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1.  Trends in incidence and mortality of nasopharyngeal carcinoma over a 20–25 year period (1978/1983–2002) in Sihui and Cangwu counties in southern China 
BMC Cancer  2006;6:178.
Background
Nasopharyngeal carcinoma (NPC) is a rare malignancy in most parts of the world but is common in southern China. A recent report from the Hong Kong Cancer Registry, a high-risk area for NPC in southern China, showed that incidence rate decreased by 29% for males and by 30% for females from 1980–1999, while mortality rate decreased by 43% for males and 50% for females. Changing environmental risk factors and improvements in diagnosis and treatment were speculated to be the major factors contributing to the downward trend of the incidence and mortality rates of NPC. To investigate the secular trends in different Cantonese populations with different socio-economic backgrounds and lifestyles, we report the incidences and mortality rates from two population-based cancer registries in Sihui and Cangwu counties from 1978–2002.
Methods
Incidence and mortality rates were aggregated by 5-year age groups and 5 calendar years. To adjust for the effect of difference in age composition for different periods, the total and age-specific rates of NPC incidence and mortality rate were adjusted by direct standardization according to the World Standard Population (1960). The Estimated Annual Percentage Change (EAPC) was used as an estimate of the trend.
Results
The incidence rate of NPC has remained stable during the recent two decades in Sihui and in females in Cangwu, with a slight increase observed in males in Cangwu from 17.81 to 19.76 per 100,000. The incidence rate in Sihui is 1.4–2.0 times higher during the corresponding years than in Cangwu, even though the residents of both areas are of Cantonese ethnicity. A progressive decline in mortality rate was observed in females only in Sihui, with an average reduction of 6.3% (p = 0.016) per five-year period.
Conclusion
To summarize, there is great potential to work in the area of NPC prevention and treatment in southern China to decrease NPC risk and improve survival risk rates in order to reduce M:I ratios. Future efforts on effective prevention, early detection and treatment strategies were also discussed in this paper. Furthermore, the data quality and completeness also need to be improved.
doi:10.1186/1471-2407-6-178
PMCID: PMC1557527  PMID: 16822324
2.  Urokinase-type plasminogen activator receptor signaling is critical in nasopharyngeal carcinoma cell growth and metastasis 
Cell Cycle  2014;13(12):1958-1969.
Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in southern China and Southeast Asia, with the highest metastasis rate among head and neck cancers. The mechanisms underlying NPC progression remain poorly understood. Genome-wide expression profiling on 18 NPC vs. 18 noncancerous nasopharyngeal tissues together with GeneGo pathway analysis and expression verification in NPC cells and tissues revealed a potential role of urokinase-type plasminogen activator receptor (uPAR) in NPC progression, which has not been investigated in NPC. We then observed that uPAR expression is increased in poorly differentiated, highly metastatic NPC cells compared with lowly metastatic cells or differentiated NPC cells. In vitro studies demonstrated that uPAR regulates NPC cell growth, colony formation, migration, and invasion and promotes the epithelial–mesenchymal transition (EMT). Additional tumor xenograft and spontaneous metastasis experiments revealed that uPAR promotes NPC cell growth and metastasis in vivo. The JAK–STAT pathway is involved in uPAR-regulated signaling in NPC cells as determined by immunoblotting. Moreover, uPAR-mediated growth and motility is partially abolished upon treatment with the Jak1/Jak2 inhibitor INCB018424. We suppressed uPA expression in uPAR-overexpressing NPC cells and found that uPAR-mediated cellular growth and motility is not exclusively dependent on uPA. In summary, uPAR is a significant regulator of NPC progression and could serve as a promising therapeutic target.
doi:10.4161/cc.28921
PMCID: PMC4111759  PMID: 24763226
uPAR; nasopharyngeal carcinoma; JAK; STAT; genome-wide expression profiling; tumor growth; metastasis
3.  Identification of new susceptibility loci for IgA nephropathy in Han Chinese 
Nature Communications  2015;6:7270.
IgA nephropathy (IgAN) is one of the most common primary glomerulonephritis. Previously identified genome-wide association study (GWAS) loci explain only a fraction of disease risk. To identify novel susceptibility loci in Han Chinese, we conduct a four-stage GWAS comprising 8,313 cases and 19,680 controls. Here, we show novel associations at ST6GAL1 on 3q27.3 (rs7634389, odds ratio (OR)=1.13, P=7.27 × 10−10), ACCS on 11p11.2 (rs2074038, OR=1.14, P=3.93 × 10−9) and ODF1-KLF10 on 8q22.3 (rs2033562, OR=1.13, P=1.41 × 10−9), validate a recently reported association at ITGAX-ITGAM on 16p11.2 (rs7190997, OR=1.22, P=2.26 × 10−19), and identify three independent signals within the DEFA locus (rs2738058, P=1.15 × 10−19; rs12716641, P=9.53 × 10−9; rs9314614, P=4.25 × 10−9, multivariate association). The risk variants on 3q27.3 and 11p11.2 show strong association with mRNA expression levels in blood cells while allele frequencies of the risk variants within ST6GAL1, ACCS and DEFA correlate with geographical variation in IgAN prevalence. Our findings expand our understanding on IgAN genetic susceptibility and provide novel biological insights into molecular mechanisms underlying IgAN.
IgA nephropathy is a major cause of end-stage renal disease in China, occurring at a high frequency in Asian populations. Here Li and colleagues conduct a four-stage genome-wide association study in a Chinese population, identifying novel loci and variants associated with disease risk.
doi:10.1038/ncomms8270
PMCID: PMC4458882  PMID: 26028593
4.  Low-density Lipoprotein Cholesterol was Inversely Associated with 3-Year All-Cause Mortality among Chinese Oldest Old: Data from the Chinese Longitudinal Healthy Longevity Survey 
Atherosclerosis  2015;239(1):137-142.
Objective
Low-density lipoprotein cholesterol (LDL-C) is a risk factor for survival in middle-aged individuals, but conflicting evidence exists on the relationship between LDL-C and all-cause mortality among the elderly. The goal of this study was to assess the relationship between LDL-C and all-cause mortality among Chinese oldest old (aged 80 and older) in a prospective cohort study.
Methods
LDL-C concentration was measured at baseline and all-cause mortality was calculated over a 3-year period. Multiple statistical models were used to adjust for demographic and biological covariates.
