Roles of PTBP1 in alternative splicing, glycolysis, and oncogensis.

Polypyrimidine tract-binding protein 1 (PTBP1) plays an essential role in splicing and is expressed in almost all cell types in humans, unlike the other proteins of the PTBP family. PTBP1 mediates several cellular processes in certain types of cells, including the growth and differentiation of neuronal cells and activation of immune cells. Its function is regulated by various molecules, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and RNA-binding proteins. PTBP1 plays roles in various diseases, particularly in some cancers, including colorectal cancer, renal cell cancer, breast cancer, and glioma. In cancers, it acts mainly as a regulator of glycolysis, apoptosis, proliferation, tumorigenesis, invasion, and migration. The role of PTBP1 in cancer has become a popular research topic in recent years, and this research has contributed greatly to the formulation of a useful therapeutic strategy for cancer. In this review, we summarize recent findings related to PTBP1 and discuss how it regulates the development of cancer cells.

The regulatory network of nasopharyngeal carcinoma metastasis with a focus on EBV, lncRNAs and miRNAs.

Metastasis of nasopharyngeal carcinoma (NPC) remains a main cause of death for NPC patients even though great advances have been made in therapeutic approaches. An in-depth study into the molecular mechanisms of NPC metastasis will help us combat NPC. Epstein-Barr virus (EBV) infection is an evident feature of nonkeratinizing NPC and is strongly associated with tumor metastasis. Recently, long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) have become a hot topic of research due to their epigenetic regulatory roles in NPC metastasis. The EBV products, lncRNAs and miRNAs can target each other and share several common signaling pathways, which form an interconnected, complex molecular regulatory network. In this review, we discuss the features of this regulatory network and summarize the molecular mechanisms of NPC metastasis, focusing on EBV, lncRNAs and miRNAs with updated knowledge.

FMNL1 mediates nasopharyngeal carcinoma cell aggressiveness by epigenetically upregulating MTA1.

It has been suggested that formin-like protein 1 (FMNL1) plays an important role in the pathogenic process of several hematopoietic malignancies. In this study, we performed a series of in vivo and in vitro assays to elucidate the biological functions of FMNL1 and underlying mechanisms in human nasopharyngeal carcinoma (NPC) pathogenesis. Herein, we report that high expression of FMNL1 in NPC is positively associated with an aggressive disease and/or poor patient survival. Ectopic overexpression of FMNL1 in NPC cells substantially promoted cell invadopodia formation, epithelial-mesenchymal transition (EMT) and invasiveness, whereas depletion of FMNL1 potently suppressed NPC cells invadopodia formation, EMT, and invasive/metastatic capacities. We further show that FMNL1 could enhance NPC cell aggressiveness by increasing a key downstream target, the metastasis-associated protein 1 (MTA1) gene. Importantly, ectopic overexpression of FMNL1 in NPC cells markedly improved the binding of HDAC1 with Profilin2 in the cytoplasm and suppressed the enrichment of HDAC1 on the promoter of MTA1 and thereby, leading to an increased MTA1 transcription and expression. Furthermore, in addition to the amplification of FMNL1 gene, decreased level of miR-16 in NPCs is another critical mechanism to upregulate FMNL1 expression. These results, collectively, provide first-line of evidences that high expression of FMNL1, resulted from decreased miR-16 and/or MTA1 amplification, has a potent oncogenic role to drive the development and aggressive process of NPC by upregulating MTA1, and FMNL1 might be employed as a new prognostic biomarker and therapeutic target for human NPC.

Research Progress on Reversing Multidrug Resistance in Tumors by Using Chinese Medicine.

Multidrug resistance (MDR) is a major cause of cancer chemotherapy failure, and it is important to develop suitable reversal agents to overcome MDR. A majority of chemical reversal agents have acceptable reversal effects. However, the toxicity and adverse reactions associated with these agents restricts their clinical use. Chinese medicines (CMs) have lower toxicities and adverse reactions and are associated with multiple components, multiple targets and reduced toxicity. CMs have several advantages and could reverse MDR, decrease drug dosage, enhance patient compliance and increase efficacy. This review summarizes the current progress of CM reversal agents..

Roles of flotillins in tumors.

