Shourong Shen

microRNA-141 is involved in a nasopharyngeal carcinoma-related genes network

microRNAs (miRNAs) are small non-coding RNAs and have been implicated in the pathology of various diseases, including cancer. Here we report that the miRNA profiles have been changed after knockdown of one of the most important oncogene c-MYC or re-expression of a candidate tumor suppressor gene SPLUNC1 in nasopharyngeal carcinoma (NPC) cells. Both c-MYC knockdown and SPLUNC1 re-expression can down-regulate microRNA-141 (miR-141). miR-141 is up-regulated in NPC specimens in comparison with normal nasopharyngeal epithelium. Inhibition of miR-141 could affect cell cycle, apoptosis, cell growth, migration and invasion in NPC cells. We found that BRD3, UBAP1 and PTEN are potential targets of miR-141, which had been confirmed following luciferase reporter assays and western blotting. BRD3 and UBAP1 are both involved in NPC carcinogenesis as confirmed through our previous studies and PTEN is a crucial tumor suppressor in many tumor types. BRD3 is involved in the regulation of the Rb/E2F pathway. Inhibition of miR-141 could affect some important molecules in the Rb/E2F, JNK2 and AKT pathways. It is well known that carcinogenesis of NPC is involved in the networks of genetic and epigenetic alteration events. We propose that miR-141- and tumor-related genes c-MYC, SPLUNC1, BRD3, UBAP1 and PTEN may constitute a gene-miRNA network to contribute to NPC development.

Circulating miR-17, miR-20a, miR-29c, and miR-223 Combined as Non-Invasive Biomarkers in Nasopharyngeal Carcinoma

Background MicroRNAs have been considered as a kind of potential novel biomarker for cancer detection due to their remarkable stability in the blood and the characteristics of their expression profile in many diseases. Methods We performed microarray-based serum miRNA profiling on the serum of twenty nasopharyngeal carcinoma patients at diagnosis along with 20 non-cancerous individuals as controls. This was followed by a real-time quantitative Polymerase Chain Reaction (RT-qPCR) in a separate cohort of thirty patients with nasopharyngeal carcinoma and thirty age- matched non-cancerous volunteers. A model for diagnosis was established by a conversion of mathematical calculation formula which has been validated by analyzing 74 cases of patients with nasopharyngeal carcinoma and 57 cases of non-cancerous volunteers. Results The profiles showed that 39 and 17 miRNAs are exclusively expressed in the serum of non-cancerous volunteers and of patients with nasopharyngeal carcinoma respectively. 4 miRNAs including miR-17, miR-20a, miR-29c, and miR-223 were found to be expressed differentially in the serum of NPC compared with that of non-cancerous control. Based on this, a diagnosis equation with Ct difference method has been established to distinguish NPC cases and non-cancerous controls and validated with high sensitivity and specificity. Conclusions We demonstrate that the serum miRNA-based biomarker model become a novel tool for NPC detection. The circulating 4-miRNA-based method may provide a novel strategy for NPC diagnosis.

IONP-PLL: a novel non-viral vector for efficient gene delivery

Non‐viral methods of gene delivery have been an attractive alternative to virus‐based gene therapy. However, the vectors that are currently available have drawbacks limiting their therapeutic application.

Lactotransferrin: a candidate tumor suppressor-Deficient expression in human nasopharyngeal carcinoma and inhibition of NPC cell proliferation by modulating the mitogen-activated protein kinase pathway.

Lactotransferrin (LTF) has been shown to regulate tumorogenesis. However, little is known about the role of LTF in regulating the development of human nasopharyngeal carcinoma (NPC). The aim of our study was to investigate whether LTF could regulate the development of NPC by characterizing the pattern of LTF expression in human NPC tissues using cDNA and tissue microarrays. Loss of LTF expression was observed in a significantly higher frequency of NPC tissues compared to that in nontumor nasopharyngeal epithelial tissues. While 61.25% of NPC tissues at the T1/T2 stage were positive for LTF expression, only 40.82% of NPC at the T3/T4 stage were stained by anti‐LTF. Similarly, 41.58% of NPC with local lymph node metastasis displayed LTF expression, a value significantly lower than the 46.36% in primary tumors (p < 0.05). These findings suggest that LTF may negatively regulate the development and metastasis of NPC in vivo. Furthermore, overexpression of or treatment with LTF inhibited the proliferation of NPC cells and promoted cell cycle arrest at the G0/G1 phase in vitro. While LTF treatment downregulated expression of cyclin D1 and phosphorylation of retinoblastoma protein (Rb), expression of p21 and p27 in 5–8F NPC cells was enhanced. Moreover, LTF treatment modulated the mitogen‐activated protein kinase (MAPK) pathway, but did not affect p53 and STAT3 expression in 5–8F NPC cells. Thus LTF is likely to be a candidate tumor suppressor and downregulates the development of NPC by inhibiting NPC proliferation through induction of cell cycle arrest and modulation of the MAPK signaling pathway. Therefore, our findings provide new insights in understanding the mechanism(s) underlying the action of LTF in regulating the development of human NPC.

