Jiping Zeng

The tumor suppressive role of miRNA-370 by targeting FoxM1 in acute myeloid leukemia

BackgroundRecent evidence has accumulated that MicroRNA (miRNA) dysregulation occurs in the majority of human malignancies including acute myeloid leukemia (AML) and may contribute to onco-/leukemo-genesis.MethodsThe expression levels of miR-370 and FoxM1 were assessed in 48 newly diagnosed AML patients, 40 AML patients in 1st complete remission (CR) and 21 healthy controls. Quantitative real-time PCR, western blots, colony formation assay, and β-Galactosidase ( SA- β-Gal) staining were used to characterize the changes induced by overexpression or inhibition of miR-370 or FoxM1.ResultsWe found that the down-regulation of miR-370 expression was a frequent event in both leukemia cell lines and primary leukemic cells from patients with de novo AML. Lower levels of miR-370 expression were found in 37 of 48 leukemic samples from AML patients compared to those in bone marrow cells derived from healthy adult individuals. Ectopic expression of miR-370 in HL60 and K562 cells led to cell growth arrest and senescence. In contrast, depletion of miR-370 expression using RNA interference enhanced the proliferation of those leukemic cells. Mechanistically, miR-370 targets the transcription factor FoxM1, a well established oncogenic factor promoting cell cycle progression. Moreover, when HL60 and K562 cells were treated with 5-aza-2′-deoxycytidine, a DNA methylation inhibitor, miR-370 expression was up-regulated, which indicates epigenetic silencing of miR-370 in leukemic cells.ConclusionsTaken together, miR-370 may function as a tumor suppressor by targeting FoxM1, and the epigenetic silence of miR-370 thus leads to derepression of FoxM1 expression and consequently contributes to AML development and progression.

RUNX3 regulates vimentin expression via miR‐30a during epithelial–mesenchymal transition in gastric cancer cells

Runt‐related transcription factor 3 (RUNX3) is a putative tumour suppressor via regulating the expression of a series of target genes. Clinical studies demonstrated that loss of RUNX3 expression is associated with gastric cancer progression and poor prognosis, but the underlying mechanism is not entirely clear. Accumulating evidence shows that the epithelial–mesenchymal transition (EMT) plays an important role in cancer relapse and metastasis. Therefore, we addressed whether RUNX3 has a role in the EMT in gastric cancer. Knockdown of RUNX3 promoted cell invasion and increased the protein expression of the mesenchymal marker vimentin in human gastric cancer cells. Overexpression of RUNX3 suppressed cell invasion and decreased the protein expression of vimentin in the cells and inhibited gastric cancer cells colonization in nude mice. Furthermore, overexpression of RUNX3 increased the expression of microRNA‐30a (miR‐30a), and miR‐30a directly targeted the 3′ untranslated region of vimentin and decreased its protein level. miR‐30a inhibitor abrogated RUNX3‐mediated inhibition of cell invasion and downregulation of vimentin. Thus, RUNX3 suppressed gastric cancer cell invasion and vimentin expression by activating miR‐30a. In gastric cancer patients, levels of RUNX3 were positively correlated with miR‐30a and negatively associated with the levels of vimentin. Collectively, our data suggest a novel molecular mechanism for the tumour suppressor activity of RUNX3. Effective therapy targeting the RUNX3 pathway may help control gastric cancer cell invasion and metastasis by inhibiting the EMT.

Histone demethylase retinoblastoma binding protein 2 is overexpressed in hepatocellular carcinoma and negatively regulated by hsa-miR-212.

Background The H3K4 demethylase retinoblastoma binding protein 2 (RBP2) is involved in the pathogenesis of gastric cancer, but its role and regulation in hepatocellular carcinoma (HCC) is unknown. We determined the function of RBP2 and its regulation in HCC in vitro and in human tissues. Methods We analyzed gene expression in 20 specimens each of human HCC and normal liver tissue by quantitative real-time PCR and immunohistochemistry. Proliferation was analyzed by foci formation and senescence by β-galactosidase staining. Promoter activity was detected by luciferase reporter assay. Results The expression of RBP2 was stronger in cancerous than non-cancerous tissues, but that of its binding microRNA, Homo sapiens miR-212 (hsa-miR-212), showed an opposite pattern. SiRNA knockdown of RBP2 significantly upregulated cyclin-dependent kinase inhibitors (CDKIs), with suppression of HCC cell proliferation and induction of senescence. Overexpression of hsa-miR-212 suppressed RBP2 expression, with inhibited cell proliferation and induced cellular senescence, which coincided with upregulated CDKIs; with low hsa-miR-212 expression, CDKIs were downregulated in HCC tissue. Inhibition of hsa-miR-212 expression upregulated RBP2 expression. Luciferase reporter assay detected the direct binding of hsa-miR-212 to the RBP2 3′ UTR. Conclusions RBP2 is overexpressed in HCC and negatively regulated by hsa-miR-212. The hsa-miR-212–RBP2–CDKI pathway may be important in the pathogenesis of HCC.

