Caiping Ren

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.

Establishment and Applications of Epstein‐Barr Virus‐Based Episomal Vectors in Human Embryonic Stem Cells

Human embryonic stem (hES) cells are capable of unlimited cell proliferation yet maintain the potential to differentiate into many cell types. Here we reported an Epstein‐Barr virus (EBV)‐based vector system used to improve transfection efficiency in hES cells. Plasmids containing oriP, the latent replication origin of EBV, can be propagated stably as episomal DNA in human cells that express the EBV nuclear antigen 1 (EBNA1), which binds to oriP and functions as the trans‐acting replication initiator. It was reported that the EBV replicon could harbor a DNA fragment of up to 330 kilobase pairs. Plasmids containing an enhanced green fluorescent protein (EGFP)/puromycin resistance gene cassette along with or without oriP were used to transfect hES cells that stably express EBNA1. The presence of oriP moderately increased the transient transfection efficiency and more importantly it elevated the stable transfection efficiency by approximately 1,000‐fold as compared with oriP‐minus plasmids. The oriP plasmid as episomal DNA and green fluorescent protein expression in hES cells was maintained for months in the presence of drug selection and gradually lost (2%–4% per cell doubling) in the absence of selection. The presence of EBNA1 did not interfere with the hES cell properties or differentiation we tested and could maintain stable EGFP expression during differentiation. In addition to transgene expression, the EBV vector system could effectively enhance the RNA interference efficiency in hES cells. Thus, the EBV vector system that allows a large DNA insert and sustained expression of transgene or small hairpin RNA will enhance basic and translational research using hES cells.

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.

An imageable metastatic treatment model of nasopharyngeal carcinoma.

Purpose: Nasopharyngeal carcinoma is highly prevalent in southern China and is often resistant to current treatment options. Experimental Design: Clinically relevant mouse models are necessary for further understanding and drug discovery in this disease. Two nasopharyngeal carcinoma cell lines, stably expressing green fluorescent protein (GFP), 5-8F-GFP and 6-10B-GFP, were established. The cells were orthotopically injected into the nasopharynx or ectopically into the subcutis of nude mice. Whole-body fluorescence imaging was used to monitor the growth of the primary tumor as well as angiogenesis and metastasis. Results: The metastatic behavior of 5-8F and 6-10B were distinct in the orthotopic model. Orthotopic implantation of highly metastatic 5-8F cells resulted in brain invasion, cervical lymph node metastases, and pulmonary metastases similar to what is often observed in patients. Cell line 6-10B was less metastatic, which occasionally resulted in pulmonary metastasis. GFP enabled imaging of micrometastasis. Neither 5-8F nor 6-10B were metastatic in the s.c. site. These results indicated that, in addition to the cancer cell type, the host microenvironment was critical for metastasis to occur consistent with the “seed-and-soil” hypothesis. 5-8F was highly sensitive to 5-fluorouracil (5-FU), whereas 6-10B was moderately sensitive. Conclusions: The imageable orthotopic model should play a critical role in elucidating the mechanisms involved in the growth, progression, metastasis, and angiogenesis of nasopharyngeal carcinoma and for evaluation of novel compounds with potential efficacy.

Underlying mechanisms for LTF inactivation and its functional analysis in nasopharyngeal carcinoma cell lines

The lactoferrin (LTF) gene, located at 3p21.3, behaves like a tumor suppressor gene in diverse tumors. To elucidate the exact role of LTF in NPC, we first detected its expression level in seven NPC cell lines by semi‐quantitative reverse transcription‐polymerase chain reaction (RT‐PCR). The results showed the mRNA level of LTF was nearly undetectable in all the seven NPC cell lines, while it could be detected in chronic nasopharyngitis tissues. Subsequently, we used methylation‐specific PCR (MSP), microsatellite assay, PCR‐single‐strand conformation polymorphism (PCR‐SSCP) and sequencing methods to examine the promoter methylation, loss of heterozygosity (LOH) and gene mutation of LTF in NPC cell lines respectively. Consequently, we found that 100% (7 of 7) of NPC cell lines were methylated in LTF promoter, only one cell line (14%, 1 of 7) had LOH and gene mutation of LTF, respectively, while LTF exhibited re‐expression in all cell lines after 5‐aza‐dC treatment, indicating promoter methylation should be the key mechanism causing LTF downregulation in NPC cell lines. Furthermore, patched methylation assay confirmed that promoter methylation could down‐regulate LTF gene expression in NPC cells. Finally, we investigated the function of LTF in NPC cell lines by gene transfection. Restoration of LTF expression in NPC cells resulted in blockage of cell cycle progression, significant inhibition of cell growth and a reduced colony‐formation capacity in vitro and obviously weaker tumor formation potential in vivo. In conclusion, our data indicate LTF may participate in NPC carcinogenesis as a negative effector, that is, a tumor suppressor gene. J. Cell. Biochem. 112: 1832–1843, 2011.

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.

GPC1 exosome and its regulatory miRNAs are specific markers for the detection and target therapy of colorectal cancer

Colorectal cancer (CRC) is the second leading cause of cancer‐related deaths worldwide. However, a biomarker for a sensitive and simple diagnostic test and highly effective target therapy of CRC is still clinically unavailable. This study is to investigate the evidence and significance of plasma GPC1 positive exosomes as a biomarker of CRC. Results showed that GPC1+ exosomes were successfully isolated from tissues and plasma. The percentage of GPC1+ exosomes and the GPC1 protein expression in exosomes from tumour tissues and plasma of CRC patients before surgical treatment was significantly elevated compared to that in the peritumoural tissues and the plasma of healthy controls. miR‐96‐5p and miR‐149 expression in tumour tissues and plasma of CRC patients as well as in the GPC1+ exosomes from CRC patients were significantly decreased compared to that in the peritumoural tissues and the plasma of healthy controls. Two months after surgical treatment, levels of all tested markers significantly normalized. Overexpression of miR‐96‐5p and miR‐149 significantly decreased GPC1 expression in HT‐29 and HCT‐116 cells, xenograft tumours, plasma in mice bearing HT‐29 and HCT‐116 tumours, and the secretion of GPC1+ exosomes from the HT‐29 and HCT‐116 cells and xenograft tumours. Overexpression of miR‐96‐5p and miR‐149 significantly decreased cell viability and increased cell apoptosis in HT‐29 and HCT‐116 cells, and inhibited the growth of xenograft HT‐29 and HCT‐116 tumours. In conclusion, the increased plasma GPC1+ exosomes and reduced plasma miR‐96‐5p and miR‐149 expression are specific markers for the diagnosis of CRC and targets for the therapy of CRC.

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.