Zheyi Han

miRNA-223 Promotes Gastric Cancer Invasion and Metastasis by Targeting Tumor Suppressor EPB41L3

Traditional research modes aim to find cancer-specific single therapeutic target. Recently, emerging evidence suggested that some micro-RNAs (miRNA) can function as oncogenes or tumor suppressors. miRNAs are single-stranded, small noncoding RNA genes that can regulate hundreds of downstream target genes. In this study, we evaluated the miRNA expression patterns in gastric carcinoma and the specific role of miR-223 in gastric cancer metastasis. miRNA expression signature was first analyzed by real-time PCR on 10 paired gastric carcinomas and confirmed in another 20 paired gastric carcinoma tissues. With the 2-fold expression difference as a cutoff level, we identified 22 differential expressed mature miRNAs. Sixteen miRNAs were upregulated in gastric carcinoma, including miR-223, miR-21, miR-23b, miR-222, miR-25, miR-23a, miR-221, miR-107, miR-103, miR-99a, miR-100, miR-125b, miR-92, miR-146a, miR-214 and miR-191, and six miRNAs were downregulated in gastric carcinoma, including let-7a, miR-126, miR-210, miR-181b, miR-197, and miR-30aa-5p. After examining these miRNAs in several human gastric originated cell lines, we found that miR-223 is overexpressed only in metastatic gastric cancer cells and stimulated nonmetastatic gastric cancer cells migration and invasion. Mechanistically, miR-223, induced by the transcription factor Twist, posttranscriptionally downregulates EPB41L3 expression by directly targeting its 3′-untranslated regions. Significantly, overexpression of miR-223 in primary gastric carcinomas is associated with poor metastasis-free survival. These findings indicate a new regulatory mode, namely, specific miRNA, which is activated by its upstream transcription factor, could suppress its direct targets and lead to tumor invasion and metastasis. Mol Cancer Res; 9(7); 824–33. ©2011 AACR.

Hypoxia‐inducible factor‐1α contributes to hypoxia‐induced chemoresistance in gastric cancer

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 colony‐forming assay revealed that hypoxia and forced HIF‐1α expression increased maximal –8.9‐fold or –14.8‐fold of IC50 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 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 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 in vivo was suppressed by blocking HIF‐1α expression with siRNA. We further demonstrated that HIF‐1 α overexpression showed a 1.85‐fold increased expression of Bcl‐2 and a 2.16‐fold decreased expression of Bax, and also showed significantly induced expression of p‐gp and MRP1, which indicated that HIF‐1α may confer hypoxia‐induced drug resistance via inhibition of drug‐induced apoptosis and decreases in intracellular drug accumulation. (Cancer Sci 2008; 99: 121–128)

In Vivo Tracking and Comparison of the Therapeutic Effects of MSCs and HSCs for Liver Injury

Background Mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) have been studied for damaged liver repair; however, the conclusions drawn regarding their homing capacity to the injured liver are conflicting. Besides, the relative utility and synergistic effects of these two cell types on the injured liver remain unclear. Methodology/Principal Findings MSCs, HSCs and the combination of both cells were obtained from the bone marrow of male mice expressing enhanced green fluorescent protein(EGFP)and injected into the female mice with or without liver fibrosis. The distribution of the stem cells, survival rates, liver function, hepatocyte regeneration, growth factors and cytokines of the recipient mice were analyzed. We found that the liver content of the EGFP-donor cells was significantly higher in the MSCs group than in the HSCs or MSCs+HSCs group. The survival rate for the MSCs group was significantly higher than that of the HSCs or MSCs+HSCs group; all surpassed the control group. After MSC-transplantation, the injured livers were maximally restored, with less collagen than the controls. The fibrotic areas had decreased to a lesser extent in the mice transplanted with HSCs or MSCs+HSCs. Compared with mice in the HSCs group, the mice that received MSCs had better improved liver function. MSCs exhibited more remarkable paracrine effects and immunomodulatory properties on hepatic stellate cells and native hepatocytes in the treatment of the liver pathology. Synergistic actions of MSCs and HSCs were most likely not observed because the stem cells in liver were detected mostly as single cells, and single MSCs are insufficient to provide a beneficial niche for HSCs. Conclusions/Significance MSCs exhibited a greater homing capability for the injured liver and modulated fibrosis and inflammation more effectively than did HSCs. Synergistic effects of MSCs and HSCs were not observed in liver injury.

Expression of delayed rectifier potassium channels and their possible roles in proliferation of human gastric cancer cells.