Results
During three years of follow-up, 447 of 935 participants died, and the overall all-cause mortality was 49.8%. Each 1 mmol/L increase of LDL-C concentration corresponded to a 19% decrease in 3-year all-cause mortality (hazard ratio [HR] 0.81, 95% confidence interval [CI] 0.71–0.92). The crude HR for abnormally higher LDL-C concentration (≥3.37 mmol/L) was 0.65 (0.41–1.03); and the adjusted HR was statistically significant around 0.60 (0.37–0.95) when adjusted for different sets of confounding factors. Results of sensitivity analysis also showed a significant association between higher LDL-C and lower mortality risk.
Conclusions
Among the Chinese oldest old, higher LDL-C level was associated with lower risk of all-cause mortality. Our findings suggested the necessity of re-evaluating the optimal level of LDL-C among the oldest old.
doi:10.1016/j.atherosclerosis.2015.01.002
PMCID: PMC4441211  PMID: 25602855
LDL-C; mortality; oldest old; epidemiology; China
5.  Pak2 regulates hematopoietic progenitor cell proliferation, survival and differentiation 
Stem cells (Dayton, Ohio)  2015;33(5):1630-1641.
p21-activated kinase 2 (Pak2), a serine/threonine kinase, has been previously shown to be essential for hematopoietic stem cell (HSC) engraftment. However, Pak2 modulation of long-term hematopoiesis and lineage commitment remain unreported. Utilizing a conditional Pak2 knock out (KO) mouse model, we found that disruption of Pak2 in HSCs induced profound leukopenia and a mild macrocytic anemia. Although loss of Pak2 in HSCs leads to less efficient short- and long-term competitive hematopoiesis than wild type (WT) cells, it does not affect HSC self-renewal per se. Pak2 disruption decreased the survival and proliferation of multi-cytokine stimulated immature progenitors. Loss of Pak2 skewed lineage differentiation toward granulocytopoiesis and monocytopoiesis in mice as evidenced by 1) a three to six-fold increase in the percentage of peripheral blood granulocytes and a significant increase in the percentage of granulocyte-monocyte progenitors (GMPs) in mice transplanted with Pak2-disrupted BM; 2) Pak2-disrupted BM and c-kit+ cells yielded higher numbers of more mature subsets of granulocyte-monocyte colonies and polymophonuclear neutrophils (PMNs), respectively, when cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF). Pak2 disruption resulted respectively in decreased and increased gene expression of transcription factors JunB and c-Myc, which may suggest underlying mechanisms by which Pak2 regulates granulocyte-monocyte lineage commitment. Furthermore, Pak2 disruption led to 1) higher percentage of CD4+CD8+ double positive T cells and lower percentages of CD4+CD8− or CD4−CD8+ single positive T cells in thymus and 2) decreased numbers of mature B cells and increased numbers of Pre-Pro B cells in BM, suggesting defects in lymphopoiesis.
doi:10.1002/stem.1951
PMCID: PMC4409559  PMID: 25586960
Pak2; hematopoietic progenitor cell; myelopoiesis; lymphopoiesis
6.  WNT5A promotes stemness characteristics in nasopharyngeal carcinoma cells leading to metastasis and tumorigenesis 
Oncotarget  2015;6(12):10239-10252.
Nasopharyngeal carcinoma (NPC) has the highest metastasis rate among head and neck cancers with unclear mechanism. WNT5A belongs to the WNT family of cysteine-rich secreted glycoproteins. Our previous high-throughput gene expression profiling revealed that WNT5A was up-regulated in highly metastatic cells. In the present study, we first confirmed the elevated expression of WNT5A in metastatic NPC tissues at both the mRNA and protein levels. We then found that WNT5A promoted epithelial-mesenchymal transition (EMT) in NPC cells, induced the accumulation of CD24-CD44+ cells and side population, which are believed to be cancer stem cell characteristics. Moreover, WNT5A promoted the migration and invasion of NPC cells in vitro, while in vivo treatment with recombinant WNT5A promoted lung metastasis. Knocking down WNT5A diminished NPC tumorigenesis in vivo. When elevated expression of WNT5A coincided with the elevated expression of vimentin in the primary NPC, the patients had a poorer prognosis. Among major signaling pathways, protein kinase C (PKC) signaling was activated by WNT5A in NPC cells. A positive feedback loop between WNT5A and phospho-PKC to promote EMT was also revealed. Taken together, these data suggest that WNT5A is an important molecule in promoting stem cell characteristics in NPC, leading to tumorigenesis and metastasis.
PMCID: PMC4496352  PMID: 25823923
nasopharyngeal carcinoma; WNT5A; metastasis; tumorigenesis; PKC
7.  Activating the Wnt/β-Catenin Pathway for the Treatment of Melanoma – Application of LY2090314, a Novel Selective Inhibitor of Glycogen Synthase Kinase-3 
PLoS ONE  2015;10(4):e0125028.
It has previously been observed that a loss of β-catenin expression occurs with melanoma progression and that nuclear β-catenin levels are inversely proportional to cellular proliferation, suggesting that activation of the Wnt/β-catenin pathway may provide benefit for melanoma patients. In order to further probe this concept we tested LY2090314, a potent and selective small-molecule inhibitor with activity against GSK3α and GSK3β isoforms. In a panel of melanoma cell lines, nM concentrations of LY2090314 stimulated TCF/LEF TOPFlash reporter activity, stabilized β-catenin and elevated the expression of Axin2, a Wnt responsive gene and marker of pathway activation. Cytotoxicity assays revealed that melanoma cell lines are very sensitive to LY2090314 in vitro (IC50 ~10nM after 72hr of treatment) in contrast to other solid tumor cell lines (IC50 >10uM) as evidenced by caspase activation and PARP cleavage. Cell lines harboring mutant B-RAF or N-RAS were equally sensitive to LY2090314 as were those with acquired resistance to the BRAF inhibitor Vemurafenib. shRNA studies demonstrated that β-catenin stabilization is required for apoptosis following treatment with the GSK3 inhibitor since the sensitivity of melanoma cell lines to LY290314 could be overcome by β-catenin knockdown. We further demonstrate that in vivo, LY2090314 elevates Axin2 gene expression after a single dose and produces tumor growth delay in A375 melanoma xenografts with repeat dosing. The activity of LY2090314 in preclinical models suggests that the role of Wnt activators for the treatment of melanoma should be further explored.
