Kaitai Yao

MiR-26a Inhibits Cell Growth and Tumorigenesis of Nasopharyngeal Carcinoma through Repression of EZH2

Several microRNAs (miRNA) have been implicated in nasopharyngeal carcinoma (NPC), a highly invasive and metastatic cancer that is widely prevalent in southern China. In this study, we report that microRNA miR-26a is commonly downregulated in NPC specimens and NPC cell lines with important functional consequences. Ectopic expression of miR-26a dramatically suppressed cell proliferation and colony formation by inducing G(1)-phase cell-cycle arrest. We found that miR-26a strongly reduced the expression of EZH2 oncogene in NPC cells. Similar to the restoring miR-26 expression, EZH2 downregulation inhibited cell growth and cell-cycle progression, whereas EZH2 overexpression rescued the suppressive effect of miR-26a. Mechanistic investigations revealed that miR-26a suppressed the expression of c-myc, the cyclin D3 and E2, and the cyclin-dependent kinase CDK4 and CDK6 while enhancing the expression of CDK inhibitors p14(ARF) and p21(CIP1) in an EZH2-dependent manner. Interestingly, cyclin D2 was regulated by miR-26a but not by EZH2, revealing cyclin D2 as another direct yet mechanistically distinct target of miR-26a. In clinical specimens, EZH2 was widely overexpressed and its mRNA levels were inversely correlated with miR-26a expression. Taken together, our results indicate that miR-26a functions as a growth-suppressive miRNA in NPC, and that its suppressive effects are mediated chiefly by repressing EZH2 expression.

Over-expression of BCAT1, a c-Myc target gene, induces cell proliferation, migration and invasion in nasopharyngeal carcinoma

BackgroundNasopharyngeal carcinoma (NPC) is a common malignant tumor in southern China and Southeast Asia, but its molecular mechanisms of pathogenesis are poorly understood. Our previous work has demonstrated that BCAT1 mRNA is over expressed in NPC and knocking down its expression in 5-8F NPC cell line can potently inhibit cell cycle progression and cell proliferation. However, the mechanism of BCAT1 up-regulation and its functional role in NPC development remain to be elucidated yet.MethodsImmunohistochemistry (IHC) method was utilized to detect the expression of BCAT1 protein in NPC at different pathological stages. The roles of gene mutation, DNA amplification and transcription factor c-Myc in regulating BCAT1 expression were analyzed using PCR-sequencing, quantitative polymerase chain reaction (qPCR), IHC, ChIP and luciferase reporter system, respectively. The functions of BCAT1 in colony formation, cell migration and invasion properties were evaluated by RNA interference (RNAi).ResultsThe positive rates of BCAT1 protein expression in normal epithelia, low-to-moderate grade atypical hyperplasia tissues, high-grade atypical hyperplasia tissues and NPC tissues were 23.6% (17/72), 75% (18/24 ), 88.9% (8/9) and 88.8% (71/80), respectively. Only one SNP site in exon1 was detected, and 42.4% (12/28) of the NPC tissues displayed the amplification of microsatellite loci in BCAT1. C-Myc could directly bind to the c-Myc binding site in promoter region of BCAT1 and up-regulate its expression. The mRNA and protein of c-Myc and BCAT1 were co-expressed in 53.6% (15/28) and 59.1% (13/22) of NPC tissues, respectively, and BCAT1 mRNA expression was also down-regulated in c-Myc knockdown cell lines. In addition, BCAT1 knockdown cells demonstrated reduced proliferation and decreased cell migration and invasion abilities.ConclusionsOur study indicates that gene amplification and c-Myc up-regulation are responsible for BCAT1 overexpression in primary NPC, and overexpression of BCAT1 induces cell proliferation, migration and invasion. The results suggest that BCAT1 may be a novel molecular target for the diagnosis and treatment of NPC.

Identification of differentially expressed genes in nasopharyngeal carcinoma by means of the Atlas human cancer cDNA expression array.

Purpose: To investigate genes of critical areas, including cell cycle/growth control, apoptosis, oncogene/tumor suppressors and growth factor/cytokines, that are differentially expressed in nasopharyngeal carcinoma. Methods: The Human Cancer cDNA Atlas, which contains 588 genes relating to tumor biology, was used to screen normal nasopharyngeal tissue, nasopharyngeal cancer (NPC). The reverse transcription/polymerase chain reaction was used to confirm the expression pattern of some genes identified by Atlas hybridization. Results: The differentially expressed cell cycle/growth control regulators in NPC showed a stronger tendency toward cell proliferation with the up-regulation of cyclin D1, cyclin D2 etc. The expression pattern of apoptosis-related genes demonstrated the up-regulation of both anti-apoptotic factors such as the BCL-2-related protein A1, TRAF3, the inhibitor of apoptosis protein A1 (IAP1) and apoptotic pathway elements such as Fas/Apo-1, Apo-2 ligand etc. Among oncogenes/tumor suppressors, MDM2, STAT1 and STAT2 were found to be up-regulated in NPC. The expression profile of growth factors/cytokines showed the up-regulation of many growth-enhancing factors such as EGR1, tumor-derived growth factor 1, platelet-derived growth factor A chain etc. as well as Th1-type cytokines e.g. interleukin-1β and interferons. A smaller number of genes were down-regulated in nasopharyngeal cancer, such as those encoding ERK1, Raf, secreted apoptosis-related protein 1, CD27BP, transforming growth factor β2, pre-B-cell-stimulating factor homologue etc. Conclusion: The consistent tendency toward cell proliferation, the possibility of a stronger anti-apoptotic force that operates on the normal apoptotic pathway, or the autocrine or paracrine growth factors may account for the development of NPC. Some genes are reported for the first time to have changed expression in nasopharyngeal carcinoma. The simple, quick, and high-throughput method of profiling gene expression by cDNA array hybridization provides us with a quick overview of key factors that may be involved in NPC, and may identify genes suitable for further study of carcinogenesis mechanism or targets for possible molecular diagnosis or therapy.

