Yanhong Zhou

miR-216b suppresses tumor growth and invasion by targeting KRAS in nasopharyngeal carcinoma

MicroRNAs (miRNAs) are small noncoding RNAs that are involved in various diseases, including cancer. In the present study, we found that miR-216b was downregulated in nasopharyngeal carcinoma (NPC) cell lines and specimens. Decreased expression of miR-216b was directly related to advanced clinical stage and lymph node metastasis. miR-216b levels correlated inversely with levels of KRAS protein during nasopharyngeal tumorigenesis. Furthermore, we demonstrated that miR-216b can bind to the 3′ untranslated region (UTR) of KRAS and inhibit expression of the KRAS protein. Both in vitro and in vivo assays revealed that miR-216b attenuated NPC cell proliferation, invasion and tumor growth in nude mice. miR-216b exerts its tumor suppressor function through inhibition of the KRAS-related AKT and ERK pathways. Our findings provide, for the first time, significant clues regarding the role of miR-216b as a tumor suppressor by targeting KRAS in NPC.

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.

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.

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.

miR-214 promotes tumorigenesis by targeting lactotransferrin in nasopharyngeal carcinoma

LTF (lactotransferrin, or lactoferrin) plays important role in innate immunity, and its anti-tumor function has also been reported in multiple cancers. We previously reported that LTF is significantly down-regulated in nasopharyngeal carcinoma (NPC) and acts as a tumor suppressor by suppressing AKT signaling. However, the exact mechanism of the down-regulation of LTF in NPC has not been revealed. In the current study, we screened and identified LTF is a bona fide target of miR-214 in NPC cells. miR-214 mimics significantly suppressed LTF mRNA and protein expression levels in NPC cells. miR-214 not only can promote NPC cell proliferation and invasion abilities in vitro, but also can accelerate tumor formation and lung metastasis in a mouse xenograft model. The pro-tumor function of miR-214 was depended on LTF suppression since LTF re-expression can reverse it. miR-214 can also activate AKT signaling by suppressing LTF expression. Furthermore, miR-214 expression level was up-regulated in NPC especially in metastasis-prone NPC tumor tissues compared with normal nasopharyngeal epithelial tissues, while the LTF expression level was negatively correlated with miR-214, suggesting that miR-214 targeting is partly responsible for LTF down-regulation in NPC specimens.

Analysis of gene expression identifies candidate molecular markers in nasopharyngeal carcinoma using microdissection and cDNA microarray

PurposeMicroarray analysis was used to bring a comprehensive insight into underlying molecular mechanisms and obtain a whole assessment of aberrant gene expression in nasopharyngeal carcinoma (NPC).MethodsCombined with microdissection, gene expression profiles in 23 NPCs and 10 nontumor nasopharyngeal epithelial tissue samples were analyzed.ResultsGene expression patterns suggested the dysregulation of the GTP/GDP-bound Ras cycle and an abnormal hyperactivity of cell cycle in NPC. Alterations in the WNT pathway suggest that this pathway may be activated in NPC. A 6-feature weighted-voting model was chosen because it represented the main characteristics of NPCs and predicted NPCs most accurately from the nontumor tissues (33 of 34 correct calls; 97.1% accuracy, Fisher’s exact test, P valuexa0=xa08.389xa0×xa010−8).ConclusionsThe data generated in this study represent a comprehensive list of genes aberrantly regulated in NPC. The 6-feature weighted-voting model may provide an extensive list of potential molecular markers for early diagnosis.

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.

Expression of LINC00312, a long intergenic non-coding RNA, is negatively correlated with tumor size but positively correlated with lymph node metastasis in nasopharyngeal carcinoma

The long intergenic non-coding RNA LINC00312, also called NAG7, was first cloned by our group. Our previous studies have found that LINC00312 could inhibit proliferation and induce apoptosis in nasopharyngeal carcinoma (NPC) cells but also stimulate NPC cell invasion. However, the relevance of LINC00312 in NPC progression or in patient outcomes has not been reported. This study aims to assess the possible correlations of LINC00312 expression with NPC progression and its potential prognostic predictive ability in NPC outcomes. A NPC tissue microarray, which included 561 normal and NPC tissue cores, was used to detect LINC00312 expression, and we found that LINC00312 was significantly down-regulated in NPC tissues compared with non-cancerous nasopharyngeal epithelium tissues. Positive expression of LINC00312 was negatively correlated with tumor size (Pxa0<xa00.001) but positively correlated with lymph node metastasis (Pxa0=xa00.002). A receiver operating characteristicxa0(ROC) analysis revealed that LINC00312 expression could distinguish non-cancerous patients from NPC patients (Pxa0<xa00.001, sensitivity: 72.1xa0%, specificity: 87.7xa0%). We also found that LINC00312 was strongly negatively correlated with EBER-1, a non-coding RNA transcribed by Epstein-Barr Virus, in NPC (rxa0=xa0−0.384, Pxa0<xa00.001). In the final logistic regression analysis model, the abnormal expression of LINC00312 and EBER-1 were found to be independent contributors to nasopharyngeal carcinogenesis (Pxa0<xa00.001, Pxa0<xa00.001, respectively). A survival analysis revealed that LINC00312 could predict a good prognosis of no lymph node metastasis (Disease Free Survival (DFS): Pxa0=xa00.005, Overall Survival (OS): Pxa0=xa00.001) and a poor prognosis of lymph node metastasis (DFS: Pxa0=xa00.011, OS: Pxa0=xa00.001) in NPC patients. Low expression of LINC00312 was an independent risk factor for OS in multivariate analyses (Pxa0=xa00.017). These observations indicated that LINC00312 could represent a potential biomarker for metastasis, progression and prognosis in NPC.

