Zhaohui Luo

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.

An in silico analysis of dynamic changes in microRNA expression profiles in stepwise development of nasopharyngeal carcinoma.

BackgroundMicroRNAs (miRNAs) are small non-coding RNAs that participate in the spatiotemporal regulation of messenger RNA (mRNA) and protein synthesis. Recent studies have shown that some miRNAs are involved in the progression of nasopharyngeal carcinoma (NPC). However, the aberrant miRNAs implicated in different clinical stages of NPC remain unknown and their functions have not been systematically studied.MethodsIn this study, miRNA microarray assay was performed on biopsies from different clinical stages of NPC. TargetScan was used to predict the target genes of the miRNAs. The target gene list was narrowed down by searching the data from the UniGene database to identify the nasopharyngeal-specific genes. The data reduction strategy was used to overlay with nasopharyngeal-specifically expressed miRNA target genes and complementary DNA (cDNA) expression data. The selected target genes were analyzed in the Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway. The microRNA-Gene-Network was build based on the interactions of miRNAs and target genes. miRNA promoters were analyzed for the transcription factor (TF) binding sites. UCSC Genome database was used to construct the TF-miRNAs interaction networks.ResultsForty-eight miRNAs with significant change were obtained by Multi-Class Dif. The most enriched GO terms in the predicted target genes of miRNA were cell proliferation, cell migration and cell matrix adhesion. KEGG analysis showed that target genes were significantly involved in adherens junction, cell adhesion molecules, p53 signalling pathway et al. Comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR-29a/c, miR-34b, miR-34c-3p, miR-34c-5p, miR-429, miR-203, miR-222, miR-1/206, miR-141, miR-18a/b, miR-544, miR-205 and miR-149 may play important roles on the development of NPC. We proposed an integrative strategy for identifying the miRNA-mRNA regulatory modules and TF-miRNA regulatory networks. TF including ETS2, MYB, Sp1, KLF6, NFE2, PCBP1 and TMEM54 exert regulatory functions on the miRNA expression.ConclusionsThis study provides perspective on the microRNA expression during the development of NPC. It revealed the global trends in miRNA interactome in NPC. It concluded that miRNAs might play important regulatory roles through the target genes and transcription factors in the stepwise development of NPC.

The microRNA-processing enzymes: Drosha and Dicer can predict prognosis of nasopharyngeal carcinoma

PurposeDysregulation of microRNA (miRNA) metabolism has been observed in a variety of human cancers, but the expression patterns of the enzymes responsible for generating miRNAs remain largely unexplored. In this study, we investigated the expression profiles of the two most important enzymes of the miRNA machinery, Drosha and Dicer, which were closely correlated with nasopharyngeal carcinoma (NPC) and patient survival.MethodsDicer and Drosha mRNA levels were detected by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) using 24 NPC tissues, 7 normal nasopharyngeal epithelium samples (NPE) and NPC cell lines. In addition, protein levels were detected by immunohistochemistry (IHC) using an NPC tissue microarray (TMA), which include 251 NPC and 105 NPE cases. For some NPC patients can not be contacted, the survival data were available only for 146 patients. Kaplan–Meier analysis was performed, and the chi-square and log-rank tests were used to detect significance levels using SPSS 15.0 software.ResultsThe mean level of Dicer and Drosha mRNA were significantly down-regulated in NPC tissue specimens and cell lines when compared with controls. The low levels of Dicer and Drosha protein were frequently seen in NPC, and the low expression of Dicer and Drosha protein was significantly correlated with shorter progression-free survival (PFS) and overall survival (OS) of NPC patients.ConclusionsWe observed that Drosha and Dicer expression was dysregulation in NPC compared with healthy control samples and was significantly correlated with shorter PFS and OS of NPC patients. Therefore, we hypothesise that the expression levels of Dicer and Drosha could be used as potential prognostic biomarkers for NPC.