doi:10.1371/journal.pone.0125028
PMCID: PMC4411090  PMID: 25915038
8.  Evaluation of non-viral risk factors for nasopharyngeal carcinoma in a high-risk population of Southern China 
To understand the role of environmental and genetic influences on nasopharyngeal carcinoma (NPC) in populations at high risk of NPC, we have performed a case-control study in Guangxi Province of Southern China in 2004-2005. NPC cases (n=1049) were compared to 785 NPC-free matched controls who were seropositive for IgA antibodies (IgA) to Epstein-Barr virus (EBV) capsid antigen (VCA)—a predictive marker for NPC in Chinese populations. A questionnaire was used to capture exposure and NPC family history data. Risk factors associated with NPC in a multivariant analysis model were the following: 1) a first, second or third degree relative with NPC [Attributable risk (AR)= 6%, Odds ratio (OR) = 3.1, 95%CI = 2.0-4.9, p < 0.001]; 2) consumption of salted fish 3 or more than 3 times per month (AR=3%, OR = 1.9, 95%CI = 1.1-3.5, p = 0.035); 3) exposure to domestic wood cooking fires for more than 10 years (AR=69%, OR = 5.8, 95%CI = 2.5-13.6, p < 0.001); and 4) exposure to occupational solvents for 10 or less years (AR=4%, OR = 2.6, 95%CI = 1.4-4.8, p = 0.002). Consumption of preserved meats or a history of tobacco smoking were not associated with NPC (P>0.05). We also assessed the contribution of EBV/IgA/VCA antibody serostatus to NPC risk—32.2% of NPC can be explained by IgA+ status. However, family history and environmental risk factors cumulatively explained only 2.7% of NPC development in NPC high risk population. These findings should have important public health implications for NPC risk reduction in endemic regions.
doi:10.1002/ijc.24293
PMCID: PMC4406046  PMID: 19296536
Nasopharyngeal Carcinoma; Risk factor; Epidemiology; Southern China; Epstein Barr Virus
9.  Corrigendum: New loci and coding variants confer risk for age-related macular degeneration in East Asians 
Cheng, Ching-Yu | Yamashiro, Kenji | Jia Chen, Li | Ahn, Jeeyun | Huang, Lulin | Huang, Lvzhen | Cheung, Chui Ming G. | Miyake, Masahiro | Cackett, Peter D. | Yeo, Ian Y. | Laude, Augustinus | Mathur, Ranjana | Pang, Junxiong | Sim, Kar Seng | Koh, Adrian H. | Chen, Peng | Lee, Shu Yen | Wong, Doric | Chan, Choi Mun | Loh, Boon Kwang | Sun, Yaoyao | Davila, Sonia | Nakata, Isao | Nakanishi, Hideo | Akagi-Kurashige, Yumiko | Gotoh, Norimoto | Tsujikawa, Akitaka | Matsuda, Fumihiko | Mori, Keisuke | Yoneya, Shin | Sakurada, Yoichi | Iijima, Hiroyuki | Iida, Tomohiro | Honda, Shigeru | Lai, Timothy Yuk Yau | Tam, Pancy Oi Sin | Chen, Haoyu | Tang, Shibo | Ding, Xiaoyan | Wen, Feng | Lu, Fang | Zhang, Xiongze | Shi, Yi | Zhao, Peiquan | Zhao, Bowen | Sang, Jinghong | Gong, Bo | Dorajoo, Rajkumar | Yuan, Jian-Min | Koh, Woon-Puay | van Dam, Rob M. | Friedlander, Yechiel | Lin, Yin | Hibberd, Martin L. | Foo, Jia Nee | Wang, Ningli | Wong, Chang Hua | Tan, Gavin S. | Park, Sang Jun | Bhargava, Mayuri | Gopal, Lingam | Naing, Thet | Liao, Jiemin | Ong, Peng Guan | Mitchell, Paul | Zhou, Peng | Xie, Xuefeng | Liang, Jinlong | Mei, Junpu | Jin, Xin | Saw, Seang-Mei | Ozaki, Mineo | Mizoguchi, Takinori | Kurimoto, Yasuo | Woo, Se Joon | Chung, Hum | Yu, Hyeong-Gon | Shin, Joo Young | Park, Dong Ho | Kim, In Taek | Chang, Woohyok | Sagong, Min | Lee, Sang-Joon | Kim, Hyun Woong | Lee, Ji Eun | Li, Yi | Liu, Jianjun | Teo, Yik Ying | Heng, Chew Kiat | Lim, Tock Han | Yang, Suk-Kyun | Song, Kyuyoung | Vithana, Eranga N. | Aung, Tin | Bei, Jin Xin | Zeng, Yi Xin | Tai, E. Shyong | Li, Xiao Xin | Yang, Zhenglin | Park, Kyu-Hyung | Pang, Chi Pui | Yoshimura, Nagahisa | Wong, Tien Yin | Khor, Chiea Chuen
Nature Communications  2015;6:6817.
doi:10.1038/ncomms7817
PMCID: PMC4400603  PMID: 25817435
10.  A common variant near TGFBR3 is associated with primary open angle glaucoma 
Li, Zheng | Allingham, R. Rand | Nakano, Masakazu | Jia, Liyun | Chen, Yuhong | Ikeda, Yoko | Mani, Baskaran | Chen, Li-Jia | Kee, Changwon | Garway-Heath, David F. | Sripriya, Sarangapani | Fuse, Nobuo | Abu-Amero, Khaled K. | Huang, Chukai | Namburi, Prasanthi | Burdon, Kathryn | Perera, Shamira A. | Gharahkhani, Puya | Lin, Ying | Ueno, Morio | Ozaki, Mineo | Mizoguchi, Takanori | Krishnadas, Subbiah Ramasamy | Osman, Essam A. | Lee, Mei Chin | Chan, Anita S.Y. | Tajudin, Liza-Sharmini A. | Do, Tan | Goncalves, Aurelien | Reynier, Pascal | Zhang, Hong | Bourne, Rupert | Goh, David | Broadway, David | Husain, Rahat | Negi, Anil K. | Su, Daniel H | Ho, Ching-Lin | Blanco, Augusto Azuara | Leung, Christopher K.S. | Wong, Tina T. | Yakub, Azhany | Liu, Yutao | Nongpiur, Monisha E. | Han, Jong Chul | Hon, Do Nhu | Shantha, Balekudaru | Zhao, Bowen | Sang, Jinghong | Zhang, NiHong | Sato, Ryuichi | Yoshii, Kengo | Panda-Jonas, Songhomita | Ashley Koch, Allison E. | Herndon, Leon W. | Moroi, Sayoko E. | Challa, Pratap | Foo, Jia Nee | Bei, Jin-Xin | Zeng, Yi-Xin | Simmons, Cameron P. | Bich Chau, Tran Nguyen | Sharmila, Philomenadin Ferdinamarie | Chew, Merwyn | Lim, Blanche | Tam, Pansy O.S. | Chua, Elaine | Ng, Xiao Yu | Yong, Victor H.K. | Chong, Yaan Fun | Meah, Wee Yang | Vijayan, Saravanan | Seongsoo, Sohn | Xu, Wang | Teo, Yik Ying | Cooke Bailey, Jessica N. | Kang, Jae H. | Haines, Jonathan L. | Cheng, Ching Yu | Saw, Seang-Mei | Tai, E-Shyong | Richards, Julia E. | Ritch, Robert | Gaasterland, Douglas E. | Pasquale, Louis R. | Liu, Jianjun | Jonas, Jost B. | Milea, Dan | George, Ronnie | Al-Obeidan, Saleh A. | Mori, Kazuhiko | Macgregor, Stuart | Hewitt, Alex W. | Girkin, Christopher A. | Zhang, Mingzhi | Sundaresan, Periasamy | Vijaya, Lingam | Mackey, David A. | Wong, Tien Yin | Craig, Jamie E. | Sun, Xinghuai | Kinoshita, Shigeru | Wiggs, Janey L. | Khor, Chiea-Chuen | Yang, Zhenglin | Pang, Chi Pui | Wang, Ningli | Hauser, Michael A. | Tashiro, Kei | Aung, Tin | Vithana, Eranga N.