Voltage-gated potassium (Kv) channels have been reported to be involved in the proliferation of many types of cells, including tumor cells. The overexpression of the Kv channels and related channel activity are involved in the neoplastic process. Our previous study has shown the existence of delayed rectifier potassium (IK) current in gastric cancer cells SGC7901. However, the expression and function of most delayed rectifier potassium (KD) channel subunits in gastric cancer cells are not completely resolved. Here we examine expression of KD channel subunits in Kv1-Kv3 families in immortalized gastric epithelial cells GES and various gastric cancer cells (including AGS, KATO¢Û, MKN28, MKN45, MGC803, SGC7901, SGC7901/ADR, and SGC7901/VCR), and their roles in cell proliferation. RT-PCR analysis reveals that all cell lines examined express Kv1.3, Kv1.5, Kv1.6, Kv2.1, and Kv2.2. However, Kv1.2 and Kv3.2 genes are barely detectable in any given cancer cell lines. Kv1.5 protein, high mRNA levels in all cell lines examined, is also expressed in some cancer cells lines and more frequently detected in gastric cancer tissues. Down-regulation of the expression of Kv1.5 in SGC7901 with RNA interference significantly inhibited the proliferation and tumorigenicity of SGC7901 cells. Moreover, in Ca2+-containing rather than Ca2+-free medium, KCl (50mM) stimulated a rapid increase in the concentration of cytosolic calcium in empty vector transfected cells that was blocked by verapamil. Likewise, decrease the expression of Kv1.5 with short interfering RNA also blocked the depolarization-induced influx of Ca2+. This finding suggests that more than one kind of KD channel subunits are expressed in various gastric cancer cell lines. Kv1.5 may involved in tumor cells proliferation by controlling Ca2+ entry, and the interference of KD channels expression and/or activity could provide a novel strategy to reverse the malignant phenotype of gastric cancer cells.

Dynamic microRNA Profiles of Hepatic Differentiated Human Umbilical Cord Lining-Derived Mesenchymal Stem Cells

Despite the extensive hepatic differentiation potential of human umbilical cord lining-derived mesenchymal stem cells (hUC-MSC), little is known about the molecular mechanisms of hUC-MSC differentiation. At the post-transcriptional level, microRNAs are key players in the control of cell fate determination during differentiation. In this study, we aimed to identify microRNAs involved in the hepatic differentiation of hUC-MSCs. After successfully isolating hUC- MSCs, we induced hepatocyte formation in vitro with growth factors. After 26 days of induction, hUC-MSCs could express hepatocyte-specific genes, synthesize urea and glycogen and uptake low-density lipoprotein. Cellular total RNA from hUC-MSCs and hepatic differentiated hUC-MSCs was collected at 7 time points, including 2 days, 6 days, 10 days, 14 days, 22 days and 26 days, for microRNA microarray analysis. Dynamic microRNA profiles were identified that did not overlap or only partially overlapped with microRNAs reported to be involved in human liver development, hepatocyte regeneration or hepatic differentiation of liver-derived progenitor cells. A total of 61 microRNAs among 1205 human and 144 human viral microRNAs displayed consistent changes and were altered at least 2-fold between hUC-MSCs and hepatic differentiated hUC-MSCs. Among these microRNAs, 25 were over-expressed; this over-expression occurred either gradually or increased sharply and was maintained at a high level. A total of 36 microRNAs were under-expressed, with an expression pattern similar to that of the over-expressed microRNAs. The expression of the altered expressed microRNAs was also confirmed by quantitative reverse-transcription polymerase chain reaction. We also found that microRNAs involved in hepatic differentiation were not enriched in hepatocyte or hepatocellular carcinoma cells and can potentially target liver-enriched transcription factors and genes. The elucidation of the microRNA profile during the hepatic differentiation of hUC-MSCs provides the basis for clarifying the role of microRNAs in hUC-MSC hepatic differentiation and specific microRNA selection for the conversion of hUC-MSCs to hepatocytes.

Runx3 suppresses gastric cancer metastasis through inactivation of MMP9 by upregulation of TIMP-1.

Recent studies have suggested that loss of RUNX3 expression is involved with gastric tumor metastasis. However, the precise mechanism of RUNX3‐mediated suppression of tumor metastasis remains elusive. We aimed to clarify the effect of RUNX3 on tumor metastasis in gastric cancer cell lines and tumors. Immunohistochemistry revealed that RUNX3 was significantly decreased in metastatic gastric cancer. Gelatin zymography and Western blot showed that instead of regulating matrix metalloproteinase 9 (MMP9) expression, RUNX3 expression inhibited MMP9 enzyme activity, and this was consistent with the upregulation of tissue inhibitor of metalloproteinases 1 (TIMP1) by RUNX3. TIMP1 siRNA treatment impaired RUNX3‐mediated suppression of gastric cancer cell invasion. Reporter assays demonstrated regulation of TIMP‐1 by RUNX3. Two RUNX3 binding sites were identified in the TIMP‐1 promoter and direct interaction of RUNX3 with the TIMP‐1 promoter was confirmed in vitro and in vivo. These findings provide evidence for RUNX3‐mediated suppression of gastric cancer invasion and metastasis and define a novel molecular mechanism that for the metastasis‐inhibiting activity of RUNX3. These data may be applied in the development of RUNX3 for gastric cancer metastasis diagnostics and therapeutics.