Epstein-Barr Virus Downregulates MicroRNA 203 through the Oncoprotein Latent Membrane Protein 1: a Contribution to Increased Tumor Incidence in Epithelial Cells

ABSTRACT The Epstein-Barr virus (EBV) is highly associated with nasopharyngeal carcinoma (NPC), and it regulates some microRNAs (miRNAs) that are involved in the development of cancer. The role of EBV in the deregulation of cellular miRNAs and how this affects the progression of NPC remain to be investigated. An analysis of the miRNA profile in an EBV-infected cell line revealed that miRNA 203 (miR-203) was downregulated. miR-203 is expressed specifically in epithelial cells. This downregulation of miR-203 was further verified and functionally analyzed. miR-203 was downregulated substantially in epithelial cells and NPC tissues that were latently infected with EBV. Downregulation of miR-203 also occurred during the early stage of EBV infection. Furthermore, the viral oncoprotein, latent membrane protein 1 (LMP1), was responsible for downregulation of miR-203. Removal of the latent EBV genome or suppression of LMP1 resulted in restoration of miR-203 expression. EBV-LMP1 mediated the downregulation of miR-203 at the primary transcript level. E2F3 and CCNG1 were identified as target genes of miR-203. Ectopic expression of miR-203 inhibited EBV-induced S-phase entry and transformation in vivo. Overexpression of the targets overcame the effects of miR-203 mimics on the cell cycle, and the expression of target genes in tumor models was inhibited by miR-203. Inhibitors of Jun N-terminal protein kinase (JNK) and NF-κB blocked miR-203 downregulation. These results imply that EBV promotes malignancy by downregulating cellular miR-203, which contributes to the etiology of NPC.

miR-149 promotes epithelial-mesenchymal transition and invasion in nasopharyngeal carcinoma cells

OBJECTIVE To investigate the function and mechanism of miR-149 in nasopharyngeal carcinoma (NPC). METHODS The expression of miR-149 was examined by real-time PCR and calculated by 2(-▵▵Ct) method. The cell proliferation was analyzed by MTT assay. The cell migration and invasion were shown by the wound healing assay and transwell migration assay, and the expression of E-cadherin was detected by Western blot. RESULTS The expression of miR-149 was higher in NPC cell lines 5-8F and 6-10B than that in normal immortalized nasopharyngeal epithelial NP69. MTT assay showed that miR-149 promoted the proliferation of NPC cell lines. The wound healing assay showed miR-149 promoted the mobility and invasion of NPC cell lines. Inhibition of miR-149 reduced the ability of NPC cell lines to proliferate and invade. miR-149 downregulated the expression of E-cadherin, whereas antagomir which mediated knockdown of miR-149 significantly upregulated the expression of E-cadherin. CONCLUSION miR-149 might be involved in the invasion and metastasis of NPC through regulation of epithelial-mesenchymal transition (EMT).

miR-181 subunits enhance the chemosensitivity of temozolomide by Rap1B-mediated cytoskeleton remodeling in glioblastoma cells

Glioblastoma multiforme (GBM) is the most malignant and frequent brain tumor, with an aggressive growth pattern and poor prognosis despite best treatment modalities. Although chemotherapy with temozolomide (TMZ) may restrain tumor growth for some months, TMZ resistance is also common and accounts for many treatment failures. Research into microRNA’s role in GBM has shown that microRNAs play a key regulatory role in the GBM, making it a potential therapeutic target. In this study, we demonstrated that the lower expression of miR-181a/b/c/d subunits contributes to astrocytoma tumorigenesis, and their overexpression could inhibit the invasive proliferation of glioblastoma cells by targeting Rap1B-mediated cytoskeleton remodeling and related molecular (Cdc42, RhoA and N-cadherin) changes, suggesting that miR-181 was a critical regulator and might be an important target for glioblastoma treatment. TMZ as a standard chemotherapeutic agent for GBM inhibited the Rap1B expression and actin cytoskeleton remodeling to exert its cell killing by upregulating miR-181a/b/c/d subunits; conversely, each miR-181a/b/c/d subunit enhanced the chemosensitivity of TMZ in glioblastoma cells.

Lactoferrin suppresses the Epstein–Barr virus-induced inflammatory response by interfering with pattern recognition of TLR2 and TLR9

Epstein–Barr virus (EBV) infection contributes to tumorigenesis of various human malignancies including nasopharyngeal carcinoma (NPC). EBV triggers innate immune and inflammatory responses partly through Toll-like receptor (TLR) signaling. Lactoferrin (LF), with its anti-inflammatory properties, is an important component of the innate immune system. We previously reported that LF protects human B lymphocytes from EBV infection by its ability to bind to the EBV receptor CD21, but whether LF can suppress EBV-induced inflammation is unclear. Here, we report that LF reduced synthesis of IL-8 and monocyte chemoattractant protein-1 (MCP-1) induced by EBV in macrophages via its suppression of NF-κB activity. LF interacted with TLR2 and interfered with EBV-triggered TLR2-NF-κB activation. LF inhibited the ability of TLR9 to recognize dsDNA by binding to its co-receptor CD14, which blocked the interaction between CD14 and TLR9. EBV-induced inflammation was thus aggravated in the presence of CD14. In addition, LF expression levels were significantly downregulated in NPC specimens, and correlated inversely with IL-8 and MCP-1 expression. These findings suggest that LF may suppress the EBV-induced inflammatory response through interfering with the activation of TLR2 and TLR9.