Helicobacter pylori CagA upregulation of CIP2A is dependent on the Src and MEK/ERK pathways

Helicobacter pylori is classified as a class I carcinogenic factor and its persistent colonization in the stomach induces gastric cancer. Cancerous Inhibitor of PP2A (CIP2A) is a newly identified oncoprotein overexpressed in gastric cancer. Serving as a key oncoprotein, CIP2A also participates in regulation of senescence and proliferation of gastric cells. The combination of aberrant CIP2A expression inducing unlimited cell proliferation, and H. pylori infection eliciting aberrant expression of some key proteins, results in the onset of gastric tumorigenesis. However, the relationship between H. pylori infection and CIP2A expression still remains undefined. The aim of our study was to verify the effect of H. pylori infection on CIP2A expression levels and identify H. pylori signalling molecules and corresponding pathways influencing CIP2A expression. Following plasmid-mediated expression of CagA in human gastric cell lines, the cells were infected with H. pylori and CIP2A expression levels were examined by immunoblotting. Signal inhibitors were used to verify which signal pathways were involved. We also performed CIP2A depletion and H. pylori infection after depletion in AGS cells. H. pylori infection-induced CIP2A expression was dependent on cagA gene expression and CagA phosphorylation. Bacterial oncoprotein CagA upregulated CIP2A expression and this upregulation effect was dependent on Src and Ras/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways. H. pylori infection-induced Myc stabilization was partially attenuated by CIP2A depletion. The results of our study provide further information for understanding the mechanism of H. pylori carcinogenesis.

Helicobacter pylori promotes epithelial-mesenchymal transition in gastric cancer by downregulating programmed cell death protein 4 (PDCD4).

Helicobacter pylori, a Gram-negative, microaerophilic bacterium found in the stomach, is assumed to be associated with carcinogenesis, invasion and metastasis in digestive diseases. Cytotoxin-associated gene A (CagA) is an oncogenic protein of H. pylori that is encoded by a Cag pathogenicity island related to the development of gastric cancer. The epithelial–mesenchymal transition (EMT) is the main biological event in invasion or metastasis of epithelial cells. H. pylori may promote EMT in human gastric cancer cell lines, but the specific mechanisms are still obscure. We explored the underlying molecular mechanism of EMT induced by H. pylori CagA in gastric cancer. In our article, we detected gastric cancer specimens and adjacent non-cancerous specimens by immunohistochemistry and found increased expression of the EMT-related regulatory protein TWIST1 and the mesenchymal marker vimentin in cancer tissues, while programmed cell death factor 4 (PDCD4) and the epithelial marker E-cadherin expression decreased in cancer specimens. These changes were associated with degree of tissue malignancy. In addition, PDCD4 and TWIST1 levels were related. In gastric cancer cells cocultured with CagA expression plasmid, CagA activated TWIST1 and vimentin expression, and inhibited E-cadherin expression by downregulating PDCD4. CagA also promoted mobility of gastric cancer cells by regulating PDCD4. Thus, H. pylori CagA induced EMT in gastric cancer cells, which reveals a new signaling pathway of EMT in gastric cancer cell lines.

Helicobacter pylori protein response to human bile stress

The ability of Helicobacter pylori to tolerate bile is likely to be important for its colonization and survival in the gastrointestinal tract of humans. As bile can be acidified after reflux into the low pH of the human stomach, the inhibitory effect of fresh human bile with normal appearance on H. pylori before and after acidification was tested first. The results showed that acidification of bile attenuated its inhibitory activity towards H. pylori. Next, the protein profiles of H. pylori under human bile and acidified bile stress were obtained by two-dimensional electrophoresis. Protein spots with differential expression were identified using tandem matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results showed that the changes in proteomic profiles under bile and acidified bile stress were similar when compared with that of normal H. pylori. Expression of 28 proteins was found to be modulated, with the majority being induced during bile or acidified bile exposure. These proteins included molecular chaperones, proteins involved in iron storage, chemotaxis protein, enzymes related to energy metabolism and flagellar protein. These results indicate that H. pylori responds to bile and acidified bile stress through multiple mechanisms involving many signalling pathways.