The identification and use of molecular biomarkers have greatly improved the diagnosis and treatment of malignant tumors. However, a much deeper understanding of oncogenic proteins is needed for the benefit to cancer patients. The lipid raft marker proteins, flotillin-1 and flotillin-2, were first found in goldfish retinal ganglion cells during axon regeneration. They have since been found in a variety of cells, mainly on the inner surface of cell membranes, and not only act as a skeleton to provide a platform for protein-protein interactions, but also are involved in signal transduction, nerve regeneration, endocytosis, and lymphocyte activation. Previous studies have shown that flotillins are closely associated with tumor development, invasion, and metastasis. In this article, we review the functions of flotillins in relevant cell processes, their underlying mechanisms of action in a variety of tumors, and their potential applications to tumor molecular diagnosis and targeted therapy.

The clinicopathological significance of miR-149 and PARP-2 in hepatocellular carcinoma and their roles in chemo/radiotherapy.

Hepatocellular carcinomas (HCC) are commonly diagnosed at an advanced stage with unresectable tumors. Although numerous non-surgical approaches have been developed to treat HCC, the prognosis of patients with HCC is still poor. This study investigated the expression of miR-149 and PARP-2 in HCC tumor tissues and their roles in sensitizing chemo/radiotherapy. The expression of miR-149 was measured by real-time PCR, and PARP-2 protein was measured by immunohistochemistry and Western blot. The xenograft HCC mouse model was established by inoculating Hep G2 cells. Increased PARP-1 and decreased miR-149 expression was observed in HCC tissues compared to peritumoral tissues. Positive PARP-2 and low miR-149 expression correlated with larger tumor mass size (P < 0.001), capsular and vascular invasion (P < 0.001), lymph node metastasis (P = 0.02), high histological grade (P < 0.001), TNM (P < 0.001), and BCLC grade (P = 0.001). The Kaplan-Meier survival analysis showed a negative correlation between high PARP-2 expression or low miR-149 expression in HCC tissues with the survival of patients. High PARP-2 and low miR-149 correlated with a low 5-year survival rate and are poor prognosis factors. Overexpression of miR-149 or inhibition of PARP-2 expression could inhibit tumor growth but was more effective in sensitizing chemotherapy and radiotherapy in xenograft HCC animal models. Increased PARP-2 expression and loss of miR-149 expression are involved in the pathogenesis of HCC and are poor prognosis factors in patients with HCC. Although both miR-149 overexpression and PARP-2 inhibitor exert some antitumoral effect, PARP-2 inhibitor is a chemo/radio sensor and can be used to enhance chemotherapy and radiotherapy in patients with HCC.

ALDH1 expression is correlated with pathologic grade and poor clinical outcome in patients with astrocytoma.

Aldehyde dehydrogenase 1 (ALDH1), a detoxifying enzyme, is a stem-like cell marker, but its expression pattern and clinical significance in astrocytoma remain unclear. In this study, we used immunohistochemical analysis to systematically investigate the expression of ALDH1 in 76 astrocytomas of different pathological grade and seven samples of normal brain tissues. We found that ALDH1 was expressed in some of the astrocytomas but was not detected in normal brain tissues. The proportion of ALDH1-expressing cells was positively correlated with the pathological grade of the astrocytomas, but not with patient age, sex or tumor size. We also collected detailed follow-up data and analyzed the correlation of ALDH1 expression with overall survival (OS) and progression-free survival (PFS) using univariate and multivariate analysis. We found that the proportion of ALDH1-positive cells was an independent prognostic factor for PFS and OS. These results show that ALDH1 is expressed in astrocytoma, and that its expression is correlated with pathological grade and patient survival.

Preparation of human scFv antibody against nasopharyngeal carcinoma and identification of its specificity.

UNLABELLED: Abstract Conclusion: The selected scFv antibody could specifically recognize and target nasopharyngeal carcinoma (NPC), and could be applied to clinical diagnosis and therapy. OBJECTIVE: The aim was to construct and screen fully human anti-NPC single chain Fv fusion phage libraries, and to identify the specificity of the scFv antibody. METHODS: Peripheral blood mononuclear cells of patients with NPC were immunized in vitro by NPC cells and transformed by Epstein-Barr virus. The total RNAwas used to construct the scFv libraries. By means of ELISA and immunochemistry, the positively bound scFv was selected and identified. The positive scFv was fused to EGFP, and was then expressed in E. coli strain BL21 (DE3) and purified. Furthermore, we observed the binding bioactivity. RESULTS: The fusion protein has the biological activity of binding the NPC cells and emitting green fluorescence. In targeting experiments in vivo, the results showed that the fusion protein can successfully target the NPC.

[Screening and preliminary analysis of the apoptosis- and proliferation-related genes in nasopharyngeal carcinoma].