Human Molecular Genetics  2015;24(13):3880-3892.
Primary open angle glaucoma (POAG), a major cause of blindness worldwide, is a complex disease with a significant genetic contribution. We performed Exome Array (Illumina) analysis on 3504 POAG cases and 9746 controls with replication of the most significant findings in 9173 POAG cases and 26 780 controls across 18 collections of Asian, African and European descent. Apart from confirming strong evidence of association at CDKN2B-AS1 (rs2157719 [G], odds ratio [OR] = 0.71, P = 2.81 × 10−33), we observed one SNP showing significant association to POAG (CDC7–TGFBR3 rs1192415, ORG-allele = 1.13, Pmeta = 1.60 × 10−8). This particular SNP has previously been shown to be strongly associated with optic disc area and vertical cup-to-disc ratio, which are regarded as glaucoma-related quantitative traits. Our study now extends this by directly implicating it in POAG disease pathogenesis.
doi:10.1093/hmg/ddv128
PMCID: PMC4459396  PMID: 25861811
11.  Highly specific in vivo gene delivery for p53-mediated apoptosis and genetic photodynamic therapies of tumour 
Nature Communications  2015;6:6456.
Anticancer therapies are often compromised by nonspecific effects and challenged by tumour environments’ inherent physicochemical and biological characteristics. Often, therapeutic effect can be increased by addressing multiple parameters simultaneously. Here we report on exploiting extravasation due to inherent vascular leakiness for the delivery of a pH-sensitive polymer carrier. Tumours’ acidic microenvironment instigates a charge reversal that promotes cellular internalization where endosomes destabilize and gene delivery is achieved. We assess our carrier with an aggressive non-small cell lung carcinoma (NSCLC) in vivo model and achieve >30% transfection efficiency via systemic delivery. Rejuvenation of the p53 apoptotic pathway as well as expression of KillerRed protein for sensitization in photodynamic therapy (PDT) is accomplished. A single administration greatly suppresses tumour growth and extends median animal survival from 28 days in control subjects to 68 days. The carrier has capacity for multiple payloads for greater therapeutic response where inter-individual variability can compromise efficacy.
Alterations of p53 are associated with more than half of all human cancers. Here the authors present a new pH-sensitive nanoparticle that is delivered via systemic circulation and combines gene delivery to restore p53 with expression of Killerred protein to induce photosensitization.
doi:10.1038/ncomms7456
PMCID: PMC4366491  PMID: 25739372
12.  A Nomogram for Predicting the Benefit of Adjuvant Cytokine-Induced Killer Cell Immunotherapy in Patients with Hepatocellular Carcinoma 
Scientific Reports  2015;5:9202.
The benefits of adjuvant cytokine-induced killer (CIK) cell immunotherapy for hepatocellular carcinoma (HCC) remain mixed among patients. Here, we constructed a prognostic nomogram to enable individualized predictions of survival benefit of adjuvant CIK cell treatment for HCC patients. Survival analysis showed that the median overall survival (OS) and progression-free survival (PFS) for patients in the hepatectomy/CIK combination group were 41 and 16 months, respectively, compared to 28 and 12 months for patients in the hepatectomy alone group (control). Based on multivariate analysis of the entire cohort, independent factors for OS were tumor size, tumor capsule, pathological grades, total bilirubin, albumin, prothrombin time, alpha-fetoprotein, and tumor number, which were incorporated into the nomogram. The survival prediction model performed well, as assessed by the c-index and calibration curve. Internal validation revealed a c-index of 0.698, which was significantly greater than the c-index value of the TNM (tumor–node–metastasis) staging systems of 0.634. The calibration curves fitted well. In conclusions, our developed nomogram resulted in more accurate individualized predictions of the survival benefit from adjuvant CIK cell treatment after hepatectomy. The model may provide valuable information to aid in the decision making regarding the application of adjuvant CIK cell immunotherapy.
doi:10.1038/srep09202
PMCID: PMC4361845  PMID: 25776856
13.  Prevalence of human papillomavirus in esophageal carcinoma in Tangshan, China 
AIM: To study the prevalence of human papillomavirus (HPV) in esophageal carcinoma in Tangshan, China, a high-incidence area.
METHODS: Formalin-fixed, paraffin-embedded tissue specimens from 198 patients who were pathologically diagnosed with esophageal squamous cell carcinoma from 2011 to 2013 were obtained from a pathology department in Tangshan. DNA was extracted from all 198 specimens to detect HPV by polymerase chain reaction (PCR). β-globin PCR was performed to check the quality of the DNA extraction procedure. PCR was performed to detect a wide range of HPV types, and type-specific PCR was performed to detect HPV types 16 and 18. Negative and positive controls were used for HPV 16 and 18 detection.
RESULTS: The DNA extraction method in this study appeared to be more effective than other previously reported methods. After DNA extraction, more than 98% of the tissue specimens had an acceptable result in the DNA qualification test (β-globin PCR). The overall prevalence of HPV in tumor tissues by GP6+/GP5+ PCR was 79.79%, and the prevalence of HPV types 16 and 18 was 40.40% and 47.47%, respectively. PCR demonstrated the presence of HPV, and direct sequencing confirmed the HPV genotypes. All HPV-positive PCR products were checked by DNA sequence analysis using DNAman and compared with the known HPV sequences listed in the Basic Local Alignment Search Tool database to evaluate the HPV types. This analysis confirmed the presence of HPV types 16 and 18.
