Xuan Xiang

microRNA-9 suppresses the proliferation, invasion and metastasis of gastric cancer cells through targeting cyclin D1 and Ets1.

Recent evidence shows that altered microRNA-9 (miR-9) expression is implicated in the progression of gastric cancer. However, the exact roles and underlying mechanisms of miR-9 in the proliferation, invasion and metastasis of gastric cancer still remain unknown. In this study, miR-9 was found to be down-regulated and inversely correlated with the expression of cyclin D1 and v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets1) in gastric cancer tissues and cell lines. Bioinformatics analysis revealed the putative miR-9 binding sites in the 3′-untranslated regions (3′-UTR) of cyclin D1 and Ets1 mRNA. Ectopic expression or knockdown of miR-9 resulted in responsively altered expression of cyclin D1, Ets1 and their downstream targets phosphorylated retinoblastoma and matrix metalloproteinase 9 in cultured gastric cancer cell lines SGC-7901 and AGS. In the luciferase reporter system, miR-9 directly targeted the 3′-UTR of cyclin D1 and Ets1, and these effects were abolished by mutating the miR-9 binding sites. Over-expression of miR-9 suppressed the proliferation, invasion, and metastasis of SGC-7901 and AGS cells in vitro and in vivo. Restoration of miR-9-mediated down-regulation of cyclin D1 and Ets1 by transient transfection, rescued the cancer cells from decrease in proliferation, migration and invasion. Furthermore, anti-miR-9 inhibitor promoted the proliferation, migration and invasion of gastric cancer cells, while knocking down of cyclin D1 or Ets1 partially phenocopied the effects of miR-9 over-expression. These data indicate that miR-9 suppresses the expression of cyclin D1 and Ets1 via the binding sites in their 3′-UTR, thus inhibiting the proliferation, invasion and metastasis of gastric cancer.

miRNA-145 targets v-ets erythroblastosis virus E26 oncogene homolog 1 to suppress the invasion, metastasis, and angiogenesis of gastric cancer cells.

Recent evidence shows that v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets1) is implicated in tumor development and progression. However, the clinical potentials and underlying mechanisms of Ets1 in gastric cancer progression and metastasis remain largely unknown. In this study, Ets1 immunostaining was identified in 56 of 84 (66.7%) gastric cancer tissues, which was correlated with tumor invasion and metastasis. In gastric cancer specimens and cell lines, miRNA-145 (miR-145) was downregulated and inversely correlated with Ets1 expression. Bioinformatics analysis and luciferase reporter assay revealed that miR-145 directly targeted the 3′-untranslated region (3′-UTR) of Ets1 mRNA. Overexpression or knockdown of miR-145 responsively altered both the mRNA and protein levels of Ets1 and its downstream genes, matrix metalloproteinase (MMP-1)-1 and -9, in gastric cancer cell lines SGC-7901 and MKN-45. Ectopic expression of miR-145 suppressed the invasion, metastasis, and angiogenesis of SGC-7901 and MKN-45 cells in vitro and in vivo. In addition, the effects of miR-145 on Ets1 expression, migration, invasion, and angiogenesis were rescued by restoration of Ets1 expression in these cells. Furthermore, anti-miR-145 inhibitor promoted the migration, invasion, and angiogenesis, whereas siRNA-mediated Ets1 knockdown phenocopied the effects of miR-145 overexpression in gastric cancer cells. These results show that miR-145 suppresses Ets1 expression via the binding site in the 3′-UTR, thus inhibiting the invasion, metastasis, and angiogenesis of gastric cancer cells. Mol Cancer Res; 11(2); 182–93. ©2012 AACR.

miRNA-558 promotes tumorigenesis and aggressiveness of neuroblastoma cells through activating the transcription of heparanase