BRD7, a novel bromodomain gene, inhibits G1–S progression by transcriptionally regulating some important molecules involved in ras/MEK/ERK and Rb/E2F pathways

Bromodomain is a 110 amino acid domain. It is evolutionally conserved and is found in proteins strongly implicated in signal‐dependent transcriptional regulation. BRD7 is a novel bromodomain gene and it is downexpressed in nasopharyngeal carcinoma (NPC) biopsies and cell lines; its function is poorly understood. In the present study, tet‐on inducible expression system was used to investigate the role of BRD7 in cell growth and cell cycle progression. We found that ectopic expression of BRD7 in NPC cells inhibited cell growth and cell cycle progression from G1 to S. We further performed cell cycle cDNA array to screen potential transcriptional targets of BRD7 in cell cycle. Thirteen important signaling molecules, mainly implicated in ras/MEK/ERK and Rb/E2F pathways, were differentially expressed by induction of BRD7. Moreover, we observed that BRD7 could regulate the promoter activity of E2F3, one of its targets. Taken together, the present study indicated that BRD7 inhibited G1–S progression by transcriptionally regulating some important molecules involved in ras/MEK/ERK and Rb/E2F pathways and suggested that BRD7 may present a promising candidate of NPC™ associated tumor suppressor gene.

Poly(l‐lysine)‐modified silica nanoparticles for the delivery of antisense oligonucleotides

Silica nanoparticles were prepared in a microemulsion system, using polyoxyethylene nonylphenyl ether/cyclohexane/ammonium hydroxide. The surface charge of the particle was modified with PLL [poly(l‐lysine)]. PAGE demonstrated the ability of PMS‐NP (PLL‐modified silica nanoparticles) to bind and protect antisense ODNs (oligonucleotides). The intracellular localization of FITC‐labelled ODN was investigated by fluorescence microscopy. The results demonstrated that ODN could be delivered to cytoplasm. Flow‐cytometry analysis showed a 20‐fold enhancement of ODN delivered by PMS‐NP compared with free ODN for a serum‐free medium. Blocking efficacy of c‐myc antisense ODN, delivered by PMS‐NP, was examined in HNE1 and HeLa cell lines. Significant down‐regulation of c‐myc mRNA levels was observed in both the cell lines. However, the cellular uptake efficiency and antisense effects on target gene decreased in the presence of serum‐containing medium. The analysis of the filtration assay showed that PMS‐NP interacted with serum proteins. These results indicated that PMS‐NP was a suitable delivery vector for antisense ODN, although its delivery efficiency decreased in the presence of a serum‐containing medium.

Identification of tissue-specific genes in nasopharyngeal epithelial tissue and differentially expressed genes in nasopharyngeal carcinoma by suppression subtractive hybridization and cDNA microarray.

Suppression subtractive hybridization (SSH) was performed for isolation of tissue‐specific genes in nasopharyngeal epithelial tissue, by use of cDNAs from human adult nasopharyngeal epithelial tissue as tester and mixed cDNAs from esophagus, lung, liver, heart, stomach, spleen, skeletal muscle, kidney, and skin as drivers. Fourteen differentially expressed genes in nasopharyngeal epithelial tissue were obtained. Among these genes, LPLUNC1 and SPLUNC1 were confirmed to be specifically expressed in nasopharyngeal epithelial tissue and the trachea. A novel transcript of SPLUNC1, which we designate NASG, was found. We also combined SSH and cDNA microarray hybridization to identify genes whose expressions were altered in nasopharyngeal carcinoma (NPC). We used NPC cell line HNE1 and primary human embryo nasopharyngeal epithelial cells in one SSH experiment, and NPC biopsies and normal adult nasopharyngeal epithelial tissue in another. Some 1,200 SSH inserts from four subtractive cDNA libraries were arrayed onto nylon membranes by use of robotic printing. Differential gene expression was verified by hybridizing of the membranes with radioactively labeled first‐strand cDNA from NPC cell line HNE1, primary human embryo nasopharyngeal epithelial cells, NPC biopsies, and normal adult nasopharyngeal epithelial tissue. Seventeen differentially expressed genes in NPC were obtained. Among these genes, we identified SPLUNC1 and LPLUNC1 to be down‐expressed in NPC biopsies (34/48, 33/48).

