Our results indicate that retrospective techniques have the potential to produce satisfactory results much of the time, but that visual inspection is necessary to guard against large errors.

MicroRNAs (miRNAs) are believed to have fundamental roles in tumorigenesis and have great potential for the diagnosis and treatment of cancer. However, the roles of miRNAs in hepatocellular carcinogenesis are still not fully elucidated. We investigated the aberrantly expressed miRNAs involved in hepatoma by comparison of miRNA expression profiles in cancerous hepatocytes with normal primary human hepatocytes, and 37 dysregulated miRNAs were screened out by twofold change with a significant difference (Po0.05). Clustering analysis based on 13 miRNAs with changes over 15-folds showed that the miRNA expression patterns between the cancerous and normal hepatocytes were clearly different. Among the 13 miRNAs, we found that miR-375 was significantly downregulated in hepatocellular carcinoma (HCC) tissues and cell lines. Overexpression of miR-375 in liver cancer cells decreased cell proliferation, clonogenicity, migration/ invasion and also induced G1 arrest and apoptosis. To unveil the molecular mechanism of miR-375-mediated phenotype in hepatoma cells described above, we examined the putative targets using bioinformatics tools and found that astrocyte elevated gene-1 (AEG-1) was a potential target of miR-375. Then we demonstrated that miR-375 bound directly to the 3 0 -untranslated region of AEG-1 and inhibited the expression of AEG-1. TaqMan quantitative reverse transcriptase-PCR and western blot analysis showed that miR-375 expression was inversely correlated with AEG-1 expression in HCC tissues. Knockdown of AEG-1 by RNAi in HCC cells, similar to miR-375 overexpression, suppressed tumor properties. Ectopic expression of AEG-1, conversely, could partially reverse the antitumor effects of miR-375. In a mouse model, therapeutic administration of cholesterol-conjugated 2 0 -O-methyl-modified miR-375 mimics (Chol-miR-375) could significantly suppress the growth of hepatoma xenografts in nude mice. In conclusion, our findings indicate that miR-375 targets AEG-1 in HCC and suppresses liver cancer cell growth in vitro and in vivo, and highlight the therapeutic potential of miR-375 in HCC treatment.

Metastasis is the predominant cause of death in breast cancer patients. Several lines of evidence have shown that microRNAs (miRs) can have an important role in cancer metastasis. Using isogenic pairs of low and high metastatic lines derived from a human breast cancer line, we have identified miR-149 to be a suppressor of breast cancer cell invasion and metastasis. We also identified GIT1 (G-protein-coupled receptor kinase-interacting protein 1) as a direct target of miR-149. Knockdown of GIT1 reduced migration/invasion and metastasis of highly invasive cells. Re-expression of GIT1 significantly rescued miR-149-mediated inhibition of cell migration/invasion and metastasis. Expression of miR-149 impaired fibronectin-induced focal adhesion formation and reduced phosphorylation of focal adhesion kinase and paxillin, which could be restored by re-expression of GIT1. Inhibition of GIT1 led to enhanced protein degradation of paxillin and α5β1 integrin via proteasome and lysosome pathways, respectively. Moreover, we found that GIT1 depletion in metastatic breast cancer cells greatly reduced α5β1-integrin-mediated cell adhesion to fibronectin and collagen. Low level of miR-149 and high level of GIT1 was significantly associated with advanced stages of breast cancer, as well as with lymph node metastasis. We conclude that miR-149 suppresses breast cancer cell migration/invasion and metastasis by targeting GIT1, suggesting potential applications of the miR-149-GIT1 pathway in clinical diagnosis and therapeutics.

A cell-in-cell process refers to the invasion of one living cell into another homotypic or heterotypic cell. Different from non-apoptotic death processes of internalized cells termed entosis or cannibalism, we previously reported an apoptotic cell-in-cell death occurring during heterotypic cell-in-cell formation. In this study, we further demonstrated that the apoptotic cell-in-cell death occurred only in internalized immune killer cells expressing granzyme B (GzmB). Vacuole wrapping around the internalized cells inside the target cells was the common hallmark during the early stage of all cell-in-cell processes, which resulted in the accumulation of reactive oxygen species and subsequent mitochondrial injury of encapsulated killer or non-cytotoxic immune cells. However, internalized killer cells mediated rapid bubbling of the vacuoles with the subsequent degranulation of GzmB inside the vacuole of the target cells and underwent the reuptake of GzmB by killer cells themselves. The confinement of GzmB inside the vacuole surpassed the lysosome-mediated cell death occurring in heterotypic or homotypic entosis processes, resulting in a GzmB-triggered caspase-dependent apoptotic cell-in-cell death of internalized killer cells. On the contrary, internalized killer cells from GzmB-deficient mice underwent a typical non-apoptotic entotic cell-in-cell death similar to that of non-cytotoxic immune cells or tumor cells. Our results thus demonstrated the critical involvement of immune cells with cytotoxic property in apoptotic cell-in-cell death, which we termed as emperitosis taken from emperipolesis and apoptosis. Whereas entosis or cannibalism may serve as a feed-on mechanism to exacerbate and nourish tumor cells, emperitosis of immune killer cells inside tumor cells may serve as an in-cell danger sensation model to prevent the killing of target cells from inside, implying a unique mechanism for tumor cells to escape from immune surveillance.