CONCLUSION: DNA of high-risk HPV types 16 and 18 is present in esophageal tumors, implicating HPV as a possible etiologic factor for esophageal squamous cell carcinoma.
doi:10.3748/wjg.v21.i10.2905
PMCID: PMC4356909  PMID: 25780287
Esophageal carcinoma; Formalin-fixed, paraffin-embedded tissue; Esophageal squamous cell carcinoma; Human papillomavirus; Polymerase chain reaction
14.  Lipopolysaccharide is Inserted into the Outer Membrane through An Intramembrane Hole, A Lumen Gate, and the Lateral Opening of LptD 
Structure(London, England:1993)  2015;23(3):496-504.
Summary
Lipopolysaccharide (LPS) is essential for the vitality of most Gram-negative bacteria and plays an important role in bacterial multidrug resistance. The LptD/E translocon inserts LPS into the outer leaflet, the mechanism of which is poorly understood. Here, we report mutagenesis, functional assays, and molecular dynamics simulations of the LptD/E complex, which suggest two distinct pathways for the insertion of LPS. The N-terminal domain of LptD comprises a hydrophobic slide that injects the acyl tails of LPS directly into the outer membrane through an intramembrane hole, while the core oligosaccharide and O-antigen pass a lumen gate that triggers the unzipping of the lateral opening between strands β1C and β26C of the barrel of LptD, to finalize LPS insertion. Mutation of the LPS transport related residues or block of the LPS transport pathways results in the deaths of Escherichia coli. These findings are important for the development of novel antibiotics.
Graphical Abstract
Highlights
•Hydrophobic residues at the N-terminal domain are essential for LPS transport•A hydrophobic intramembrane hole of LptD is critical for LPS insertion•A lumenal gate of LptD is important for translocation of LPS
Through molecular dynamics simulations, mutagenesis, and functional assays, Gu et al. reveal key residues of the N-terminal domain, a hydrophobic intramembrane hole, and a luminal gate of LptD for LPS transport, insertion, and translocation. These findings are significant not just for understanding the function of LptD, but also to develop novel antibiotics.
doi:10.1016/j.str.2015.01.001
PMCID: PMC4353691  PMID: 25684578
15.  Recurrent gain-of-function USP8 mutations in Cushing's disease 
Cell Research  2015;25(3):306-317.
Cushing's disease, also known as adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas (PAs) that cause excess cortisol production, accounts for up to 85% of corticotrophin-dependent Cushing's syndrome cases. However, the genetic alterations in this disease are unclear. Here, we performed whole-exome sequencing of DNA derived from 12 ACTH-secreting PAs and matched blood samples, which revealed three types of somatic mutations in a candidate gene, USP8 (encoding ubiquitin-specific protease 8), exclusively in exon 14 in 8 of 12 ACTH-secreting PAs. We further evaluated somatic USP8 mutations in additional 258 PAs by Sanger sequencing. Targeted sequencing further identified a total of 17 types of USP8 variants in 67 of 108 ACTH-secreting PAs (62.04%). However, none of these mutations was detected in other types of PAs (n = 150). These mutations aggregate within the 14-3-3 binding motif of USP8 and disrupt the interaction between USP8 and 14-3-3 protein, resulting in an elevated capacity to protect EGFR from lysosomal degradation. Accordingly, PAs with mutated USP8 display a higher incidence of EGFR expression, elevated EGFR protein abundance and mRNA expression levels of POMC, which encodes the precursor of ACTH. PAs with mutated USP8 are significantly smaller in size and have higher ACTH production than wild-type PAs. In surgically resected primary USP8-mutated tumor cells, USP8 knockdown or blocking EGFR effectively attenuates ACTH secretion. Taken together, somatic gain-of-function USP8 mutations are common and contribute to ACTH overproduction in Cushing's disease. Inhibition of USP8 or EGFR is promising for treating USP8-mutated corticotrophin adenoma. Our study highlights the potentially functional mutated gene in Cushing's disease and provides insights into the therapeutics of this disease.
doi:10.1038/cr.2015.20
PMCID: PMC4349249  PMID: 25675982
Cushing's disease; pituitary adenomas; USP8; mutation; whole-exome sequencing
16.  Neuropilin 1 is an entry factor that promotes EBV infection of nasopharyngeal epithelial cells 
Nature Communications  2015;6:6240.
Epstein–Barr virus (EBV) is implicated as an aetiological factor in B lymphomas and nasopharyngeal carcinoma. The mechanisms of cell-free EBV infection of nasopharyngeal epithelial cells remain elusive. EBV glycoprotein B (gB) is the critical fusion protein for infection of both B and epithelial cells, and determines EBV susceptibility of non-B cells. Here we show that neuropilin 1 (NRP1) directly interacts with EBV gB23–431. Either knockdown of NRP1 or pretreatment of EBV with soluble NRP1 suppresses EBV infection. Upregulation of NRP1 by overexpression or EGF treatment enhances EBV infection. However, NRP2, the homologue of NRP1, impairs EBV infection. EBV enters nasopharyngeal epithelial cells through NRP1-facilitated internalization and fusion, and through macropinocytosis and lipid raft-dependent endocytosis. NRP1 partially mediates EBV-activated EGFR/RAS/ERK signalling, and NRP1-dependent receptor tyrosine kinase (RTK) signalling promotes EBV infection. Taken together, NRP1 is identified as an EBV entry factor that cooperatively activates RTK signalling, which subsequently promotes EBV infection in nasopharyngeal epithelial cells.
Epstein–Barr virus (EBV) is involved in the development of some cancers including nasopharyngeal carcinoma. Here, the authors show that a direct interaction between the viral protein gB and a host protein, neuropilin 1, is required for EBV infection of nasopharyngeal epithelial cells.