Akt1/protein Kinase Bα is Involved in Gastric Cancer Progression and Cell Proliferation

Akt (also known as protein kinase B, PKB) is involved in a variety of biological processes, for example cell development, proliferation, and angiogenesis. Clinical studies in support of the idea that increased activity of Akt could contribute directly to gastric carcinogenesis are rare, however. In this study we discovered that phospho-Akt1 was overexpressed in human gastric cancers and its levels correlated with tumor differentiation and pTNM. Akt1 activation promoted cell survival, because the phosphatidylinositol 3-kinase(PI3K) inhibitor LY294002 inhibited Akt1 phosphorylation and inhibited cell growth, especially in cells with active Akt1. Dominant negative Akt inhibited proliferation of gastric cancer cells and induced G1 cell-cycle arrest whereas constitutively active Akt increased cell proliferation. We have therefore identified Akt1 as an active kinase that contributes to gastric cancer progression and promotes proliferation of gastric cancer cells.

Bone marrow-derived stem cells ameliorate hepatic fibrosis by down-regulating interleukin-17.

BackgroundAccumulating evidences have identified the immunoregulatory features of stem cells. In this study, the immunoregulation of bone marrow-derived stem cells (BMSCs) transplanted into patients with HBV-related decompensated cirrhosis and mouse model of liver injury induced by carbon tetrachloride (CCl4) administration was observed.ResultsCompared with healthy controls, patients with HBV-related decompensated cirrhosis showed significantly higher levels of TNF-alpha, IL-12, TGF-beta1, IL-17, and IL-8. However, only IL-17 was markedly decreased after autologous BMSCs transplantation during their follow-up. The same results were found in the CCl4-treated mice. Furthermore, we found that exogenous IL-17 partly abolished the therapeutic effect of BMSCs whereas IL-17-specific antibody promoted improvement of liver injury in CCl4-treated mice, resembling the therapeutic effect of BMSCs transplantation.ConclusionsThese data suggested that BMSCs transplantation induces a decrease of IL-17 level, which at least in part delineates the mechanisms of stem cells-mediated therapeutic benefit on liver disease.

Detection of potassium currents and regulation of multidrug resistance by potassium channels in human gastric cancer cells

The present study aimed to investigate the potassium currents and further explore the role of potassium channels in drug response of gastric cancer cells. By patch‐clamp technique, potassium currents of human gastric cancer cell SGC7901 were recorded in the mode of voltage clamp. Both 4‐aminopyridine (4‐AP) and tetraethylammonium (TEA) could almost completely block this current. The chemotherapeutic drugs, adriamycin or 5‐fluorouracil could significantly increase the K+ current density on SGC7901 cells in a dose‐dependent manner. 4‐AP or TEA was found to restrain adriamycin‐induced apoptosis and enhance multidrug‐resistant phenotype of SGC7901 cells. Up‐regulation of Kv1.5, which has been found widely expressed in gastric cancer cells including SGC7901, increased the K+ current density and sensitivity of SGC7901 cells to multiple chemotherapeutic drugs, whereas down‐regulation of Kv1.5 enhanced the drug‐resistant phenotype of SGC7901 cells. In conclusion, potassium channels may exert regulatory effects on multidrug resistance by regulating drug‐induced apoptosis in gastric cancer cells.

Reversal of Multidrug Resistance of Gastric Cancer Cells by Downregulation of TSG101 with TSG101siRNA

We have previously identified tumor susceptibility gene101 (TSG101), overexpressed in vincristine-resistant human gastric adenocarcinoma cell line SGC7901/VCR using a modified subtractive hybridization method and Western blot. In the present study, we constructed two siRNA eukaryotic expression vectors of TSG101 and transfected them into SGC7901/VCR cells to examine whether the down-regulation of TSG101 increased cell sensitivity towards chemotherapeutic drugs. After transfection, the expression of TSG101 was dramatically decreased in TSG101 siRNA transfectants compared with that in parental cells and empty vector control cells. The down-regulation of TSG101 could significantly enhance the sensitivity of SGC7901/VCR cells to vincristine and adriamycin. The capacity of cells to efflux adriamycin markedly decreased in TSG101 siRNA transfectants. These observations suggested that the siRNA constructs of TSG101 we made could effectively down-regulate the expression of TSG101 and reverse the resistant phenotype of gastric cancer cells. The preliminary study suggested that TSG101 might play an important role in multidrug resistance of gastric carcinoma.