Heparanase (HPSE) is the endogenous endoglycosidase that degrades heparan sulfate proteoglycans and promotes the tumor growth, invasion, metastasis and angiogenesis. Our previous studies have shown that HPSE is highly expressed in neuroblastoma (NB), the most common extracranial solid tumor in childhood. However, the underlying regulatory mechanisms remain largely unknown. In this study, we identified one binding site of microRNA-558 (miR-558) within the HPSE promoter. In NB tissues and cell lines, miR-558 was up-regulated and positively correlated with HPSE expression. Gain- and loss-of-function studies demonstrated that miR-558 facilitated the transcript and protein levels of HPSE and its downstream gene, vascular endothelial growth factor, in NB cell lines. In addition, miR-558 enhanced the promoter activities of HPSE, and these effects were abolished by the mutation of the miR-558-binding site. Mechanistically, miR-558 induced the enrichment of the active epigenetic marker and RNA polymerase II on the HPSE promoter in NB cells in an Argonaute 1-dependent manner, which was abolished by repressing the miR-558-promoter interaction. Knockdown of endogenous miR-558 decreased the growth, invasion, metastasis and angiogenesis of NB cells in vitro and in vivo. In contrast, over-expression of miR-558 promoted the growth, invasion, metastasis and angiogenesis of SH-SY5Y and SK-N-SH cells. Restoration of HPSE expression prevented the NB cells from changes in these biological features induced by knockdown or over-expression of miR-558. These data indicate that miR-558 induces the transcriptional activation of HPSE via the binding site within promoter, thus facilitating the tumorigenesis and aggressiveness of NB.

Methyl jasmonate abolishes the migration, invasion and angiogenesis of gastric cancer cells through down-regulation of matrix metalloproteinase 14

BackgroundRecent evidence indicates that methyl jasmonate (MJ), a plant stress hormone, exhibits anti-cancer activity on human cancer cells. The aim of this study is to determine whether sub-cytotoxic MJ can abolish the migration, invasion and angiogenesis gastric cancer cells.MethodsHuman gastric cancer cell lines SGC-7901 and MKN-45 were treated with diverse concentrations of MJ. Cell viability, proliferation, migration, invasion and angiogenesis capabilities of cancer cells were measured by MTT colorimetry, EdU incorporation, scratch assay, matrigel invasion assay, and tube formation assay. Gene expression was detected by western blot and real-time quantitative RT-PCR. Binding of transcription factor on gene promoter was detected by chromatin immunoprecipitation.ResultsSub-cytotoxic (0.05 to 0.2 mM) MJ attenuated the migration, invasion and angiogenesis, but not the cell viability or proliferation, of gastric cancer cells in a time- and dose-dependent manner, with down-regulation of matrix metalloproteinase 14 (MMP-14) and its downstream gene vascular endothelial growth factor. Restoration of MMP-14 expression rescued the SGC-7901 and MKN-45 cells from sub-cytotoxic MJ-inhibited migration, invasion and angiogenesis. In addition, sub-cytotoxic MJ decreased the specificity protein 1 (Sp1) expression and binding on MMP-14 promoter, while restoration of Sp1 expression rescued the cancer cells from sub-cytotoxic MJ-mediated defects in MMP-14 expression, migration, invasion and angiogenesis.ConclusionsSub-cytotoxic MJ attenuates the MMP-14 expression via decreasing the Sp1 expression and binding on MMP-14 promoter, thus inhibiting the migration, invasion and angiogenesis of gastric cancer cells.

miRNA-337-3p inhibits gastric cancer progression through repressing myeloid zinc finger 1-facilitated expression of matrix metalloproteinase 14.

Matrix metalloproteinase 14 (MMP-14), a membrane-anchored MMP that promotes the tumorigenesis and aggressiveness, is highly expressed in gastric cancer. However, the transcriptional regulators of MMP-14 expression in gastric cancer still remain largely unknown. In this study, through mining computational algorithm programs and chromatin immunoprecipitation datasets, we identified adjacent binding sites of myeloid zinc finger 1 (MZF1) and miRNA-337-3p (miR-337-3p) within the MMP-14 promoter. We demonstrated that MZF1 directly bound to the MMP-14 promoter to facilitate its nascent transcription and expression in gastric cancer cell lines. In contrast, endogenous miR-337-3p suppressed the MMP-14 expression through recognizing its binding site within MMP-14 promoter. Mechanistically, miR-337-3p repressed the binding of MZF1 to MMP-14 promoter via recruiting Argonaute 2 and inducing repressive chromatin remodeling. Gain- and loss-of-function studies demonstrated that miR-337-3p suppressed the growth, invasion, metastasis, and angiogenesis of gastric cancer cells in vitro and in vivo through repressing MZF1-facilitated MMP-14 expression. In clinical specimens and cell lines of gastric cancer, MZF1 was highly expressed and positively correlated with MMP-14 expression. Meanwhile, miR-337-3p was under-expressed and inversely correlated with MMP-14 levels. miR-337-3p was an independent prognostic factor for favorable outcome of gastric cancer, and patients with high MZF1 or MMP-14 expression had lower survival probability. Taken together, these data indicate that miR-337-3p directly binds to the MMP-14 promoter to repress MZF1-facilitatd MMP-14 expression, thus suppressing the progression of gastric cancer.