doi:10.1038/ncomms7240
PMCID: PMC4339892  PMID: 25670642
17.  Genome-wide association analyses identify three new susceptibility loci for primary angle closure glaucoma 
Vithana, Eranga N | Khor, Chiea-Chuen | Qiao, Chunyan | Nongpiur, Monisha E | George, Ronnie | Chen, Li-Jia | Do, Tan | Abu-Amero, Khaled | Huang, Chor Kai | Low, Sancy | Tajudin, Liza-Sharmini A | Perera, Shamira A | Cheng, Ching-Yu | Xu, Liang | Jia, Hongyan | Ho, Ching-Lin | Sim, Kar Seng | Wu, Ren-Yi | Tham, Clement C Y | Chew, Paul T K | Su, Daniel H | Oen, Francis T | Sarangapani, Sripriya | Soumittra, Nagaswamy | Osman, Essam A | Wong, Hon-Tym | Tang, Guangxian | Fan, Sujie | Meng, Hailin | Huong, Dao T L | Wang, Hua | Feng, Bo | Baskaran, Mani | Shantha, Balekudaru | Ramprasad, Vedam L | Kumaramanickavel, Govindasamy | Iyengar, Sudha K | How, Alicia C | Lee, Kelvin Y | Sivakumaran, Theru A | Yong, Victor H K | Ting, Serena M L | Li, Yang | Wang, Ya-Xing | Tay, Wan-Ting | Sim, Xueling | Lavanya, Raghavan | Cornes, Belinda K | Zheng, Ying-Feng | Wong, Tina T | Loon, Seng-Chee | Yong, Vernon K Y | Waseem, Naushin | Yaakub, Azhany | Chia, Kee-Seng | Allingham, R Rand | Hauser, Michael A | Lam, Dennis S C | Hibberd, Martin L | Bhattacharya, Shomi S | Zhang, Mingzhi | Teo, Yik Ying | Tan, Donald T | Jonas, Jost B | Tai, E-Shyong | Saw, Seang-Mei | Hon, Do Nhu | Al-Obeidan, Saleh A | Liu, Jianjun | Chau, Tran Nguyen Bich | Simmons, Cameron P | Bei, Jin-Xin | Zeng, Yi-Xin | Foster, Paul J | Vijaya, Lingam | Wong, Tien-Yin | Pang, Chi-Pui | Wang, Ningli | Aung, Tin
Nature genetics  2012;44(10):1142-1146.
Primary angle closure glaucoma (PACG) is a major cause of blindness worldwide. We conducted a genome-wide association study including 1,854 PACG cases and 9,608 controls across 5 sample collections in Asia. Replication experiments were conducted in 1,917 PACG cases and 8,943 controls collected from a further 6 sample collections. We report significant associations at three new loci: rs11024102 in PLEKHA7 (per-allele odds ratio (OR) = 1.22; P = 5.33 × 10−12), rs3753841 in COL11A1 (per-allele OR = 1.20; P = 9.22 × 10−10) and rs1015213 located between PCMTD1 and ST18 on chromosome 8q (per-allele OR = 1.50; P = 3.29 × 10−9). Our findings, accumulated across these independent worldwide collections, suggest possible mechanisms explaining the pathogenesis of PACG.
doi:10.1038/ng.2390
PMCID: PMC4333205  PMID: 22922875
18.  Systemic Delivery of MicroRNA-101 Potently Inhibits Hepatocellular Carcinoma In Vivo by Repressing Multiple Targets 
PLoS Genetics  2015;11(2):e1004873.
Targeted therapy based on adjustment of microRNA (miRNA)s activity takes great promise due to the ability of these small RNAs to modulate cellular behavior. However, the efficacy of miR-101 replacement therapy to hepatocellular carcinoma (HCC) remains unclear. In the current study, we first observed that plasma levels of miR-101 were significantly lower in distant metastatic HCC patients than in HCCs without distant metastasis, and down-regulation of plasma miR-101 predicted a worse disease-free survival (DFS, P<0.05). In an animal model of HCC, we demonstrated that systemic delivery of lentivirus-mediated miR-101 abrogated HCC growth in the liver, intrahepatic metastasis and distant metastasis to the lung and to the mediastinum, resulting in a dramatic suppression of HCC development and metastasis in mice without toxicity and extending life expectancy. Furthermore, enforced overexpression of miR-101 in HCC cells not only decreased EZH2, COX2 and STMN1, but also directly down-regulated a novel target ROCK2, inhibited Rho/Rac GTPase activation, and blocked HCC cells epithelial-mesenchymal transition (EMT) and angiogenesis, inducing a strong abrogation of HCC tumorigenesis and aggressiveness both in vitro and in vivo. These results provide proof-of-concept support for systemic delivery of lentivirus-mediated miR-101 as a powerful anti-HCC therapeutic modality by repressing multiple molecular targets.
Author Summary
Human hepatocellular carcinoma (HCC) is one of the most common malignancy worldwide and among the leading causes of cancer-related death. HCC is often diagnosed at an advanced stage and there is still no effective therapeutic strategy for non-resectable HCCs. It has been suggested that the therapeutic delivery of certain miRNA(s) has a unique advantage in clinical use. We first find that the plasma levels of miR-101 are significantly down-regulated in HCC patients with distant metastasis and associated closely with HCCs progression and/or worse disease-free survival (DFS). Next, we identify that systemic delivery of lentivirus-mediated miR-101 in an orthotopic liver implanted HCC model of mouse, not only suppresses tumor xenograft growth in the liver, but also substantially blocks intrahepatic metastasis and distant metastasis to the lung and to the mediastinum, resulting in a dramatic abrogation of HCC tumorigenesis and progression in mice without toxicity. Furthermore, functional and/or mechanistic studies of miR-101 demonstrate that miR-101 in HCC cells inhibits Rho/Rac GTPase activation, and blocks HCC cells epithelial-mesenchymal transition (EMT) and angiogenesis, inducing a strong abrogation of HCC tumorigenesis and aggressiveness both in vitro and in vivo.
doi:10.1371/journal.pgen.1004873
PMCID: PMC4334495  PMID: 25693145
19.  Disrupting the Interaction of BRD4 with Di-acetylated Twist Suppresses Tumorigenesis in Basal-like Breast Cancer 
Cancer cell  2014;25(2):210-225.
Summary
Twist is a key transcription activator of epithelial-mesenchymal transition (EMT). It remains unclear how Twist induces gene expression. Here we reported a mechanism by which Twist recruits BRD4 to direct WNT5A expression in basal-like breast cancer (BLBC). Twist contains a “histone H4 mimic” GK-X-GK motif that is di-acetylated by Tip60. The di-acetylated Twist binds the second bromodomain of BRD4, whose first bromodomain interacts with acetylated H4, thereby constructs an activated Twist/BRD4/P-TEFb/RNA-PolII complex at the WNT5A promoter and enhancer. Pharmacologic inhibition of the Twist-BRD4 association reduced WNT5A expression and suppressed invasion, cancer stem cell (CSC)-like properties, and tumorigenicity of BLBC cells. Our study indicates that the interaction with BRD4 is critical for the oncogenic function of Twist in BLBC.
