Qiangsong Tong

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.

Laparoscopic Versus Open Pyeloplasty for Ureteropelvic Junction Obstruction in Children: A Systematic Review and Meta-Analysis

PURPOSE To comprehensively review the available evidences in the literature on the use of laparoscopic pyeloplasty (LP) vs open pyeloplasty (OP) for the repair of ureteropelvic junction (UPJ) obstruction in children. MATERIALS AND METHODS Published studies until the end of October 2010 were searched from Medline, Embase, Web of Science, Ovid, and Cochrane databases. The literature search, quality assessment, and data extraction were independently performed by two reviewers. A systematic review and meta-analysis were performed by using Review Manager 4.2.8 software. RESULTS Of 1403 studies, one randomized controlled trial (RCT), two prospective comparative studies, and six retrospective observational studies were eligible for inclusion criteria, comprising 694 cases of LP and 7334 cases of OP. The OP has significantly reduced operative time (weighted mean difference [WMD] = 59.00; 95% confidence interval [CI] = 41.15 to 76.85; P < 0.00001) and higher stent placement rate (odds ratio [OR] = 5.97; 95% CI=3.17 to 11.26; P<0.00001) compared with LP, whereas the duration of hospital stay was shorter in the LP group (WMD = -0.40; 95% CI = -0.77 to -0.03; P=0.03). No difference was observed between LP and OP regarding complications (OR = 0.78; 95% CI = 0.46 to 1.34; P = 0.37) or success rate (OR = 1.76; 95% CI = 0.71 to 4.36; P = 0.22). CONCLUSIONS LP is a minimally invasive, safe, and effective therapy method for UPJ obstruction in children, with shorter hospital stay and excellent outcomes, and without additional risk of postoperative complications. Because of the publishing bias, a series of RCTs are necessary to explore the efficiencies of LP in the management of UPJ obstruction in children.

MicroRNA-145 inhibits the growth, invasion, metastasis and angiogenesis of neuroblastoma cells through targeting hypoxia-inducible factor 2 alpha.

Recent evidence shows that hypoxia-inducible factor 2 alpha (HIF-2α) may have critical roles in the growth and progression of neuroblastoma (NB) under non-hypoxic conditions. However, the underlying mechanisms and clinical potentials of normoxic HIF-2α expression in NB still remain largely unknown. In this study, HIF-2α immunostaining was identified in 26/42 NB tissues, which was correlated with clinicopathological features. In subtotal 20 NB cases, microRNA-145 (miR-145) was downregulated and inversely correlated with HIF-2α expression. Bioinformatics analysis revealed a putative miR-145 binding site in the 3′-untranslated region (3′-UTR) of HIF-2α messenger RNA (mRNA). Overexpression or knockdown of miR-145 responsively altered both the mRNA and protein levels of HIF-2α and its downstream genes, cyclin D1, matrix metalloproteinase 14 and vascular endothelial growth factor, in normoxically cultured NB cell lines SH-SY5Y and SK-N-SH. In a luciferase reporter system, miR-145 downregulated the luciferase activity of HIF-2α 3′-UTR, and these effects were abolished by a mutation in the putative miR-145-binding site. Overexpression of miR-145 suppressed the growth, invasion, metastasis and angiogenesis of SH-SY5Y and SK-N-SH cells in vitro and in vivo, while restoration of HIF-2α expression rescued the tumor cells from miR-145-mediated defects in these biological features. Furthermore, anti-miR-145 inhibitor rescued the HIF-2α knockdown-mediated repression on the growth, migration, invasion and angiogenesis of NB cells. These data indicate that miR-145 suppresses HIF-2α expression via the binding site in the 3′-UTR under normoxic conditions, thus inhibiting the aggressiveness and angiogenesis of NB.

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.

Small RNAs Targeting Transcription Start Site Induce Heparanase Silencing through Interference with Transcription Initiation in Human Cancer Cells

Heparanase (HPA), an endo-h-D-glucuronidase that cleaves the heparan sulfate chain of heparan sulfate proteoglycans, is overexpressed in majority of human cancers. Recent evidence suggests that small interfering RNA (siRNA) induces transcriptional gene silencing (TGS) in human cells. In this study, transfection of siRNA against −9/+10 bp (siH3), but not −174/−155 bp (siH1) or −134/−115 bp (siH2) region relative to transcription start site (TSS) locating at 101 bp upstream of the translation start site, resulted in TGS of heparanase in human prostate cancer, bladder cancer, and gastric cancer cells in a sequence-specific manner. Methylation-specific PCR and bisulfite sequencing revealed no DNA methylation of CpG islands within heparanase promoter in siH3-transfected cells. The TGS of heparanase did not involve changes of epigenetic markers histone H3 lysine 9 dimethylation (H3K9me2), histone H3 lysine 27 trimethylation (H3K27me3) or active chromatin marker acetylated histone H3 (AcH3). The regulation of alternative splicing was not involved in siH3-mediated TGS. Instead, siH3 interfered with transcription initiation via decreasing the binding of both RNA polymerase II and transcription factor II B (TFIIB), but not the binding of transcription factors Sp1 or early growth response 1, on the heparanase promoter. Moreover, Argonaute 1 and Argonaute 2 facilitated the decreased binding of RNA polymerase II and TFIIB on heparanase promoter, and were necessary in siH3-induced TGS of heparanase. Stable transfection of the short hairpin RNA construct targeting heparanase TSS (−9/+10 bp) into cancer cells, resulted in decreased proliferation, invasion, metastasis and angiogenesis of cancer cells in vitro and in athymic mice models. These results suggest that small RNAs targeting TSS can induce TGS of heparanase via interference with transcription initiation, and significantly suppress the tumor growth, invasion, metastasis and angiogenesis of cancer cells.

Small RNA interference-mediated gene silencing of heparanase abolishes the invasion, metastasis and angiogenesis of gastric cancer cells.

BackgroundHeparanase facilitates the invasion and metastasis of cancer cells, and is over-expressed in many kinds of malignancies. Our studies indicated that heparanase was frequently expressed in advanced gastric cancers. The aim of this study is to determine whether silencing of heparanase expression can abolish the malignant characteristics of gastric cancer cells.MethodsThree heparanase-specific small interfering RNA (siRNAs) were designed, synthesized, and transfected into cultured gastric cancer cell line SGC-7901. Heparanase expression was measured by RT-PCR, real-time quantitative PCR and Western blot. Cell proliferation was detected by MTT colorimetry and colony formation assay. The in vitro invasion and metastasis of cancer cells were measured by cell adhesion assay, scratch assay and matrigel invasion assay. The angiogenesis capabilities of cancer cells were measured by tube formation of endothelial cells.ResultsTransfection of siRNA against 1496-1514 bp of encoding regions resulted in reduced expression of heparanase, which started at 24 hrs and lasted for 120 hrs post-transfection. The siRNA-mediated silencing of heparanase suppressed the cellular proliferation of SGC-7901 cells. In addition, the in vitro invasion and metastasis of cancer cells were attenuated after knock-down of heparanase. Moreover, transfection of heparanase-specific siRNA attenuated the in vitro angiogenesis of cancer cells in a dose-dependent manner.ConclusionsThese results demonstrated that gene silencing of heparanase can efficiently abolish the proliferation, invasion, metastasis and angiogenesis of human gastric cancer cells in vitro, suggesting that heparanase-specific siRNA is of potential values as a novel therapeutic agent for human gastric cancer.

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.