MicroRNA let-7 suppresses nasopharyngeal carcinoma cells proliferation through downregulating c-Myc expression

Aims: This study aimed at evaluating the potential anti-proliferative effects of the microRNA let-7 family in nasopharyngeal carcinoma (NPC) cells. In addition, the association between let-7 suppression and DNA hypermethylation is examined. Materials and methods: Levels of mature let-7 family members (-a,-b,-d,-e,-g, and-i) in normal nasopharyngeal cells (NP69 and NP460) and nasopharyngeal carcinoma cells (HK1 and HONE1) were measured by real-time quantitative PCR. Cell-proliferation assay and c-Myc immunohistochemical staining were performed on NPC cells transfected with let-7 precursor molecules. In addition, expression changes in let-7 family members in response to demethylating agents (5-azacytidine and zebularine) were also examined. Results: In comparison with the normal nasopharyngeal cells, let-7 (-a,-b,-d,-e,-g, and-i) levels were reduced in nasopharyngeal carcinoma cells. Ectopic expression of the let-7 family in nasopharyngeal carcinoma cells resulted in inhibition of cell proliferation through downregulation of c-Myc expression. Demethylation treatment of nasopharyngeal carcinoma cells caused activation of let-7 expression in poorly differentiated nasopharyngeal carcinoma cells only. Conclusion: Our results suggested that miRNA let-7 might play a role in the proliferation of NPC. DNA methylation is a potential regulatory pathway, which is affected when let-7 is suppressed in NPC cells. However, the extent of DNA hypermethylation/hypomethylation in regulating let-7 expression requires further elucidation. © The Author(s) 2010. This article is published with open access at Springerlink.com.published_or_final_versionSpringer Open Choice, 21 Feb 201

Serum amyloid A 1 (SAA1) polymorphisms are associated with variation in tumor suppressive activities in esophageal squamous cell carcinoma (ESCC)

Esophageal cancer (EC), a malignancy originated from the epithelium of the esophagus, ranks eighth in incidence rate of cancer, sixth as the most common cause of death worldwide, and causes the eighth highest mortality rate in Hong Kong cancer patients. High prevalence areas, called “esophageal cancer belt”, are located along Northern Iran through the Central Asian Republics to North-Central China. Esophageal squamous cell carcinoma (ESCC) occurs as the dominant type of EC worldwide. Using the functional complementation approach, SAA1 was identified as one of the candidate tumor suppressive genes in ESCC. SAA1 is expressed as a secretary protein and is present in most histologically normal human epithelial tissues. The three SAA1 isoforms (SAA1.1, 1.3, 1.5) with two single nucleotide polymorphisms were observed in the ESCC patients and healthy individuals. The expression of SAA1 was frequently down-regulated in ESCC cell lines. Therefore, the functional roles of the three SAA1 isoforms in tumor suppression in ESCC were investigated. The restoration of SAA1.1 and 1.3 isoforms in the SAA1-null ESCC cell lines showed suppression of tumor growth and angiogenesis. The proteins of these two isoforms could inhibit tumor angiogenesis by their strong binding affinity to block the integrin αVβ3 and the downstream activation of FAK of the vascular endothelial cells. After that, these two SAA1 variant proteins abolished the assembly of stress fiber and focal adhesions, and led to the detachment of the vascular endothelial cells and eventually resulted in cell apoptosis. In contrast, SAA1.5 had weaker binding affinity to the integrin αVβ3 of the vascular endothelial cell surface and it could only delay the cytoskeleton arrangement and cell adhesion but could not induce cell death, demonstrating that SAA1.5 is a defective in the anti-angiogenic function. The restoration of SAA1.1 and 1.3 gene expression in the ESCC cell lines could down-regulate the integrin αV (ITGAV) expression, and subsequently resisted to the epithelial-mesenchymal transition (EMT) induced by TGF-β. On the other hand, the SAA1.5 expression could not reverse this effect induced by TGF-β. In the reverse experiments, when SAA1 was depleted in the SAA1-positive cell lines, both the expression of ITGAV and N-cadherin (CDH2) were induced, showing that ITGAV might play a vital role in EMT in ESCC. Silencing of ITGAV gene expression resisted to the TGF-β-induced EMT morphology in ESCC. At the same time, the ITGAV could positively regulate the CDH2 expression in ESCC to control the cell migration. It is likely that SAA1.1 and 1.3 might suppress the ITGAV-mediated EMT by down-regulation of CDH2. In summary, this slightly change of amino acid of the current SAA1 polymorphisms can significantly affect their function in tumor suppression, tumor angiogenesis, and EMT in ESCC.published_or_final_versionClinical OncologyDoctoralDoctor of Philosoph

A comparative study of circulating microRNAs in nasopharyngeal carcinoma patients