Intelectin 1 suppresses the growth, invasion and metastasis of neuroblastoma cells through up-regulation of N-myc downstream regulated gene 2

BackgroundRecent studies have revealed the potential roles of intelectin 1 (ITLN1) in tumorigenesis. However, its functions and underlying mechanisms in neuroblastoma (NB), the most common extracranial solid tumor in childhood, still remain largely unknown.MethodsHuman neuroblastoma cell lines were treated with recombinant ITLN1 protein or stably transfected with ITLN1 expression and short hairpin RNA vectors. Gene expression and signaling pathway were detected by western blot and real-time quantitative RT-PCR. Gene promoter activity and transcription factor binding were detected by luciferase reporter and chromatin immunoprecipitation assays. Growth and aggressiveness of tumor cells were measured by MTT colorimetry, colony formation, scratch assay, matrigel invasion assay, and nude mice model.ResultsMining of public microarray databases revealed that N-myc downstream regulated gene 2 (NDRG2) was significantly correlated with ITLN1 in NB. Gain- and loss-of-function studies indicated that secretory ITLN1 facilitated the NDRG2 expression, resulting in down-regulation of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP-9), in NB cell lines SH-SY5Y, SK-N-BE(2), and SK-N-SH. Krüppel-like factor 4 (KLF4), a transcription factor crucial for NDRG2 expression, was up-regulated by ITLN1 in NB cells via inactivation of phosphoinositide 3-kinase (PI3K)/AKT signaling. Ectopic expression of ITLN1 suppressed the growth, invasion and metastasis of NB cells in vitro and in vivo. Conversely, knockdown of ITLN1 promoted the growth, invasion, and metastasis of NB cells. In addition, rescue experiments in ITLN1 over-expressed or silenced NB cells showed that restoration of NDRG2 expression prevented the tumor cells from ITLN1-mediated changes in these biological features. In clinical NB tissues, ITLN1 was down-regulated and positively correlated with NDRG2 expression. Patients with high ITLN1 or NDRG2 expression had greater survival probability.ConclusionsThese findings indicate that ITLN1 functions as a tumor suppressor that affects the growth, invasion and metastasis of NB through up-regulation of NDRG2.

miRNA-584-5p exerts tumor suppressive functions in human neuroblastoma through repressing transcription of matrix metalloproteinase 14

Matrix metalloproteinase 14 (MMP-14) is a membrane-anchored MMP crucial for tumorigenesis and aggressiveness, and is highly expressed in neuroblastoma (NB), the most common extracranial solid tumor in childhood. Recent evidence shows the emerging roles of endogenous promoter-targeting microRNAs (miRNAs) in regulating gene transcription. However, the roles of miRNAs in the transcription of MMP-14 still remain largely unknown. In this study, through mining computational algorithm program and Argonaute-chromosome interaction dataset, we identified one binding site of miRNA-584-5p (miR-584-5p) within the MMP-14 promoter. In NB tissues, miR-584-5p was under-expressed and inversely correlated with MMP-14 expression, and was an independent prognostic factor for favorable outcome of patients. miR-584-5p precursor attenuated the expression of MMP-14 in a Dicer-dependent manner, resulting in decreased levels of vascular endothelial growth factor, in cultured NB cell lines. In addition, miR-584-5p suppressed the promoter activity of MMP-14, and mutation of miR-584-5p binding site abolished these effects. Mechanistically, miR-584-5p recruited Argonaute 2 to facilitate the enrichment of enhancer of zeste homolog 2, histone H3 lysine 27 trimethylation, and histone H3 lysine 9 dimethylation on MMP-14 promoter in NB cells, which was abolished by repressing the miR-584-5p-promoter interaction. Gain- and loss-of-function studies demonstrated that miR-584-5p suppressed the growth, invasion, metastasis, and angiogenesis of NB cells in vitro and in vivo. Moreover, restoration of MMP-14 expression rescued the NB cells from changes in these biological features. Taken together, these results indicate that promoter-targeting miR-584-5p exerts tumor suppressive functions in NB through repressing the transcription of MMP-14.