doi:10.1016/j.ccr.2014.01.028
PMCID: PMC4004960  PMID: 24525235
20.  New loci and coding variants confer risk for age-related macular degeneration in East Asians 
Cheng, Ching-Yu | Yamashiro, Kenji | Jia Chen, Li | Ahn, Jeeyun | Huang, Lulin | Huang, Lvzhen | Cheung, Chui Ming G. | Miyake, Masahiro | Cackett, Peter D. | Yeo, Ian Y. | Laude, Augustinus | Mathur, Ranjana | Pang, Junxiong | Sim, Kar Seng | Koh, Adrian H. | Chen, Peng | Lee, Shu Yen | Wong, Doric | Chan, Choi Mun | Loh, Boon Kwang | Sun, Yaoyao | Davila, Sonia | Nakata, Isao | Nakanishi, Hideo | Akagi-Kurashige, Yumiko | Gotoh, Norimoto | Tsujikawa, Akitaka | Matsuda, Fumihiko | Mori, Keisuke | Yoneya, Shin | Sakurada, Yoichi | Iijima, Hiroyuki | Iida, Tomohiro | Honda, Shigeru | Lai, Timothy Yuk Yau | Tam, Pancy Oi Sin | Chen, Haoyu | Tang, Shibo | Ding, Xiaoyan | Wen, Feng | Lu, Fang | Zhang, Xiongze | Shi, Yi | Zhao, Peiquan | Zhao, Bowen | Sang, Jinghong | Gong, Bo | Dorajoo, Rajkumar | Yuan, Jian-Min | Koh, Woon-Puay | van Dam, Rob M. | Friedlander, Yechiel | Lin, Ying | Hibberd, Martin L. | Foo, Jia Nee | Wang, Ningli | Wong, Chang Hua | Tan, Gavin S. | Park, Sang Jun | Bhargava, Mayuri | Gopal, Lingam | Naing, Thet | Liao, Jiemin | Guan Ong, Peng | Mitchell, Paul | Zhou, Peng | Xie, Xuefeng | Liang, Jinlong | Mei, Junpu | Jin, Xin | Saw, Seang-Mei | Ozaki, Mineo | Mizoguchi, Takanori | Kurimoto, Yasuo | Woo, Se Joon | Chung, Hum | Yu, Hyeong-Gon | Shin, Joo Young | Park, Dong Ho | Kim, In Taek | Chang, Woohyok | Sagong, Min | Lee, Sang-Joon | Kim, Hyun Woong | Lee, Ji Eun | Li, Yi | Liu, Jianjun | Teo, Yik Ying | Heng, Chew Kiat | Lim, Tock Han | Yang, Suk-Kyun | Song, Kyuyoung | Vithana, Eranga N. | Aung, Tin | Bei, Jin Xin | Zeng, Yi Xin | Tai, E. Shyong | Li, Xiao Xin | Yang, Zhenglin | Park, Kyu-Hyung | Pang, Chi Pui | Yoshimura, Nagahisa | Yin Wong, Tien | Khor, Chiea Chuen
Nature Communications  2015;6:6063.
Age-related macular degeneration (AMD) is a major cause of blindness, but presents differently in Europeans and Asians. Here, we perform a genome-wide and exome-wide association study on 2,119 patients with exudative AMD and 5,691 controls, with independent replication in 4,226 patients and 10,289 controls, all of East Asian descent, as part of The Genetics of AMD in Asians (GAMA) Consortium. We find a strong association between CETP Asp442Gly (rs2303790), an East Asian-specific mutation, and increased risk of AMD (odds ratio (OR)=1.70, P=5.60 × 10−22). The AMD risk allele (442Gly), known to protect from coronary heart disease, increases HDL cholesterol levels by 0.17 mmol l−1 (P=5.82 × 10−21) in East Asians (n=7,102). We also identify three novel AMD loci: C6orf223 Ala231Ala (OR=0.78, P=6.19 × 10−18), SLC44A4 Asp47Val (OR=1.27, P=1.08 × 10−11) and FGD6 Gln257Arg (OR=0.87, P=2.85 × 10−8). Our findings suggest that some of the genetic loci conferring AMD susceptibility in East Asians are shared with Europeans, yet AMD in East Asians may also have a distinct genetic signature.
Age-related macular degeneration (AMD) is a major cause of blindness worldwide. Here, the authors carry out a two-stage genome-wide association study for AMD and identify three new AMD risk loci, highlighting the shared and distinct genetic basis of the disease in East Asians and Europeans.
doi:10.1038/ncomms7063
PMCID: PMC4317498  PMID: 25629512
21.  A Comparison between the Sixth and Seventh Editions of the UICC/AJCC Staging System for Nasopharyngeal Carcinoma in a Chinese Cohort 
PLoS ONE  2014;9(12):e116261.
Background
The International Union Against Cancer/American Joint Committee on Cancer (UICC/AJCC) TNM staging system of nasopharyngeal carcinoma (NPC) is the most important system for survival prediction. The TNM 7th edition UICC/AJCC TNM staging system for NPC was adopted in January 2009, and is now internationally recommended. In comparison with the TNM 6th edition, there were several revisions in the new edition staging system. This study aims to evaluate the prognostic value of the TNM 7th edition for NPC patients in comparison with the TNM 6th edition.
Method
Clinical data of 2,629 NPC patients from the Sun Yat-sen University Cancer Center between January 2006 and December 2010 were retrospectively collected and all the patients were restaged according to the criteria of the TNM 6th edition and TNM 7th edition UICC/AJCC staging manual. Univariate and multivariate COX proportional hazards analyses were applied to evaluate the prognostic values between adjacent stage categories of the TNM 6th edition and TNM 7th edition.
Results
In comparison with the TNM 6th edition, a significant alteration of the distribution of N categories was observed when the TNM 7th edition was applied (χ2 = 20.589, P<0.001), with 119 (119/670, 17.8%) patients up-staging from N0 to N1. With regard to T and overall stage, 37 (37/561, 6.6%) patients were down-staged from T2a with the TNM 6th edition to T1 with the TNM 7th edition, and finally two patients were up-staged to overall stage II (2/118, 1.7%). Moreover, the survival curves were significantly segregated (P<0.05) between T1 and T2 as well as N1 and N2 with the TNM 7th edition.
Conclusions
The TNM 7th edition led to a significant alteration in the distribution of N categories and it is superior to the TNM 6th edition in predicting the frequency of overall survival and distant metastasis-free survival.
doi:10.1371/journal.pone.0116261
PMCID: PMC4275293  PMID: 25536307
22.  Large-scale genetic study in East Asians identifies six new loci associated with colorectal cancer risk 
Nature genetics  2014;46(6):533-542.