UNLABELLED: To screen and analyze the apoptosis- and proliferation-related genes in human nasopharyngeal carcinoma (NPC). METHODS: According to gene ontology classification, the abnormal expressions of the genes related to cell apoptosis and proliferation were identified in the NPC gene chip data. The cell apoptosis- and proliferation-related genes expressed in each of the 3 stages, as defined by the tree model for the pathogenesis and progression of NPC, were screened, and with literature review, their distribution in the tree model were analyzed. RESULTS: Nineteen genes related to cell apoptosis were found in NPC, among which 9 were down-regulated (such as DNASE1L3) and located in the chromosome deletion regions, and 10 were up-regulated (such as DEDD) in the chromosome amplification regions. Twenty-one cell proliferation-related genes were identified, including 8 down-regulated genes (such as TUSC2) in the chromosome deletion regions and 13 up-regulated ones (such as EMP1) in the chromosome amplification regions. In the chromosome deletion regions, the down-regulated cell apoptosis-related genes participated mostly in inducing and regulating cell apoptosis, and the up-regulated cell proliferation-related genes in the chromosome amplification regions were mostly associated with the positive regulation of cell proliferation. CONCLUSION: NPC occurs possibly through two pathways by inhibiting cell apoptosis or by promoting excessive cell proliferation.

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.

[PTX1 in nasopharyngeal carcinoma by RNAi technology].

OBJECTIVE: To explore the expression and the role of PTX1 located at the amplified 12p12-p11 region in nasopharyngeal carcinoma (NPC). METHODS: Semi-quantitative RT-PCR and real-time RT-PCR were applied to detect the expression level of PTX1 in 36 NPC and 8 chronic nasopharyngitis (NP) biopsies. RNAi vector targeting PTX1 was constructed and transfected into NPC cell line 6-10B. The RNAi effect was determined by detecting the expression level of PTX1 in transfected 6-10B cell line. Finally, the cell biological characteristics were compared between transfected 6-10B and parental 6-10B by analyzing the cell cycle distribution and apoptosis status using flow cytometry. RESULTS: RT-PCR and real-time RT-PCR revealed that PTX1 gene was over-expressed in NPC tissues (P<0.05). PTX1 expression was suppressed in NPC cell line 6-10B by approximately 65% by RNAi, confirmed by RT-PCR. The depletion of PTX1 could effectively block the proliferation and induce the apoptosis of NPC cells. CONCLUSION: Blocking the expression of PTX1 on mRNA level changed the characterization of NPC cell line 6-10B by RNAi, suggesting that PTX1 identified in the amplified 12p12-p11 region may be involved in the genesis and development of NPC via promoting the cell proliferation and inhibiting the cell apoptosis.

[Expression, loss of heterozygosity, and methylation of GNAT1 gene in nasopharyngeal carcinoma].

BACKGROUND & OBJECTIVE: In nasopharyngeal carcinoma (NPC), chromosome 3p21.3 is a high frequency deletion region. Evidences from both loss of heterozygosity (LOH) and functional studies showed that there may exists NPC-related tumor suppressor genes on 3p21.3. This study was to investigate the expression, LOH, and methylation of GNAT1 gene, which locates at 3p21.3, in NPC. METHODS: The expression of GNAT1 in 33 specimens of primary NPC and 15 specimens of chronic nasopharyngitis tissue was detected by reverse transcription-polymerase chain reaction (RT-PCR). LOH and promoter methylation status of GNAT1 were examined by microsatellites analysis and methylation-specific polymerase chain reaction (MSP). RESULTS: GNAT1 was expressed stably in all chronic nasopharyngitis tissues, while absent or down-regulated in 24 (72.7%) specimens of NPC (P=0.022). LOH analysis showed allele loss of GNAT1 in 3 (15%) specimens of NPC. LOH of GNAT1 was correlated to its expression level (P=0.016). Methylation analysis showed hypermethylation of GNAT1 promoter in all primary NPC tissues and in 12 (80%) specimens of chronic nasopharyngitis tissues. CONCLUSIONS: Expression of GNAT1 gene is down-regulated or absent in NPC tissues, which may relate to allele loss of GNAT1 in NPC, but not relate to its promoter hypermethylation. Hypermethylation of GNAT1 may not be oncogenic mechanisms of 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 (approximately 2% of cells) were detected in the adult mice nasopharyngeal epithelia by immunostaining with BrdU antibody and some of LRCs (approximately 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 (approximately 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 nasopharynx 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.