Nasopharyngeal carcinoma (NPC) is squamous cell carcinoma derived from the epithelial layer of nasopharynx. The incidence is high in Southern China and South-east Asia. In Hong Kong, the prevalent of NPC subtype is undifferentiated NPC and is in close association with Epstein-Barr virus (EBV). MicroRNAs (miRNAs) are small non-coding RNAs. They play vital roles in regulating gene expression at post-transcriptional level. EBV also expresses viral miRNAs but the function remains unclear. In NPC diagnosis and monitoring, circulating EBV DNA level has been commonly used. However, in some cases, EBV DNA is below the detection threshold in the plasma of NPC patients making it impossible to be used in continuous monitoring of the patients. This study aimed to evaluate whether miRNAs (both NPC-derived and EBV-derived miRNAs) could be used as candidate circulating markers for disease monitoring. Candidate gene approach was used to select suitable circulating miRNA markers for NPC patients. Four candidate miRNAs including miR-21, miR-1301, miRBART7 and miR-BART22 were examined. The expression levels were first validated in paired NPC tissues and normal counterparts. Furthermore, circulating miRNA levels were evaluated in the plasma of NPC and normal individuals. To examine the changes of miRNA in response to radiotherapy, changes of circulating miRNA were monitored in 13 NPC patients before and after radiotherapy. In addition, functional assay in cell proliferation was performed to validate the potential role of the candidate miRNA in the pathogenesis of undifferentiated NPC. Of the 4 candidate miRNAs, miR-BART7 was consistently over-expressed in both tumor tissues and plasma samples of NPC. In addition, circulating miRBART7 was also detected in NPC patients in case of the plasma EBV DNA levels below the detection threshold. In response to radiotherapy, 10 of 13 (76.92%) patients had decreasing circulating miR-BART7 in the plasma samples collected at 3 month after radiotherapy. Furthermore, introducing miR-BART7 mimics into the undifferentiated NPC cell line HONE1 and normal nasopharyngeal-derived epithelial cell cultures NP69 and NP460 resulted in significant increases in cell proliferation rates of all the 3 cell lines. To summarize, miR-BART7 expression was significantly higher in NPC patients as a potential oncogenic miRNA. Evaluating the miR-BART7 levels is a possible screening approach in NPC diagnosis and post-treatment monitoring. The oncogenic role of miR-BART7 in the development of undifferentiated NPC deserves further investigation.published_or_final_versionSurgeryMasterMaster of Philosoph

Effect of polybrominated diphenyl ether (PBDE) treatment on the composition and function of the bacterial community in the sponge Haliclona cymaeformis

Marine sponges play important roles in benthic environments and are sensitive to environmental stresses. Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants since the 1970s and are cytotoxic and genotoxic to organisms. In the present study, we studied the short-period effect of PBDE-47 (2,2',4,4'-tetrabromodiphenyl ether) treatment on the community structure and functional gene composition of the bacterial community inhabiting the marine sponge Haliclona cymaeformis. Our results showed that the bacterial community shifted from an autotrophic bacteria-dominated community to a heterotrophic bacteria-dominated community in response to PBDE-47 in a time- and concentration-dependent manner. A potentially symbiotic sulfur-oxidizing bacterium (SOB) was dominant (>80% in abundance) in the untreated sponge. However, exposure to a high concentration (1 µg/L) of PBDE-47 caused a substantial decrease in the potential symbiont and an enrichment of heterotrophic bacteria like Clostridium. A metagenomic analysis showed a selective effect of the high concentration treatment on the functional gene composition of the enriched heterotrophic bacteria, revealing an enrichment for the functions responsible for DNA repair, multidrug efflux pumping, and bacterial chemotaxis and motility. This study demonstrated that PBDE-47 induced a shift in the composition of the community and functional genes in the sponge-associated bacterial community, revealing the selective effect of PBDE-47 treatment on the functions of the bacterial community in the microenvironment of the sponge

Palladium-Catalyzed Direct Arylation of Polyfluoroarenes for Accessing Tetra-<i>ortho</i>-Substituted Biaryls: Buchwald-type Ligand Having Complementary −PPh₂ Moiety Exhibits Better Efficiency

The first general examples of direct C–H arylation of electron-deficient polyfluoroarenes with challenging di-<i>ortho</i>-substituted aryl­(heteroaryl) chlorides for tetra-<i>ortho</i>-substituted biaryl synthesis are reported. Key to success is the use of Buchwald-type biaryl phosphine ligand, notably with inexpensive −PPh₂ moiety (instead of −PCy₂ group). Pd­(OAc)₂ associated with ligand <b>L9</b> exhibits even higher efficiency than the corresponding SPhos toward this reaction. A wide range of sterically hindered di-<i>ortho</i>-substituted chloroarenes bearing electron-donating or -withdrawing groups are found applicable. Excellent product yields are obtained under mild reaction conditions, and the catalyst loading down to 0.25 mol % of Pd can also be achieved

