Upregulation of miR-31* Is Negatively Associated with Recurrent/Newly Formed Oral Leukoplakia

BACKGROUND: Oral leukoplakia (OLK) is a potentially malignant disorder of the oral cavity. However, the underlying mechanism of OLK is still unclear. In this study, we explore possible miRNAs involved in OLK. METHODOLOGY/PRINCIPAL FINDINGS: Using miRNA microarrays, we profiled miRNA expression in OLK and malignantly transformed OLK (mtOLK) tissue samples. The upregulation of miR-31*, miR-142-5p, miR-33a, miR-1259, miR-146b-5p, miR-886-3p, miR-886-5p, miR-519d, and miR-301a along with the downregulation of miR-572, miR-611, miR-602, miR-675, miR-585, miR-623, miR-637, and miR-1184 in mtOLK were new observations. Fluorescence in situ hybridization (FISH) analyses confirmed that miR-31* is highly expressed in mtOLK. There was a significant difference between the FISH score (p<0.05) in patients with or without recurrent/newly formed OLK. Functional analyses demonstrated that a miR-31* inhibitor decreased apoptosis in the Leuk-1, which is an immortalized oral epithelial cell line spontaneously derived from an oral leukoplakia lesion. miR-31* regulated apoptosis, cell proliferation, migration, and invasion in the HOIEC, which is a HPV E6/E7-immortalized oral epithelial cell line. Furthermore, miR-31* modulated the biological functions of apoptosis, cell proliferation, cell cycle, migration, and invasion in the oral squamous cell carcinoma cell line, Cal-27. Using bioinformatic analyses and dual luciferase reporter assays, we determined that the 3' untranslated region of fibroblast growth factor 3 (FGF3) is the target of miR-31*. Expression of FGF3 was downregulated or upregulated in the presence of a miR-31* mimic or inhibitor, respectively. CONCLUSIONS/SIGNIFICANCE: Upregulation of miR-31* is negatively associated with recurrent/newly formed OLK. MiR-31* may exert similar but distinguishable effects on biological function in oral cells with different malignant potential. FGF3 is the target of miR-31*. miR-31* may play an important role during OLK progression through regulating FGF3. MiRNA* strands may also have prominent roles in oral carcinogenesis

Characterization of a new EBV-associated nasopharyngeal carcinoma cell line

Only a few reports exist of cytogenetic studies in nasophoryngeal carcinoma (NPC), probably because of the technical limitations of culturing solid tumors. We established a cell line (cell-666) that was derived from an NPC xenograft growing in athymic nude mice. Epstein-Barr virus (EBV) infection is one of the important etiologic factors of NPC. This cell line has been shown to have the consistent presence of EBV by Epstein-Barr virus encoded RNAs (EBER) in situ hybridization. Furthermore, aberrant methylation of 5'CpG island of p16 gene was detected. Thus, this cell line represents a valuable model for the study of NPC tumorigenecity. The karyotype of this newly established cell line is: 45,X,del(X)(q24), - 5der(5)(5pter→5q31::5q13→5q31::5p12→5pter),der(6;?)(q10;p10),add(7)(p22),+ 9, del(11)(q13),add(12)(p13),-14,-18,-21, +2mar. Fluorescence in situ hybridization analyses were performed using whole chromosome painting probes of chromosomes 5, 6, 11, and 12.link_to_subscribed_fulltex

Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein-Barr virus

We have established a cell line (C666-1) from undifferentiated nasopharyngeal carcinoma (NPC). This cell line consistently carries the Epstein-Barr virus (EBV) in long-term cultures. C666-1 is a subclone of its parental cell line, C666, derived from an NPC xenograft of southern Chinese origin. It grows as an adherent culture and lacks contact inhibition. In addition, it is tumorigenic in athymic nude mice. The cells consistently express EBV-encoded RNAs and are positively stained for cytokeratin, an epithelial marker. In addition, they express EBNA1 protein, LMP1 and LMP2 transcripts and thus resemble the EBV latency II pattern. The virus genotype is EBV-1 with the latent membrane protein 1 gene showing a 30-bp deletion at the carboxyl terminus, both consistent with findings in southern Chinese NPC tumours. Cytogenetic analysis revealed a sub-diploid status with a chromosomal modal number of 45. C666-1 is unique among NPC cell lines in that it carries EBV. These cells may serve as a good investigative tool as the viral latency pattern and genotype are observed in the majority of primary NPC biopsies from Chinese patients.link_to_subscribed_fulltex