LOC401317, a p53-regulated long non-coding RNA, inhibits cell proliferation and induces apoptosis in the nasopharyngeal carcinoma cell line HNE2

Recent studies have revealed that long non-coding RNAs participate in all steps of cancer initiation and progression by regulating protein-coding genes at the epigenetic, transcriptional, and post-transcriptional levels. Long non-coding RNAs are in turn regulated by other genes, forming a complex regulatory network. The regulation networks between the p53 tumor suppressor and these RNAs in nasopharyngeal carcinoma remains unclear. The aims of this study were to investigate the regulatory roles of the TP53 gene in regulating long non-coding RNA expression profiles and to study the function of a TP53-regulated long non-coding RNA (LOC401317) in the nasopharyngeal carcinoma cell line HNE2. Long non-coding RNA expression profiling indicated that 133 long non-coding RNAs were upregulated in the human NPC cell line HNE2 cells following TP53 overexpression, while 1057 were downregulated. Among these aberrantly expressed long non-coding RNAs, LOC401317 was the most significantly upregulated one. Further studies indicated that LOC401317 is directly regulated by p53 and that ectopic expression of LOC401317 inhibits HNE2 cell proliferation in vitro and in vivo by inducing cell cycle arrest and apoptosis. LOC401317 inhibited cell cycle progression by increasing p21 expression and decreasing cyclin D1 and cyclin E1 expression and promoted apoptosis through the induction of poly(ADP-ribose) polymerase and caspase-3 cleavage. Collectively, these results suggest that LOC401317 is directly regulated by p53 and exerts antitumor effects in HNE2 nasopharyngeal carcinoma cells.

Evaluation of the prognostic value of TGF-β superfamily type I receptor and TGF-β type II receptor expression in nasopharyngeal carcinoma using high-throughput tissue microarrays

Gene expression profiling had revealed that TGF-β superfamily type I receptor (also known as activin receptor-like kinase-1, ALK1) and TGFβR2 (TGF-β type II receptor) were down-regulated in nasopharyngeal carcinoma (NPC) (Pxa0<xa00.05, respectively). However, no study with significantly large clinical samples to address the relevance of ALK1 and TGFβR2 in NPC progression or in patient outcomes has been reported. This study aims to assess the possible correlations of ALK1 and TGFβR2 expression with NPC progression and their potential prognostic predictive ability in NPC outcomes. ALK1 and TGFβR2 mRNA and protein levels were detected by qRT-PCR and NPC tissue microarray (TMA), which included 742 tissue cores. Both mRNA and protein levels of ALK1 and TGFβR2 were significantly lower in the cancer tissues compared with the non-cancerous tissues (Pxa0<xa00.05). Epstein-Barr virus small RNA (EBER-1) hybridization signals in NPC showed significant associations with ALK1 and TGFβR2 proteins (Pxa0=xa00.000 and 0.003, respectively). In the final logistic regression analysis model, the abnormal expression of ALK1 and TGFβR2 were found to be independent contributors to nasopharyngeal carcinogenesis (Pxa0=xa00.000 and 0.000, respectively). A survival analysis revealed that ALK1 (Disease Free Survival (DFS): Pxa0=xa00.002, Overall Survival (OS): Pxa0=xa00.007) and TGFβR2 (DFS: Pxa0=xa00.072, OS: Pxa0=xa00.045) could predict the prognosis of NPC patients. The positive expression of ALK1 and TGFβR2 were independent risk factors for DFS and OS in multivariate analyses (DFS: Pxa0=xa00.001 and 0.420, respectively; OS: Pxa0=xa00.018 and 0.047, respectively). These results suggest that ALK1 and TGFβR2 may be useful prognostic biomarkers in NPC.