Identification of genes related to nasopharyngeal carcinoma with the help of pathway-based networks.

cDNA microarray is a powerful tool to analyze simultaneously the expression levels of tens of thousands of genes. Compared with normal nasopharynx (NP) tissues, 2210 genes were highly differentially expressed in nasopharyngeal carcinoma (NPC) tissues detected by cDNA microarray. Since signal pathway is widely used to describe the complex relationship between genes, a pathway-based network was constructed to visualize the connection between the genes obtained from microarray data in this report. We analyzed the targeted genes that may have more important influence on this gene network with statistical methods and found that some genes might have significant influence on this network, especially Ras-related nuclear protein (RAN), carboxyl ester lipase (CEL), v-rel reticuloendotheliosis viral oncogene homolog A (RELA) genes. To verify the results from pathway-based selection, reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR were performed to detect the expression levels of RAN, CEL and RELA genes and it was found that the RAN and CEL genes were significantly up-regulated in more than 80% of NPC tissues. To further elucidate the function of the RAN gene, RAN expression was specifically suppressed in a 5-8F NPC cell line by RNA interference (RNAi). As expected, the depletion of RAN could effectively block the proliferation of tumor cells. Therefore, our study may open up a new way to analyze the vast microarray data.

Genetic and epigenetic alterations of DLC-1, a candidate tumor suppressor gene, in nasopharyngeal carcinoma.

The DLC-1 gene, located at the human chromosome region 8p22, behaves like a tumor suppressor gene and is frequently deleted in diverse tumors. The deletion of 8p22 is not an uncommon event in nasopharyngeal carcinoma (NPC), therefore we explored the expression levels of the DLC-1 gene in NPCs and NPC cell lines by reverse transcription-polymerase chain reaction. The results showed the mRNA level of DLC-1 was downregulated. To identify the mechanism of DLC-1 downregulation in NPC, we investigated the methylation status of the DLC-1 gene using methylation-specific PCR, and found that 79% (31 of 39) of the NPC tissues and two DLC-1 nonexpressing NPC cell lines, 6-10B and 5-8F, were methylated in the DLC-1 CpG island. Microsatellite PCR was also carried out, and loss of heterozygosity was found at four microsatellite sites (D8S552, D8S1754, D8S1790 and D8S549) covering the whole DLC-1 gene with ratios of 33% (4 of 12 informative cases), 18% (2 of 11), 5% (1 of 18), and 25% (3 of 12), respectively. Taken together, our results suggest that DLC-1 might be an NPC-related tumor suppressor gene affected by aberrant promoter methylation and gene deletion.

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.

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.

Reexploring the possible roles of some genes associated with nasopharyngeal carcinoma using microarray-based detection.

In gene expression profiling, nasopharyngeal carcinoma (NPC) 5-8F cells differ from 6-10B cells in terms of their high tumorigenicity and metastatic ability. Differentially expressed genes from the two cell types were analyzed by combining with MILANO (the automatic custom annotation of microarray results which is based on all the available published work in PubMed). The results showed that five genes, including CTSD, P63, CSE1L, BPAG1 and EGR1, have been studied or mentioned in published work on NPC. Subsequently, we reevaluated the roles of these genes in the pathogenesis of NPC by combining the data of gene chips from NPCs versus NPs and pooled cells from 5-8F, 6-10B and CNE2 versus NPs. The results suggested that the roles of BPAG1 and EGR1 are possibly different from those reported in previous NPC studies. These five genes are likely to be involved in the proliferation, apoptosis, invasion and metastasis of NPC. A reexploration of the genes will further define their roles in the pathogenesis of NPC.

Critical role of Cys168 in noggin protein's biological function.

Previous studies have indicated that noggin exerts its neural inducing effect by binding and antagonizing bone morphogenetic protein 4 (BMP4). In order to further clarify the relationship between the structure and the function of noggin, and elucidate the possible mechanism responsible for noggin-BMP4 interaction, we generated three noggin mutants, C168S, C174S and C197S, by using a site-directed mutagenesis method. Ectopic expression of wild-type (WT) noggin, C174S or C197S, in Xenopus animal caps (ACs) by mRNA injection converted the explants (prospective ectoderm) into neural tissue, as indicated by the neural-like morphology and expression of the neural cell adhesion molecule (NCAM) in the ACs. In contrast, ACs expressing C168S suffered an epidermal fate similar to the control caps. Similarly, among the three mutants, only C168S lost the dorsalizing function. These studies highlight the critical role played by Cys168 in noggin's biological activities. It probably participates in the formation of an intermolecular disulfide bridge.