Hepatocyte nuclear factor 4 alpha promotes the invasion, metastasis and angiogenesis of neuroblastoma cells via targeting matrix metalloproteinase 14

Matrix metalloproteinase 14 (MMP-14) is the only membrane-anchored MMP that plays critical roles in tumorigenesis and aggressiveness. However, the regulatory mechanisms underlying the high MMP-14 expression in neuroblastoma (NB), a highly malignant tumor in childhood, still remain unclear. Herein, we applied an integrative approach to analyze the public datasets, and identified hepatocyte nuclear factor 4 alpha (HNF4α) as a crucial transcription factor facilitating the MMP-14 expression in NB. In clinical NB tissues, HNF4α was up-regulated and positively correlated with MMP-14 expression, and was an independent prognostic factor for unfavorable outcome of patients. Luciferase reporter and chromatin immunoprecipitation assays indicated that HNF4α directly targeted the binding site within the MMP-14 promoter to facilitate its transcription. Knockdown of HNF4α suppressed the invasion, metastasis and angiogenesis of NB cells in vitro and in vivo. Conversely, ectopic expression of HNF4α promoted the invasion, metastasis and angiogenesis of NB cells. Importantly, restoration of MMP-14 expression prevented the tumor cells from HNF4α-mediated changes in these biological features. Taken together, HNF4α exhibits oncogenic activity that affects the aggressiveness and angiogenesis of NB through activating the transcription of MMP-14.

Association of Ureaplasma urealyticum colonization with development of bronchopulmonary dysplasia: a systemic review and meta-analysis.

There is controversy regarding the roles of Ureaplasma urealyticum (U. urealyticum) colonization in the development of bronchopulmonary dysplasia (BPD). This study explored the association between U. urealyticum and bronchopulmonary dysplasia at 36 weeks post-menstrual age (BPD36). Studies published before December 31, 2013 were searched from Medline, Embase, Ovid, Web of Science, and Cochrane databases, with the terms “Ureaplasma urealyticum”, “chronic lung disease”, or “BPD36” used, and English language as a limit. The association between U. urealyticum colonization and BPD36 was analyzed with RevMan 4.2.10 software, using the odds ratio (OR) and relative risk (RR) for dichotomous variables. Out of the enrolled 81 studies, 11 investigated the BPD36 in total 1193 infants. Pooled studies showed no association between U. urealyticum colonization and subsequent development of BPD36, with the OR and RR being 1.03 (95% CI=0.78–1.37; P=0.84) and 1.01 (95% CI= 0.88–1.16, P=0.84), respectively. These findings indicated no association between U. urealyticum colonization and the development of BPD36.SummaryThere is controversy regarding the roles of Ureaplasma urealyticum (U. urealyticum) colonization in the development of bronchopulmonary dysplasia (BPD). This study explored the association between U. urealyticum and bronchopulmonary dysplasia at 36 weeks post-menstrual age (BPD36). Studies published before December 31, 2013 were searched from Medline, Embase, Ovid, Web of Science, and Cochrane databases, with the terms “Ureaplasma urealyticum”, “chronic lung disease”, or “BPD36” used, and English language as a limit. The association between U. urealyticum colonization and BPD36 was analyzed with RevMan 4.2.10 software, using the odds ratio (OR) and relative risk (RR) for dichotomous variables. Out of the enrolled 81 studies, 11 investigated the BPD36 in total 1193 infants. Pooled studies showed no association between U. urealyticum colonization and subsequent development of BPD36, with the OR and RR being 1.03 (95% CI=0.78–1.37; P=0.84) and 1.01 (95% CI= 0.88–1.16, P=0.84), respectively. These findings indicated no association between U. urealyticum colonization and the development of BPD36.