LRRC4 inhibits human glioblastoma cells proliferation, invasion, and proMMP‐2 activation by reducing SDF‐1α/CXCR4‐mediated ERK1/2 and Akt signaling pathways

Gliomas take a number of different genetic routes in the progression to glioblastoma multiforme, a highly invasive variant that is mostly unresponsive to current therapies. The α‐chemokine stromal cell‐derived factor (SDF)‐1α binds to the seven transmembrane G‐protein‐coupled CXCR‐4 receptor and acts to modulate cell migration and proliferation by activating multiple signal transduction pathways. Leucine‐rich repeats containing 4 (LRRC4), a putative glioma suppressive gene, inhibits glioblastoma cells tumorigenesis in vivo and cell proliferation and invasion in vitro. We also previously demonstrated that LRRC4 controlled glioblastoma cells proliferation by ERK/AKT/NF‐κB signaling pathway. In the present study, we demonstrate that CXC chemokine receptor 4 (CXCR4) is expressed in human glioblastoma U251 cell line, and that SDF‐1α increases the proliferation, chemotaxis, and invasion in CXCR4+ glioblastoma U251 cells through the activation of ERK1/2 and Akt. The reintroduction of LRRC4 in U251 cells inhibits the expression of CXCR4 and SDF‐1α/CXCR4 axis‐mediated downstream intracellular pathways such as ERK1/2 and Akt leading to proliferate, chemotactic and invasive effects. Furthermore, we provide evidence for proMMP‐2 activation involvement in the SDF‐1α/CXCR4 axis‐mediated signaling pathway. LRRC4 significantly inhibits proMMP‐2 activation by SDF‐1α/CXCR4 axis‐mediated ERK1/2 and Akt signaling pathway. Collectively, these results suggest a possible important “cross‐talk” between LRRC4 and SDF‐1α/CXCR4 axis‐mediated intracellular pathways that can link signals of cell proliferation, chemotaxis and invasion in glioblastoma, and may represent a new target for development of new therapeutic strategies in glioma. J. Cell. Biochem. 103: 245–255, 2008.

Differential miRNA expression and their target genes between NGX6-positive and negative colon cancer cells

Nasopharyngeal carcinoma-associated gene 6 (NGX6) was shown to be a novel putative tumor suppressor gene in colon cancer. The purpose of this study is to investigate its role in regulation of miRNA expression for in the hopes of translating this data into a novel strategy in control of colon cancer. In this study colon cancer HT-29 cells were stably transfected with NGX6 or vector-only plasmid and then subjected to miRNA array analysis, and Q-RT-PCR was then used to verify miRNA array data. Then bioinformatic analyses using Sanger, Target Scan, and MicroRNA software were performed to obtain data on the target genes of each miRNA and define their function. Our results showed that 14 miRNAs were found to be differentially expressed in NGX6-transfected cells compared to the control cells. In particular, miR-126, miR-142-3p, miR-155, miR-552, and miR-630 were all upregulated, whereas miR-146a, miR-152, miR-205, miR-365, miR-449, miR-518c, miR-584, miR-615, and miR-622 were downregulated after NGX6 transfection. Q-RT-PCR confirmed all of these miRNAs, and invalidated miR-552 and miR-630. Furthermore, bioinformatic analyses of these 12 miRNAs, among these miRNAs, target genes of miR-615 are unclear, another 11 miRNAs produced a total of 254 potential target genes and further study showed that these genes together formed a regulatory network that contributes to apoptosis, mobility/migration, hydrolysis activity, and molecular signaling through targeting JNK and Notch pathways. Taken together, these results have suggested that NGX6 plays an important role in regulation of apoptosis, mobility/migration, and hydrolase as well as activity of JNK and Notch pathways through NGX6-mediated miRNA expression. Further investigation will reveal the function of these differentially expressed miRNAs and verify expression of the miRNA-targeted genes for development of novel strategies for better control of colon cancer.

Family-based association analysis validates chromosome 3p21 as a putative nasopharyngeal carcinoma susceptibility locus

Purpose: Nasopharyngeal carcinoma (NPC) poses one of the serious health problems in southern Chinese, with an incidence rate ranging from 15 to 50/100,000. In our previously linkage analysis, a locus on 3p21 was identified to link to NPC. In this study, family-based association analysis was performed to test the transmission disequilibrium of chromosome 3p in 18 high-risk nasopharyngeal carcinoma families of Hunan province in southern China.Methods: Single locus and multi-point of transmission disequilibrium test was performed by Genehunter program package with 15 microsatellite markers on chromosome 3p in 18 nasopharyngeal carcinoma pedigrees.Results: A major transmission disequilibrium peak was observed near D3S1568, which possessed 20 alleles or haplotypes of 6 loci, spanning a 12.4 cM region from D3S1298 to D3S1289 on chromosome 3p21.31-3p21.2, and 3 alleles or haplotypes reached high significantly difference (P < 0.01).Conclusion: These results reflected a link disequilibrium between this chromosome region and a nasopharyngeal carcinoma susceptibility locus, and provided further evidence that a novel nasopharyngeal carcinoma susceptibility gene may be located in this chromosome region. These alleles or haplotypes transmitting disequilibrium in nasopharyngeal carcinoma pedigrees may act as the highly risk molecular markers after verified in large population.