Identification of a novel 12p13.3 amplicon in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer commonly occurring in southern China. To decipher the molecular basis of this cancer, we performed high-resolution array CGH analysis on eight tumour lines and 10 primary tumours to identify the genes involved in NPC tumorigenesis. In this study, multiple regions of gain were consistently found at 1q21-q24, 7q11-12, 7q21-22., 11q13, 12p13, 12q13, 19p13 and 19q13. Importantly, a 2.1 Mb region at 12p13.31 was highly amplified in a NPC xenograft, xeno-2117. By FISH mapping, we have further delineated the amplicon to a 1.24 region flanked by RP11-319E16 and RP11-433J6. Copy number gains of this amplicon were confirmed in 21/41 (51%) primary tumours, while three cases (7.3%) showed high copy number amplification. Among the 13 genes within this amplicon, three candidate genes, lymphotoxin beta receptor (LTβR), tumour necrosis factor receptor superfamily memeber 1A (TNFRSF1R) and FLJ10665, were specifically over-expressed in the NPC xenograft with 12p13.3 amplification. However, only LTβR was frequently over-expressed in primary tumours. LTβR is a member of the TNF family of receptors, which can modulate NF-κB signalling pathways. Over-expression of LTβR in nasopharyngeal epithelial cells resulted in an increase of NF-κB activity and cell proliferation. In vivo study showed that suppression of LTβR by siRNA led to growth inhibition in the NPC tumour with 12p13.3 amplification. These findings implied that LTβR is a potential NPC-associated oncogene within the 12p13.3 amplicon and that its alteration is important in NPC tumorigenesis. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.link_to_subscribed_fulltex

Array-based comparative genomic hybridization analysis identified cyclin D1 as a target oncogene at 11q13.3 in nasopharyngeal carcinoma

Nasopharyegeal carcinoma is highly prevalent in Southern China and Southeast Asia. To unveil the molecular basis of this endemic disease, high-resolution comparative genomic hybridization arrays were used for systematic investigation of genomic abnormalities in 26 nasopharyngeal carcinoma samples. A comprehensive picture of genetic lesions associated with tumorigenesis of nasopharyngeal carcinoma was generated. Consistent chromosomal gains were frequently found on 1q, 3q, 8q, 11q, 12p, and 12q. High incidences of nonrandom losses were identified on chromosomes 3p, 9p, 11q, 14q, and 16q. In addition to previously characterized regions, we have identified several novel minimal regions of gains, including 3q27.3-28, 8q21-24, 11q13.1-13.3, and 12q13, which may harbor candidate nasopharyngeal carcinoma-associated oncogenes. In this study, gain of 11q13.1-13.3 was the most frequently detected chromosomal aberration and a 5.3-Mb amplicon was delineated at this region. Within this 11q13 amplicon, concordant amplification and overexpression of cyclin D1 (CCND1) oncogene was found in nasopharyngeal carcinoma cell lines, xenografts, and primary tumors. Knockdown of cyclin D1 by small interfering RNA in nasopharyngeal carcinoma cell lines led to significant decrease of cell proliferation. The findings suggest that cyclin D1 is a target oncogene at 11q13 in nasopharyngeal carcinoma and its activation plays a significant role in nasopharyngeal carcinoma tumorigenesis. ©2005 American Association for Cancer Research.link_to_OA_fulltex

Inhibiting tumorigenic potential by restoration of p16 in nasopharyngeal carcinoma

The p16 gene, encodes a key checkpoint protein p16 in the cell cycle, has been reported inactivation in a wide variety of human cancers. We have previously demonstrated high frequency of p16 alterations in primary nasopharyngeal carcinoma (NPC), xenografts and cell lines. The finding implied that inactivation of the p16 gene may play an important role in the NPC development. To investigate the tumour suppressor function of p16 in NPC, we tranfected p16-deficient NPC cell line, NPC/HK-1, with a wild-type p16 expression construct, and evaluated growth and tumorigenic properties of the clones stably expressing exogenous p16. Expression of the exogenous wild-type p16 significantly inhibited cell growth by more than 70% when compared to that of the parental and empty vector-transfected cells. This growth inhibition was attributable to a significant proportion of p16-expressing cells arrested at G1 phase in the cell cycle as revealed by flow cytometric analysis. By anchorage-independent colony forming assay, we found that the ability to form colonies in soft agar was highly reduced in cells expressing p16. NPC/HK1 cells expressing functional p16 also showed suppressed tumorigenicity in athymic nude mice. Taken together, our results provide strong evidence for a tumour suppressor role of p16 in NPC.link_to_subscribed_fulltex

Establishment and characterization of new tumor xenografts and cancer cell lines from EBV-positive nasopharyngeal carcinoma

The lack of representative nasopharyngeal carcinoma (NPC) models has seriously hampered research on EBV carcinogenesis and preclinical studies in NPC. Here we report the successful growth of five NPC patient-derived xenografts (PDXs) from fifty-eight attempts of transplantation of NPC specimens into NOD/SCID mice. The take rates for primary and recurrent NPC are 4.9% and 17.6%, respectively. Successful establishment of a new EBV-positive NPC cell line, NPC43, is achieved directly from patient NPC tissues by including Rho-associated coiled-coil containing kinases inhibitor (Y-27632) in culture medium. Spontaneous lytic reactivation of EBV can be observed in NPC43 upon withdrawal of Y-27632. Whole-exome sequencing (WES) reveals a close similarity in mutational profiles of these NPC PDXs with their corresponding patient NPC. Whole-genome sequencing (WGS) further delineates the genomic landscape and sequences of EBV genomes in these newly established NPC models, which supports their potential use in future studies of NPC