Critical role of histone demethylase RBP2 in human gastric cancer angiogenesis

BackgroundThe molecular mechanisms responsible for angiogenesis and abnormal expression of angiogenic factors in gastric cancer, including vascular endothelial growth factor (VEGF), remain unclear. The histone demethylase retinoblastoma binding protein 2 (RBP2) is involved in gastric tumorgenesis by inhibiting the expression of cyclin-dependent kinase inhibitors (CDKIs).MethodsThe expression of RBP2, VEGF, CD31, CD34 and Ki67 was assessed in 30 human gastric cancer samples and normal control samples. We used quantitative RT-PCR, western blot analysis, ELISA, tube-formation assay and colony-formation assay to characterize the change in VEGF expression and associated biological activities induced by RBP2 silencing or overexpression. Luciferase assay and ChIP were used to explore the direct regulation of RBP2 on the promoter activity of VEGF. Nude mice and RBP2-targeted mutant mice were used to detect the role of RBP2 in VEGF expression and angiogenesis in vivo.ResultsRBP2 and VEGF were both overexpressed in human gastric cancer tissue, with greater microvessel density (MVD) and cell proliferation as compared with normal tissue. In gastric epithelial cell lines, RBP2 overexpression significantly promoted the expression of VEGF and the growth and angiogenesis of the cells, while RBP2 knockdown had the reverse effect. RBP2 directly bound to the promoter of VEGF to regulate its expression by histone H3K4 demethylation. The subcutis of nude mice transfected with BGC-823 cells with RBP2 knockdown showed reduced VEGF expression and MVD, with reduced carcinogenesis and cell proliferation. In addition, the gastric epithelia of RBP2 mutant mice with increased H3K4 trimethylation showed reduced VEGF expression and MVD.ConclusionsThe promotion of gastric tumorigenesis by RBP2 was significantly associated with transactivation of VEGF expression and elevated angiogenesis. Overexpression of RBP2 and activation of VEGF might play important roles in human gastric cancer development and progression.

MiR-320a inhibits gastric carcinoma by targeting activity in the FoxM1-P27 KIP1 axis

MicroRNAs (miRNAs) regulate tumorigenesis by inhibiting gene expression. In this study, we showed that miR-320a expression is decreased in human gastric cancer tissues and correlates inversely with expression of FoxM1, a key cell cycle regulator involved in gastric carcinoma. By contrast, the expression of P27KIP1, a downstream effector of FoxM1, correlates positively with miR-320a levels. Luciferase assays indicate that miR-320a suppresses FoxM1 expression, and in vitro recovery tests using FoxM1 siRNA indicate miR-320a inhibits gastric cancer cell proliferation by suppressing activity in the FoxM1-P27KIP1 axis. In vivo, nude mice injected with BGC-823 gastric cancer cells expressing a miR-320a inhibitor exhibit faster tumor growth than mice injected with control cells. Analysis of FoxM1 and P27KIP1 expression in tumor tissues indicates that miR-320a suppression increases the tumor growth by enhancing FoxM1-P27KIP1 signaling. These results thus reveal the crucial role played by miR-320a in limiting gastric carcinoma by directly targeting FoxM1- P27KIP1 axis.

MiR-370 sensitizes chronic myeloid leukemia K562 cells to homoharringtonine by targeting Forkhead box M1

BackgroundHomoharringtonine (HHT) is a kind of cephalotaxus alkaloid used in traditional Chinese medicine. Although HHT has been successfully used as a therapeutic agent for leukemia, the drug resistance and toxicity are major concerns. MicroRNAs (miRNAs) have been identified to modulate cellular sensitivity to anticancer drugs. We examined the synergistic action between miR-370 and HHT in vitro and in vivo.MethodsThe synergistic action between miR-370 and HHT was examined by flow cytometry. The effect of HHT on miR-370 expression was determined by quantitative RT-PCR (qRT-PCR). The expression of miR-370 and Forkhead box M1 (FoxM1) in 23 patients with newly diagnosed chronic-phase chronic myeloid leukemia (CML-CP) and 10 patients with blast-crisis CML (CML-BP) as well as miR-370–targeted FoxM1 was determined by qRT-PCR and western blot analysis.ResultsEctopic expression of miR-370 sensitized the CML K562 cell line to HHT by targeting FoxM1, the major regulator in cell proliferation and apoptosis. miR-370 significantly promoted HHT-mediated cell apoptosis and miR-370 and HHT cooperated in affecting FoxM1 expression. As well, miR-370 was moderately upregulated after HHT treatment in K562 cells. In addition, the expression of miR-370 was significantly reduced in CML patients as compared with healthy controls. Furthermore, the expression of miR-370 was lower in CML-BP than CML-CP patients.ConclusionsMiR-370 sensitized K562 cells to HHT by inducing apoptosis in part by downregulation of FoxM1 expression. These findings may provide further information for CML treatment with HHT.

RUNX3 inhibits survivin expression and induces cell apoptosis in gastric cancer

Transcription factor RUNX3 is associated with gastric tumorigenesis and progression through regulating the expression of its target genes. Survivin is a member of the inhibitor of apoptosis (IAP) family and has been shown to inhibit cell apoptosis and promote cell proliferation. Increased survivin expression has been found in various cancer types, including gastric cancer. In this study, we found that restoration of RUNX3 promotes cell apoptosis through inhibiting the survivin expression, while RUNX3 inhibition increases the expression of survivin in gastric cancer cell lines. Moreover, RUNX3 over-expression inhibits,whereas its inhibition increases, the promoter activity of survivin gene, respectively. RUNX3-R122C, a mutation located in the conserved Runt domain, has no effect on the promoter activity of survivin gene. We further identified a RUNX3-binding site in the promoter of survivin gene and the binding of RUNX3 on survivin promoter leads to significantly inhibition of survivin expression. Finally, we confirmed the negative correlation of RUNX3 and survivin expression in clinical specimens of gastric cancer. These findings reveal a novel mechanism of RUNX3 for the induction of cell apoptosis in human gastric cancer.