FLJ10540 is associated with tumor progression in nasopharyngeal carcinomas and contributes to nasopharyngeal cell proliferation, and metastasis via osteopontin/CD44 pathway

BACKGROUND: Nasopharyngeal carcinoma (NPC) is well-known for its highly metastatic characteristics, but little is known of its molecular mechanisms. New biomarkers that predict clinical outcome, in particular the ability of the primary tumor to develop metastatic tumors are urgently needed. The aim of this study is to investigate the role of FLJ10540 in human NPC development. METHODS: A bioinformatics approach was used to explore the potentially important regulatory genes involved in the growth/metastasis control of NPC. FLJ10540 was chosen for this study. Two co-expression strategies from NPC microarray were employed to identify the relationship between FLJ10540 and osteopontin. Quantitative-RT-PCR, immunoblotting, and immunohistochemistry analysis were used to investigate the mRNA and protein expression profiles of FLJ10540 and osteopontin in the normal and NPC tissues to confirm microarray results. TW01 and Hone1 NPC cells with overexpression FLJ10540 or siRNA to repress endogenous FLJ10540 were generated by stable transfection to further elucidate the molecular mechanisms of FLJ10540-elicited cell growth and metastasis under osteopontin stimulation. RESULTS: We found that osteopontin expression exhibited a positive correlation with FLJ10540 in NPC microarray. We also demonstrated comprehensively that FLJ10540 and osteopontin were not only overexpressed in NPC specimens, but also significantly correlated with advanced tumor and lymph node-metastasis stages, and had a poor 5-year survival rate, respectively. Stimulation of NPC parental cells with osteopontin results in an increase in FLJ10540 mRNA and protein expressions. Functionally, FLJ10540 transfectant alone, or stimulated with osteopontin, exhibited fast growth and increased metastasis as compared to vehicle control with or without osteopontin stimulation. Conversely, knockdown of FLJ10540 by siRNA results in the suppression of NPC cell growth and motility. Treatment with anti-CD44 antibodies in NPC parental cells not only resulted in a decrease of FLJ10540 protein, but also affected the abilities of FLJ10540-elicited cell growth and motility in osteopontin stimulated-NPC cells. CONCLUSIONS: These findings suggest that FLJ10540 may be critical regulator of disease progression in NPC, and the underlying mechanism may involve in the osteopontin/CD44 pathway

Comparing factors affecting commencement and cessation of betel quid chewing behavior in Taiwanese adults

<p>Abstract</p> <p>Background</p> <p>Betel quid is the fourth most common used substance in the world after tobacco, alcohol and caffeine. Although factors related to betel quid chewing or cessation of behaviors were reported previously, few studies simultaneously compared both behaviors in the same population. In addition, it is essential to consider time-to-event concept, since the chance of developing or stopping habit may vary over time. The purpose of this study was to compare the risk factors for commencement and cessation of betel quid chewing behaviors in a time-to-event setting.</p> <p>Methods</p> <p>A stratified multi-stage cluster sampling with selection probabilities proportional to size (PPS) was designed for Taiwanese adults with aged 18 years old and above. Kaplan-Meier estimates and Cox proportional hazard regression models were used to compare and calculate the hazard rate ratios for related factors to commencement or cessation of chewing habits.</p> <p>Results</p> <p>In Taiwan, men had a higher betel quid chewing rate (M: 20.9%, W: 1.2%), but woman chewers had a lower cessation rate (M: 27.5%, W: 12.7%). The hazard rate ratio (HRR) of having chewing habit changed from 4.22 (men vs women) univariately to 1.38 multivariablely, which indicated gender differences were confounded by other factors. In multivariable analysis, the risk factors of gender, education and ethnicity were significantly associated with both starting and cessation of betel quid chewing behavior. The factors of occupation, cigarette smoking and alcohol drinking were only associated with starting habit.</p> <p>Conclusion</p> <p>Commencement or cessation of chewing behavior involves a scenario of time, hence it is preferable to use a time-to-event approach for the comparison. The cessation rates of betel quid chewing were decreasingly associated with the daily consumption of betel quid. Hence, reducing of daily amount in betel quid cessation program may be associated with future stopping habit.</p

Studies on the Interfacial Reactions and Whisker Growth in Ce-Doped Sn3Ag0.5Cu Pb-free Solder Ball Grid Array Package

