aberrant expression of miR-21 may contribute to the malignant phenotype of laryngeal carcinoma by maintaining a low level of BTG2. The identification of the oncogenic miR-21 and its target gene, BTG2, in laryngeal carcinoma is potentially valuable for cancer diagnosis and therapy.

SummaryMicroRNAs are a group of endogenously expressed, singlestranded, 18-24 nt RNAs that regulate diverse cellular pathways. Although documented evidence indicates that some microRNAs can function as oncogenes or tumor-suppressors, the role of miR-214 in regulating human cervical cancer cells remains unexplored. We determined the expression level of miR-214 and found it is downregulated in cervical cancer compared with normal tissue. Overexpression of miR-214 in HeLa cells, a human cervical cancer cell line, significantly inhibited cell proliferation according to the MTT and colony forming assays. HeLa cells that stably overexpress miR-214 downregulate the expression of MEK3 and JNK1 at both mRNA and protein levels. Further investigation revealed that miR-214 regulates the expression of MEK3 and JNK1 by targeting the 3 0 UTRs of these genes. Collectively, these results suggest that miR-214 negatively regulates HeLa cell proliferation by targeting the noncoding regions of MEK3 and JNK1 mRNAs.

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MicroRNAs (miRNAs) constitute a class of noncoding RNAs that post‐transcriptionally regulate gene expression. Recent evidence indicates that many miRNAs function as oncogenes or tumor suppressors by negatively regulating their target genes. In our previous study, using miRNA microarray analysis, we found that miRNA‐182 (miR‐182) was significantly downregulated in human gastric adenocarcinoma tissue samples. Here, we confirmed the downregulation of miR‐182 in a larger sample of gastric tissue samples. Overexpression of miR‐182 suppressed the proliferation and colony formation of gastric cancer cells. An oncogene, encoding cAMP‐responsive element binding protein 1 (CREB1), serves as a direct target gene of miR‐182. A fluorescent reporter assay confirmed that miR‐182 binds specifically to the predicted site of the CREB1 mRNA 3′‐UTR. When miR‐182 was overexpressed in gastric cancer cell lines, both the mRNA and protein levels of CREB1 were depressed. Furthermore, CREB1 was present at a high level in human gastric adenocarcinoma tissues, and this was inversely correlated with miR‐182 expression. Ectopic expression of CREB1 overcame the suppressive phenotypes of gastric cancer cells caused by miR‐182. These results indicate that miR‐182 targets the CREB1 gene and suppresses gastric adenocarcinoma cell growth, suggesting that miR‐182 shows tumor‐suppressive activity in human gastric cancer.

Matrine has potent antitumor activities in HepG2 cells and may be used as a novel effective reagent in treatment of hepatocellular carcinoma.