Palladium-Catalyzed Direct Arylation of Polyfluoroarenes for Accessing Tetra-<i>ortho</i>-Substituted Biaryls: Buchwald-type Ligand Having Complementary −PPh₂ Moiety Exhibits Better Efficiency

The first general examples of direct C–H arylation of electron-deficient polyfluoroarenes with challenging di-<i>ortho</i>-substituted aryl­(heteroaryl) chlorides for tetra-<i>ortho</i>-substituted biaryl synthesis are reported. Key to success is the use of Buchwald-type biaryl phosphine ligand, notably with inexpensive −PPh₂ moiety (instead of −PCy₂ group). Pd­(OAc)₂ associated with ligand <b>L9</b> exhibits even higher efficiency than the corresponding SPhos toward this reaction. A wide range of sterically hindered di-<i>ortho</i>-substituted chloroarenes bearing electron-donating or -withdrawing groups are found applicable. Excellent product yields are obtained under mild reaction conditions, and the catalyst loading down to 0.25 mol % of Pd can also be achieved

Modulation of LMP2A Expression by a Newly Identified Epstein-Barr Virus-Encoded MicroRNA miR-BART2212

Infection with the Epstein-Barr virus (EBV) is a strong predisposing factor in the development of nasopharyngeal carcinoma (NPC). Many viral gene products including EBNA1, LMP1, and LMP2 have been implicated in NPC tumorigenesis, although the de novo control of these viral oncoproteins remains largely unclear. The recent discovery of EBV-encoded viral microRNA (miRNA) in lymphoid malignancies has prompted us to examine the NPC-associated EBV miRNA. Using large-scale cloning analysis on EBV-positive NPC cells, two novel EBV miRNA, now named miR-BART21 and miR-BART22, were identified. These two EBV-encoded miRNA are abundantly expressed in most NPC samples. We found two nucleotide variations in the primary transcript of miR-BART22, which we experimentally confirmed to augment its biogenesis in vitro and thus may underline the high and consistent expression of miR-BART22 in NPC tumors. More importantly, we determined that the EBV latent membrane protein 2A (LMP2A) is the putative target of miR-BART22. LMP2A is a potent immunogenic viral antigen that is recognized by the cytotoxic T cells; down-modulation of LMP2A expression by miR-BART22 may permit escape of EBV-infected cells from host immune surveillance. Taken together, we demonstrated that two newly identified EBV-encoded miRNA are highly expressed in NPC. Specific sequence variations on the prevalent EBV strain in our locality might contribute to the higher miR-BART22 expression level in our NPC samples. Our findings emphasize the role of miR-BART22 in modulating LMP2A expression, which may facilitate NPC carcinogenesis by evading the host immune response

SOX9 is a dose-dependent metastatic fate determinant in melanoma

Abstract Background In this research, we aimed to resolve contradictory results whether SOX9 plays a positive or negative role in melanoma progression and determine whether SOX9 and its closely related member SOX10 share the same or distinct targets in mediating their functions in melanoma. Methods Immunofluorescence, TCGA database and qPCR were used to analyze the correlation between the expression patterns and levels of SOX9, SOX10 and NEDD9 in melanoma patient samples. AlamarBlue, transwell invasion and colony formation assays in melanoma cell lines were conducted to investigate the epistatic relationship between SOX10 and NEDD9, as well as the effects of graded SOX9 expression levels. Lung metastasis was determined by tail vein injection assay. Live cell imaging was conducted to monitor dynamics of melanoma migratory behavior. RHOA and RAC1 activation assays measured the activity of Rho GTPases. Results High SOX9 expression was predominantly detected in patients with distant melanoma metastases whereas SOX10 was present in the different stages of melanoma. Both SOX9 and SOX10 exhibited distinct but overlapping expression patterns with metastatic marker NEDD9. Accordingly, SOX10 was required for NEDD9 expression, which partly mediated its oncogenic functions in melanoma cells. Compensatory upregulation of SOX9 expression in SOX10-inhibited melanoma cells reduced growth and migratory capacity, partly due to elevated expression of cyclin-dependent kinase inhibitor p21 and lack of NEDD9 induction. Conversely, opposite phenomenon was observed when SOX9 expression was further elevated to a range of high SOX9 expression levels in metastatic melanoma specimens, and that high levels of SOX9 can restore melanoma progression in the absence of SOX10 both in vitro and in vivo. In addition, overexpression of SOX9 can also promote invasiveness of the parental melanoma cells by modulating the expression of various matrix metalloproteinases. SOX10 or high SOX9 expression regulates melanoma mesenchymal migration through the NEDD9-mediated focal adhesion dynamics and Rho GTPase signaling. Conclusions These results unravel NEDD9 as a common target for SOX10 or high SOX9 to partly mediate their oncogenic events, and most importantly, reconcile previous discrepancies that suboptimal level of SOX9 expression is anti-metastatic whereas high level of SOX9 is metastatic in a heterogeneous population of melanoma