Known genetic loci explain only a small proportion of the familial relative risk of colorectal cancer (CRC). We conducted the largest genome-wide association study in East Asians with 14,963 CRC cases and 31,945 controls and identified six new loci associated with CRC risk (P = 3.42 × 10−8 to 9.22 × 10−21) at 10q22.3, 10q25.2, 11q12.2, 12p13.31, 17p13.3 and 19q13.2. Two of these loci map to genes (TCF7L2 and TGFB1) with established roles in colorectal tumorigenesis. Four other loci are located in or near genes involved in transcription regulation (ZMIZ1), genome maintenance (FEN1), fatty acid metabolism (FADS1 and FADS2), cancer cell motility and metastasis (CD9) and cell growth and differentiation (NXN). We also found suggestive evidence for three additional loci associated with CRC risk near genome-wide significance at 8q24.11, 10q21.1 and 10q24.2. Furthermore, we replicated 22 previously reported CRC loci. Our study provides insights into the genetic basis of CRC and suggests new biological pathways.
doi:10.1038/ng.2985
PMCID: PMC4068797  PMID: 24836286
23.  The association of polymorphisms of TLR4 and CD14 genes with susceptibility to sepsis in a Chinese population 
BMC Medical Genetics  2014;15:123.
Background
Sepsis is now the leading cause of death in the non-cardiovascular intensive care unit (ICU). Recent research suggests that sepsis is likely to be due to an interaction between genetic and environmental factors. Genetic mutations of toll-like receptor 4 (TLR4) and cluster of differentiation 14 (CD14) genes are involved in the immune and (or) inflammatory response. These may contribute to the susceptibility to sepsis in patients. This study was designed to evaluate whether the TLR4 and cluster CD14 gene polymorphisms are associated with susceptibility to sepsis.
Methods
The single nucleotide polymorphisms (SNPs) of TLR4 (rs10759932, rs11536889, rs7873784, rs12377632, rs1927907, rs1153879) and CD14 (rs2569190 and rs2563298) in patients with sepsis and control subjects in the Guangxi Province were analyzed by using the polymerase chain reaction-single base extension (PCR-SBE) and DNA sequencing methods.
Results
The rs11536889 polymorphism in TLR4 and rs2563298 polymorphism in CD14 were significantly associated with the risk of sepsis when compared to the control group. The frequencies of rs11536889 and rs2563298 polymorphisms in the group with sepsis were higher than that in the control group (OR = 1.430, 95% CI, 1.032-1.981, P<0.05; OR = 2.454, 95% CI, 1.458-4.130, P<0.05, respectively). Followed up haplotype analysis suggested that there were two haplotypes in which increased risk factors for sepsis were indicated.
Conclusions
The rs11536889 polymorphism in TLR4 and rs2563298 polymorphism in CD14, and two haplotypes were associated with increased susceptibility to sepsis.
doi:10.1186/s12881-014-0123-4
PMCID: PMC4411696  PMID: 25394369
Sepsis; Single nucleotide polymorphism; TLR4; CD14
24.  Tea Consumption and Mortality Among Oldest-Old Chinese 
Journal of the American Geriatrics Society  2013;61(11):10.1111/jgs.12498.
Objectives
To investigate the association between tea consumption and mortality among oldest-old Chinese.
Design
Population-based longitudinal data from The Chinese Longitudinal Healthy Longevity Survey (CLHLS) was analyzed using Cox semi-parametric proportional hazard model.
Setting
631 randomly selected counties and cities of China’s 22 provinces.
Participants
9,093 old adults aged 80 and above who provided complete data at baseline survey (year 1998).
Measurements
Self-reported current frequency of tea drinking and past frequency around age 60 were ascertained at baseline survey, and follow-up survey was conducted respectively in years 2000, 2002 and 2005.
Results
Among oldest-old Chinese, tea consumption was associated with reduced risk of mortality after adjusting for demographic characteristics, socioeconomic status, health practices, and health status. Compared with non-tea drinkers, the adjusted hazard ratio (HR) was 0.90 (95% CI 0.84–0.96) for daily tea drinkers (at the baseline survey, 1998) and 1.00 (95% CI 1.01–1.07) for occasional tea drinkers respectively (P for linear trend=0.003). Similar results were found when tea drinking status around age 60 was used in analysis. Further analysis showed that compared to consistently infrequent tea drinkers, subjects who reported frequent tea drinking at both age 60 and at baseline survey had a 10% reduction in mortality (HR=0.90, 95%CI 0.84–0.97).
Conclusion
Tea consumption is associated reduced risk of mortality among oldest-old Chinese.
doi:10.1111/jgs.12498
PMCID: PMC3830687  PMID: 24117374
Tea; Oldest-old Chinese; mortality; longitudinal study
25.  Inhibition of Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication by HIV-1 Nef and Cellular MicroRNA hsa-miR-1258 
Journal of Virology  2014;88(9):4987-5000.
ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to several AIDS-related malignancies, including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. The interaction of human immunodeficiency virus type 1 (HIV-1) and KSHV has a central role in promoting the aggressive manifestations of AIDS-KS. We have previously shown that negative factor (Nef), a secreted HIV-1 protein, synergizes with KSHV viral interleukin-6 (vIL-6) to promote angiogenesis and tumorigenesis by activating the AKT pathway (X. Zhu, et al., Oncogene, 22 April 2013, http://dx.doi.org/10.1038/onc.2013.136). Here, we further demonstrated the role of soluble and ectopic Nef in the regulation of KSHV latency. We found that both soluble Nef protein and ectopic expression of Nef by transfection suppressed the expression of KSHV viral lytic mRNA transcripts and proteins and the production of infectious viral particles. MicroRNA (miRNA) microarray analysis identified a number of Nef-regulated miRNAs. Bioinformatics and luciferase reporter analyses showed that one of the Nef-upregulated miRNAs, cellular miRNA 1258 (hsa-miR-1258), directly targeted a seed sequence in the 3′ untranslated region (UTR) of the mRNA encoding the major lytic switch protein (RTA), which controls KSHV reactivation from latency. Ectopic expression of hsa-miR-1258 impaired RTA synthesis and enhanced Nef-mediated inhibition of KSHV replication, whereas repression of hsa-miR-1258 has the opposite effect. Mutation of the seed sequence in the RTA 3′UTR abolished downregulation of RTA by hsa-miR-1258. Collectively, these novel findings demonstrate that, by regulating cellular miRNA, Nef may inhibit KSHV replication to promote viral latency and contribute to the pathogenesis of AIDS-related malignancies.
IMPORTANCE This study found that Nef, a secreted HIV-1 protein, suppressed KSHV lytic replication to promote KSHV latency. Mechanistic studies indicated that a Nef-upregulated cellular miRNA, hsa-miR-1258, inhibits KSHV replication by directly targeting a seed sequence in the KSHV RTA 3′UTR. These results illustrate that, in addition to viral miRNAs, cellular miRNAs also play an important role in regulating the life cycle of KSHV. Overall, this is the first study to report the involvement of Nef in KSHV latency, implying its likely important role in the pathogenesis of AIDS-related malignancies.
doi:10.1128/JVI.00025-14
PMCID: PMC3993842  PMID: 24554664

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