近年來許多相關研究報導在無鉛銲錫添加微量稀土元素可改善其潤濕性與機械性質，中國大陸有意將其納入China ROHS規範，然而在運用到真實的接點時，還有許多效應仍待進一步確認，以評估此無鉛銲錫的取代性。因此本論文使用Sn3Ag0.5Cu-XCe(X=0.1,0.25, 0.5,1.0wt%)無鉛銲錫之球格陣列構裝接點作為研究，第一部份探討添加不同含量稀土元素添加對銲錫熔點、界面介金屬反應及高溫時效後推球強度之影響，第二部份為錫鬚成長特性研究，以更進一步建立此稀土添加之無鉛銲錫的錫鬚成長機構。 研究結果顯示，添加稀土元素Ce可以減緩Ag3Sn顆粒粗大化並抑制時效後推球強度下降，有效改善此銲錫的機械性質，其中以0.25 wt%添加效果最好。在固固反應中，稀土元素添加提高了Cu6Sn5與Cu3Sn成長活化能，使Cu6Sn5與Cu3Sn的成長受溫度影響較大。在添加稀土元素後，錫球內部可觀察到CeSn3介金屬，CeSn3氧化後可觀察到錫鬚形成，導致此種無鉛銲錫在使用上將因可靠度而受到限制。 本研究中的錫鬚型態可分為細長狀(fiber)、柱狀(column)、粗短花狀(flower-cluster)與山丘狀(hillock)，其中細長狀錫鬚中又分為直徑較細的TypeI錫鬚(0.1-0.5μm)與較粗的TypeII錫鬚(1μm)兩種。而提高相對濕度會使錫鬚型態由細長狀轉變成短花狀，提高溫度轉變成山丘狀，提高溫度也會使細長狀錫鬚數目減少、成長速度變快、直徑變大與平均長度變短，而低溫度5℃下與真空10-2torr下則可抑止CeSn3氧化與錫鬚生成。本研究亦藉由橫截面與氧化性質歸納出此稀土添加之無鉛銲錫的錫鬚成長機構為CeSn3氧化導致體積膨脹與外來氧原子擴散，使周圍銲錫基地與CeSn3區域承受壓應力所致。Recently, the addition of a trace amount of rare earth elements is reportedly able to considerably improve the wetting and mechanical properties of mast of these Pb-free solder, related researches have just gotten underway is very recent years and China government are willing to arrange this Ce-doped solder into China ROHS. However, to apply this Ce-doped solder into a real package production, a lot of these efforts are still to be clarified through further experiments so that this Ce-doped solder will be confirmed as the Pb-free solder replacer. In the first part of this study is concerned with the Sn3Ag0.5Cu-XCe(X=0.1, 0.25, 0.5, 1.0wt%) Pb-free solder joints in Ball Grid Array (BGA) Package, while focusing on the melting point, interfacial intermetallic reactions and shear test after high temperature aging. The second part of this study is to clarify the mechanism of tin whisker formation through the whisker growth properties. From the experimental results, it is evidenced that the Ce-dope is effectively to slow down the coarsening of Ag3Sn precipitates in the solder matrix and the degradation of bonding strengths after high temperature aging. For the solid interfacial reactions, Ce-dope increases the growth activation energy of Cu6Sn5 and Cu3Sn, which implies that the growth reaction of Cu6Sn5 and Cu3Sn-intermetallics is much more sensitive to the aging temperature. In the microstructure of solder matrix, Ce-dope results in the formation of precipitated CeSn3 clusters in the reflowed solder matrix and the oxidation of CeSn3 clusters makes the tin whisker grow. However, this Ce-doped solder will be restricted by the reliable issue of tin whisker growth. In this study, the morphology of whiskers observed can be summarized as fiber, column, flower-cluster and hillock. Different diameter fiber whiskers are Types I and II, which can coexist on the same CeSn3 oxide layer. In