Tumor-Conditioned Mesenchymal Stem Cells Display Hematopoietic Differentiation and Diminished Influx of Ca2+

Mesenchymal stem cells (MSCs) that are present in many adult tissues can generate new cells either continuously or in response to injury/cancer. An increasing number of studies demonstrated that MSCs have the ability to differentiate into cells of mesodermal origin and transdifferentiate into cells such as hepatocytes, neural cells. There has been growing interest in the application of MSCs to cancer therapy. The relationship between MSCs and cancer cells remains highly controversial. In this study, we analyzed the interaction of bone marrow-derived MSCs and cancer cells by cell-cell contact and transwell culture system. The flow cytometry and real-time polymerase chain reaction showed that after coculture of MSCs and cancer cells, MSCs displayed the hematopoietic cell markers such as CD34, CD45, and CD11b. The CD68, MRCI, and CSF1R were dramatically upregulated after coculture. The cytokine array showed that MSCs after coculture secreted monokines and chemokines much more than that of intact MSCs. The MSCs under tumor conditions were responsive to stimulation with lipopolysaccharide by cytokines release. The tumor-conditioned MSCs showed phagocytic ability and enhanced release of nitric oxide, which are the characteristics of macrophages. Calcium ion is an important intracellular messenger responsible for differentiation and gene expression regulations. The influx of Ca(2+) into MSCs was obviously reduced after coculture. The blocking of calcium channel with verapamil obviously increased the expression of CD34, CD45, and CD11b, thus indicating that the diminished calcium ion influx is coupled with the hematopoietic differentiation of MSCs under tumor conditions. Taken together, in a cancer environment, MSCs could effectively differentiate into immune hematopoietic cells, precisely macrophages. Diminished transient influx of Ca(2+) may mediate the hematopoietic differentiation of MSCs.

Mesenchymal stem cells regulate cytoskeletal dynamics and promote cancer cell invasion through low dose nitric oxide.

Bone marrow-derived mesenchymal stem cells (BMSCs) can be recruited to tumor sites and integrate into the stroma of tumors. When co-cultured with BMSCs, otherwise weakly metastatic nasopharyngeal carcinoma cells (NPC) showed improved metastatic ability. BMSCs in the tumor environment displayed the characteristics of macrophages. Nitric oxide produced by BMSCs in tumor environment could translocate caldesmon to podosome in Ca2+/calmodulin manner and promoted metastatic ability of NPC cells through invadopodia formation, with which the NPC cells degrade the extracellular matrix. Thus, we concluded that the BMSCs promoted cell migration and invasion through nitric oxide-induced paracrine signals.

Prohibitin is an important biomarker for nasopharyngeal carcinoma progression and prognosis.

Prohibitin-1 (PHB, also known as PHB1) is a pleiotropic protein in cells. PHB is a cell-surface receptor and is involved in the regulation of proliferation, apoptosis, transcription, and mitochondrial protein folding. PHB is upregulated in 5-8F cells, which overexpress LPLUNC1 (long palate, lung, nasal epithelium clone 1, a candidate tumour suppressor gene), and was identified using two-dimensional fluorescence difference gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI–TOF/TOF–MS/MS). Thus, we examined PHB mRNA levels using 24 nasopharyngeal carcinoma (NPC) and eight normal nasopharyngeal epithelium (NPE) tissues. Protein levels were detected using immunohistochemistry with a tissue microarray consisting of 323 NPC and NPE tissues. A Kaplan–Meier analysis was carried out, and the log-rank test was used to determine the statistical significance of the results using SPSS 15.0 software. PHB mRNA and protein expression levels were significantly downregulated in NPC tissue specimens compared with the NPE samples (P<0.01). In addition, decreased PHB expression correlates significantly with a poor prognosis, whereas decreased PHB protein expression is closely associated with advanced clinical stage and metastasis in NPC lesions. Therefore, we favour the hypothesis that the expression level of PHB could be used as a potential prognostic biomarker for NPC.