microRNAs are endogenous small noncoding RNAs that regulate gene expression negatively at posttranscriptional level. This latest addition to the complex gene regulatory circuitry revolutionizes our way to understanding physiological and pathological processes in the human body. Here we investigated the possible role of microRNAs in the development of multidrug resistance (MDR) in gastric cancer cells. microRNA expression profiling revealed a limited set of microRNAs with altered expression in multidrugresistant gastric cancer cell line SGC7901/VCR compared to its parental SGC7901 cell line. Among the downregulated microRNAs are miR-15b and miR-16, members of miR-15/16 family, whose expression was further validated by qRT-PCR. In vitro drug sensitivity assay demonstrated that overexpression of miR15b or miR-16 sensitized SGC7901/VCR cells to anticancer drugs whereas inhibition of them using antisense oligonucleotides conferred SGC7901 cells MDR. The downregulation of miR-15b and miR-16 in SGC7901/VCR cells was concurrent with the upregulation of Bcl-2 protein. Enforced mir-15b or miR-16 expression reduced Bcl-2 protein level and the luciferase activity of a BCL2 3 0 untranslated region-based reporter construct in SGC7901/VCR cells, suggesting that BCL2 is a direct target of miR-15b and miR-16. Moreover, overexpression of miR-15b or miR-16 could sensitize SGC7901/VCR cells to VCR-induced apoptosis. Taken together, our findings suggest that miR-15b and miR-16 could play a role in the development of MDR in gastric cancer cells at least in part by modulation of apoptosis via targeting BCL2.

MicroRNAs play key roles in tumor metastasis. Here, we describe the regulation and function of miR-218 in gastric cancer (GC) metastasis. miR-218 expression is decreased along with the expression of one of its host genes, Slit3 in metastatic GC. However, Robo1, one of several Slit receptors, is negatively regulated by miR-218, thus establishing a negative feedback loop. Decreased miR-218 levels eliminate Robo1 repression, which activates the Slit-Robo1 pathway through the interaction between Robo1 and Slit2, thus triggering tumor metastasis. The restoration of miR-218 suppresses Robo1 expression and inhibits tumor cell invasion and metastasis in vitro and in vivo. Taken together, our results describe a Slit-miR-218-Robo1 regulatory circuit whose disruption may contribute to GC metastasis. Targeting miR-218 may provide a strategy for blocking tumor metastasis.

Hypoxia induced drug resistance is a major obstacle in the development of effective cancer therapy. Our previous study revealed that hypoxia-inducible factor-1 (HIF-1), the major transcriptional factor significantly activated by hypoxia, was overexpressed in gastric vincristine-resistant cells SGC7901/vincristine (VCR) under normoxic conditions, which suggested that it was associated with drug resistance in gastric cancer cells. In the present study, a colonyforming assay revealed that hypoxia and forced HIF-1α α α α expression increased maximal -8.9-fold or -14.8-fold of IC 50 toward vincristine in gastric cancer cell lines SGC7901 and SGC7901/VCR, respectively (P < 0.01). Annexin-V/propidium iodide staining analysis revealed hypoxia or forced HIF-1α α α α expression reduced apoptosis by 24% or 18% in SGC7901 cells (P < 0.05). Flow cytometry analysis of intracellular adriamycin revealed that hypoxia and forced expression of HIF-1α α α α increased -1.79-fold or -2.36-fold of the adriamycin releasing index, respectively (P < 0.05). However, resistance acquisition subject to hypoxia in vitro and H ypoxia, frequently found in the center of solid tumors, is a major obstacle in the development of effective cancer chemotherapy.(1,2) It was reported that hypoxia decreased the efficacy of cisplatin, etoposide, bleomycin, and mitomycin C in testicular germ cell tumors, (3) and the colorectal cancer cell line maintained anti-apoptotic potential under hypoxia. In pancreatic cancer cells, hypoxia increases resistance to apoptosis induced by gemcitabine.(4) Growing evidence suggests that hypoxia in tumors selects for cells with decreased potential for apoptosis through the overexpression of Bcl-2 and decreased killing effect through upregulation of drug transporter protein, (5,6) may contribute to resistance to standard radiation therapy and chemotherapy. (7)(8)(9)

Alzheimer’s disease (AD) can be divided into sporadic AD (SAD) and familial AD (FAD). Most AD cases are sporadic and result from multiple etiologic factors, including environmental, genetic and metabolic factors, whereas FAD is caused by mutations in the presenilins or amyloid-β (Aβ) precursor protein (APP) genes. A commonly used animal model for AD is the 3xTg-AD transgenic mouse model, which harbors mutated presenilin 1, APP and tau genes and thus represents a model of FAD. There is an unmet need to in the field to characterize animal models representing different AD mechanisms, so that potential drugs for SAD can be evaluated preclinically in these animal models. A mouse model generated by intracerebroventricular (icv) administration of streptozocin (STZ), the icv-STZ mouse, shows many aspects of SAD. In this study, we compared the non-cognitive and cognitive behaviors as well as biochemical and immunohistochemical alterations between the icv-STZ mouse and the 3xTg-AD mouse. We found that both mouse models showed increased exploratory activity as well as impaired learning and spatial memory. Both models also demonstrated neuroinflammation, altered synaptic proteins and insulin/IGF-1 (insulin-like growth factor-1) signaling, and increased hyperphosphorylated tau in the brain. The most prominent brain abnormality in the icv-STZ mouse was neuroinflammation, and in the 3xTg-AD mouse it was elevation of hyperphosphorylated tau. These observations demonstrate the behavioral and neuropathological similarities and differences between the icv-STZ mouse and the 3xTg-AD mouse models and will help guide future studies using these two mouse models for the development of AD drugs.