Identification of differentially expressed genes in metastatic and non-metastatic nasopharyngeal carcinoma cells by suppression subtractive hybridization

Background & Objective: Nasopharyngeal carcinoma (NPC) is an epithelial neoplasm with high occurrence rates in southern China. The disease often metastasizes to regional lymphnodes at a very early stage. Local recurrences and metastasis occur frequently in patients with NPC and are a leading cause of death, despite improvements on treatment modalities. The molecular mechanism underlying the metastasis of nasopharyngeal carcinoma remains poorly understood, however, and requires additional elucidation. The aim of this study was to explore possible NPC gene candidates that may play key roles in NPC metastasis. Methods: Subtractive suppression hybridization (SSH) was performed to isolate differentially expressed clones between the metastatic 5-8F and non-metastatic 6-10B nasopharyngeal carcinoma cell lines. Differentially expressed clones were screened and confirmed by reverse Northern blotting. The sequences of cDNA fragments were subsequently analyzed and compared to known sequences in Genbank. Results & Discussion: The SSH library contained thousands of positive clones. Random analysis of 300 clones by PCR demonstrated that 269 clones contained inserted fragments. Reverse Northern blot confirmed that 20 out of 192 clones examined were significantly up-regulated in the 5-8F cell line. Among these 20 clones, 16 were previously identified genes (flotilin-2, ezrin, pim-3, fli-1, mel, neugrin, znf216, ASB1, raly, UBE2A, keratin6A, TMED7, EIF3S9, FTL, two ribosomal proteins RPL21 and RPL16), two were predicted genes (c9orf74 and MDS006), and two sequences shared no homology with known genes listed in GenBank and may represent novel genes. The proposed functions of the genes identified in this study include cell signal transduction, cell survival, transcription regulation, cell mobility, protein synthesis, and DNA damage repair. Flotillin-2, fli-1, pim-3 and ezrin have previously been reported to be associated with tumor metastasis and progression. The remaining up-regulated genes identified in this study have not been reported to be markers of metastasis and may represent new candidates of NPC metastasis-related genes. The Results of this study may provide novel points of therapeutic intervention for NPC.

Identification of label-retaining cells in nasopharyngeal epithelia and nasopharyngeal carcinoma tissues

Adult stem cells can be identified by label-retaining cell (LRC) approach based on their ability to retain nucleoside analog, such as bromodeoxyuridine (BrdU). We hypothesized that mouse nasopharynx contains a small population of epithelial stem/progenitor cells that may be detected by the LRC technique. To identify LRCs in mice nasopharyngeal epithelia, neonatal mice were intraperitoneally injected with BrdU twice daily for 3 consecutive days. After an 8-week chase, long-term BrdU-labeled LRCs (∼2% of cells) were detected in the adult mice nasopharyngeal epithelia by immunostaining with BrdU antibody and some of LRCs (∼12% of cells) were found to be recruited into the S phase of cell cycle with an additional radioactive thymidine-labeling technique, indicating that the stem cells also divide, most likely asymmetrically. To further investigate whether the LRCs existed in human nasopharyngeal carcinoma (NPC) tissues, three NPC cell lines (5-8F, 6-10B and TMNE) were labeled with BrdU in vitro and then individually engrafted into the back of nude mice, which developed tumors. Again, label-retaining stem cells were found in all the three kinds of NPC xenograft tumors (∼0.3% of cells), around 16% of which were also labeled with radioactive thymidine. Thus, this study has demonstrated for the first time the presence of epithelial LRCs in mouse nasopharyngx and human NPC tissues and these stem-like LRCs are not completely quiescent, as they will be recruited into the cell cycle to participate physiological or pathological process at any moment. More importantly, our data showed that NPC also contained stem cells, which are most likely the cause for NPC spread, metastasis and recurrence.