The role of cytosolic calcium oscillation has long been recognized in the regulation of cellular and molecular interactions. Information embedded in calcium oscillation can provide molecular cues for cell behavior such as cell differentiation. Although calcium dynamics are versatile and likely to depend on the cell type, the calcium dynamics in human mesenchymal stem cells (hMSCs) and its role in differentiation are yet to be fully elucidated. In the present study we characterized the calcium oscillation profiles in hMSCs before and after subjecting the cells to the osteoinductive factors. Our findings indicate that the calcium spikes decreased rapidly with osteodifferentiation to a level observed in terminally differentiated human osteoblasts. In addition, the calcium oscillations appear to serve as a bidirectional signal during hMSC differentiation. While an altered calcium oscillation pattern may be an indicator for hMSC differentiation, it is also likely to be involved in directing hMSC differentiation. Treatment of hMSCs with a noninvasive electrical stimulation, for example, not only altered the calcium oscillations but also facilitated osteodifferentiation. Regulation of calcium oscillation by external physical stimulation could amplify hMSC differentiation into a tissue-specific lineage and may offer an alternate biotechnology to harness the unique properties of stem cells.

The activation of cochlear progenitor cells is a promising approach for hair cell (HC) regeneration and hearing recovery. The mechanisms underlying the initiation of proliferation of postnatal cochlear progenitor cells and their transdifferentiation to HCs remain to be determined. We show that Notch inhibition initiates proliferation of supporting cells (SCs) and mitotic regeneration of HCs in neonatal mouse cochlea in vivo and in vitro. Through lineage tracing, we identify that a majority of the proliferating SCs and mitotic-generated HCs induced by Notch inhibition are derived from the Wnt-responsive leucine-rich repeat-containing G proteincoupled receptor 5 (Lgr5

Epithelial-to-mesenchymal transition (EMT) induced by chronic hypoxia is one of the critical causes of renal fibrosis. Twist, a basic helix-loop-helix transcription factor, is believed to be important in promoting EMT. We found that the expression of Twist was increased in human tubule cell lines (HK-2 and HKC) grown under hypoxic conditions. This was accompanied by reduced expression of the epithelial markers E-cadherin and ZO-1 and enhanced expression of the mesenchymal markers vimentin and alpha-smooth muscle actin. When Twist was overexpressed in these cells it induced a mesenchymal phenotype, whereas its knockdown by short interfering RNA (siRNA) effectively reversed hypoxia-induced EMT. We showed that transfection with siRNA to hypoxia-inducible factor-1alpha (HIF-1alpha), another basic helix-loop-helix transcription factor, reduced Twist expression. Twist promoters contain HIF1-alpha-binding sites and transfection of reporter constructs using the promoter showed increased transcription in cells subjected to hypoxia. Electrophoretic mobility shift and chromatin immunoprecipitation assays identified the presence of a functional HIF-1alpha-binding site within the proximal Twist gene promoter. In an in vivo assay using the rat remnant kidney we found that both Twist and HIF-1alpha were overexpressed in tubular epithelial cells showing EMT. These studies suggest that HIF-1alpha induces Twist expression in hypoxic tubular cells and that this plays a role in EMT during renal fibrogenesis.

Metabolism of S-nitrosoglutathione (GSNO), a major biologically active nitric oxide (NO) species, is catalyzed by the evolutionally conserved GSNO reductase (GSNOR). Previous studies showed that the Arabidopsis GSNOR1/ HOT5 gene regulates salicylic acid signaling and thermotolerance by modulating the intracellular S-nitrosothiol level. Here, we report the characterization of the Arabidopsis paraquat resistant2-1 (par2-1) mutant that shows an anti-cell death phenotype. The production of superoxide in par2-1 is comparable to that of wild-type plants when treated by paraquat (1,1′-dimethyl-4,4′-bipyridinium dichloride), suggesting that PAR2 acts downstream of superoxide to regulate cell death. PAR2, identified by positional cloning, is shown to be identical to GSNOR1/HOT5. The par2-1 mutant carries a missense mutation in a highly conserved glycine, which renders the mutant protein unstable. Compared to wild type, par2-1 mutant has a higher NO level, as revealed by staining with 4,5-diaminofluorescein diacetate. Consistent with this result, wild-type plants treated with an NO donor display resistance to paraquat. Interestingly, the GSNOR1/HOT5/PAR2 protein level, other than its steady-state mRNA level, is induced by paraquat, but is reduced by NO donors. Taken together, these results suggest that GSNOR1/HOT5/PAR2 plays an important role in regulating cell death in plant cells through modulating intracellular NO level.