Genetic and epigenetic alterations of LTF at 3p21.3 in nasopharyngeal carcinoma

To investigate the roles of lactotransferrin gene (LTF, also referred to as the lactoferrin gene, LF), located at 3p21.3 within the common minimal deletion region, in the pathogenesis of nasopharyngeal carcinoma (NPC), we first detected its expression level in 33 primary NPC tissues and 15 chronic nasopharyngitis tissues. Absent expression or downregulation of LTF were observed in 76% (25 of 33) of primary NPC tissues. We further found that 25% (5 of 20) of NPC specimens had loss of heterozygosity (LOH) at the LTF locus. LTF mutation assessed by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing was noted in 30% (6 of 20) of primary NPC tissues. In addition, hyper-methylation of LTF promoter region was found in 63.6% (21 of 33) of primary NPC samples but not in chronic nasopharyngitis tissues. The LTF transcripts in NPC cell lines increased upon treatment with the demethylation compound, 5-aza-2-deoxycytidine. In conclusion, our data indicate that two-hit silencing of LTF through genetic and epigenetic changes may be a common and important event in the carcinogenesis of NPC.

Functional evidence for a nasopharyngeal carcinoma-related gene BCAT1 located at 12p12.

Nasopharyngeal carcinoma (NPC) is a malignancy that is prevalent among populations from Southeast Asia. The carcinogenesis of NPC is thought to be a multistep process involving several genetic changes. Our previous study based on distance and branching-tree models for NPC carcinogenesis indicated +12p11-p12 was an early event and should play an important role in NPC development. To understand the role of +12p11-p12 as the tree model predicted and evaluate which gene located at 12p11-p12 might be involved in NPC development, semiquantitative RT-PCR was applied to examine the expression status of 18 genes selected from 12p11-p12 in 36 NPC and 8 normal nasopharynx (NP) biopsies. The results revealed that BCAT1, KCNJ8, PTX1, and KRAS2 genes were overexpressed in NPC tissues and BCAT1 was of particular interest based on its function reported in other tumors. To further elucidate the function of BCAT1 gene in NPC, BCAT1 expression was specifically suppressed in 5-8F NPC cell line by RNA interference (RNAi), confirmed by RT-PCR and Western blotting. As expected, the depletion of BCAT1 could effectively block the proliferation of NPC cells. The BCAT1 identified in the amplified 12p11-p12 region may play a certain role in NPC development.

Epigenetic and Genetic Alterations of the EDNRB Gene in Nasopharyngeal Carcinoma

Background: Loss of heterozygosity (LOH) at 13q22 is a common event in nasopharyngeal carcinoma (NPC). EDNRB gene located at 13q22 has been demonstrated to be hypermethylated in some kinds of tumors. In the current study, we focused on the epigenetic and genetic alterations of EDNRB in NPC. Methods: The mRNA expression of EDNRB was detected by semiquantitative RT-PCR and real-time quantitative PCR in 49 NPC and 12 chronic nasopharyngitis biopsies. The methylation and LOH status of EDNRB were examined by methylation-specific polymerase chain reaction, microsatellite PCR and sequencing. We also examined the mRNA expression of EDNRB in four NPC cell lines after 5-aza-2′-deoxycytidine treatment. Results:EDNRB was downregulated in primary NPC tissues and NPC cell lines, and a relatively higher methylation level of EDNRB was found in NPC biopsies (84%) compared to that in chronic nasopharyngitis biopsies (42%). Treatment of NPC cell lines with 5-aza-2′-deoxycytidine activated EDNRB expression. LOH of EDNRB gene was also found at two microsatellite sites with ratios of 6.25 and 16.67% in NPC. Conclusion: Our results suggested that EDNRB expression may be affected by aberrant promoter methylation and gene deletion and may play a role in the development of NPC.

The DLC-1 −29A/T Polymorphism Is Not Associated with Nasopharyngeal Carcinoma Risk in Chinese Population

Deleted in liver cancer-1 (DLC-1), encoding a Rho GTPase-activating protein (GAP), is considered as a promising candidate tumor suppressor gene in nasopharyngeal carcinoma (NPC). The single-nucleotide polymorphism (SNP) -29A/T upstream of ATG start codon was found when gene mutation profile of DLC-1 in NPC was analyzed. To evaluate the correlation between SNP -29A/T in the promoter region of DLC-1 gene and risk of NPC, a total of 521 samples from a Chinese population, including 320 healthy individuals and 201 NPC patients, were collected for SNP analysis by PCR-single-strand conformation polymorphism and sequencing. The differences in allele and genotype frequencies between NPC patients and controls were tested using logistic regression statistical method. No significant differences were found in allele or genotype frequencies between NPC patients and controls or among different NPC clinical stages. Hence, our data indicate that the SNP -29A/T of DLC-1 gene is not associated with NPC susceptibility.

Cloning of a novel gene associated with human nasopharyngeal carcinoma

One EST N27741 with high expression in normal adult nasopharynx tissues but low expression in adult poorly differentiated squamous nasopharyngeal carcinoma has been selected out by the high-density cDNA array expression profiling technique. The differential expression has been confirmed by RT-PCR. One novel gene of 1096 bp has been cloned based on this EST. Bioinformatics analysis found that the new gene sequence contains a whole reading frame encoding 256 amino acids. There is a stop codon TAA in front of the 5′ end start codon, and a tailing signal AATAAA and poly A tail at the 3′ end. There is no homologous known gene found after searching by blasting this sequence to non-redundancy nucleotide database. Therefore it is considered a novel gene related to nasopharyngeal carcinoma.