addition, the morphology of whiskers will transfer from fiber into flower-cluster whisker with increasing related humidity and into hillock whisker with increasing temperature. The growth rate and diameter of fiber whisker will be increased by increasing temperature, but the average length decreased. Lower temperature at 5℃ and 10-2 torr vacuum can inhibit the whisker formation. Through the relationship between cross section observation and oxidation behavior at various temperatures, the mechanism of abnormal tin whisker formation in this Ce-doped solder can be attributed to the compressive stress induced by the diffusion of oxygen into the CeSn3 precipitate clusters in the solder, which squeezes the Sn atoms in the Ce-depleted region of CeSn3 phase out of the oxide layer.目錄 ......Ⅰ 圖目錄 ......Ⅳ 表目錄 ......Ⅹ 壹、研究動機 ......1 貳、理論基礎 ......4 2.1 電子構裝之演進與球格陣列構裝 ......4 2.2 無鉛銲錫之發展 ......6 2.2.1 銲錫無鉛化b ......6 2.2.2無鉛銲錫添加第四元元素之研究 ......7 2.2.2.1 添加Ni元素之影響 ......7 2.2.2.2 添加Sb元素之影響 ......8 2.2.2.3 添加Bi元素之影響 ......9 2.2.2.4 其添加它元素之影響 ......11 2.2.3無鉛銲錫添加稀土元素之研究 ......13 2.2.3.1添加稀土元素對銲錫物理特性之影響 ......13 2.2.3.2添加稀土元素對介金屬成長之影響 ......14 2.2.3.3添加稀土元素對銲錫機械性質之影響 ......16 2.2.3.4 高含量稀土的添加 ......17 2.3 界面反應理論 ......18 2.4 錫鬚理論 ......19 2.4.1 錫鬚發展 ......19 2.4.2 錫鬚之成長機構 ......20 2.4.2.1 差排理論 ......21 2.4.2.2 再結晶理論 ......22 2.4.2.3 壓應力理論 ......23 2.4.2.3.1 界面擴散與反應引起之應力 ......24 2.4.2.3.2 錫層與基材熱膨脹係數不匹配所引起之應力 ......28 2.4.2.3.3 電遷移導致原子擴散所產生之壓應力 ......30 2.4.2.4 氧化層理論 ......30 2.4.3錫鬚測試規範 ......32 参、實驗方法 ......39 3.1 銲錫之配製與分別 ......39 3.2 球格陣列構裝之界面反應與推球強度 ......39 3.3 錫鬚成長觀察 ......41 肆、結果與討論 ......45 4.1 Sn-3Ag-0.5Cu-XCe (X=0、0.1、0.25、0.5、1.0)銲錫球格陣列構裝研究 ......45 4.1.1 Sn-3Ag-0.5Cu-XCe銲錫特性 ......45 4.1.1.1 Sn-3Ag-0.5Cu-XCe銲錫熔點 ......45 4.1.1.2 Sn-3Ag-0.5Cu-XCe銲錫金相組織 ......45 4.1.2 Sn-3Ag-0.5Cu-XCe銲錫球格陣列化銀基板構裝研究 ......46 4.1.2.1 Sn-3Ag-0.5Cu-XCe銲球接點之界面反應 ......46 4.1.2.2 Sn-3Ag-0.5Cu-XCe銲球接點界面反應之動力學分析 ......49 4.1.2.3 Sn-3Ag-0.5Cu-XCe銲球接點之推球強度 ......50 4.2 Sn-3Ag-0.5Cu稀土元素添加銲錫之錫鬚成長研究 ......52 4.2.1 室溫下錫鬚成長 ......52 4.2.1.1 Sn-3Ag-0.5Cu-0.5Ce銲錫經迴銲後之室溫下錫鬚成長 ......52 4.2.1.2 Sn-3Ag-0.5Cu-X(=0.1、0.25、1.0)Ce銲錫經迴銲後之室溫下錫鬚成長 ......56 4.2.1.3 Sn6.6X(=La、Lu、M)銲錫經迴銲後之室溫下錫鬚成長 ......57 4.2.1.4 Sn-3Ag-0.5Cu-0.5Ce銲錫經時效後之室溫下錫鬚成長 ......58 4.2.1.5 Sn-3Ag-0.5Cu-0.5Ce銲錫球於化金基板在室溫下錫鬚成長 ......58 4.2.2 不同溫度下錫鬚成長 ......60 4.2.2.1 Sn-3Ag-0.5Cu-0.5Ce銲錫經迴銲後之高溫50℃下錫鬚成長 ......60 4.2.2.2 Sn-3Ag-0.5Cu-0.5Ce銲錫經迴銲後之高溫100℃下錫鬚成長......62 4.2.2.3 Sn-3Ag-0.5Cu-0.5Ce銲錫經迴銲後之高溫150℃下錫鬚成長 ......63 4.2.2.4 Sn-3Ag-0.5Cu-0.5Ce銲錫經迴銲後之低溫5℃下錫鬚成長 ......64 4.2.3 不同氣氛下錫鬚成長 ......66 4.2.3.1 Sn-3Ag-0.5Cu-0.5Ce銲錫經迴銲後之相對濕度90%室溫下錫鬚成長 ......66 4.2.3.2 Sn-3Ag-0.5Cu-0.5Ce銲錫經迴銲後之室溫下真空10-2torr中錫鬚成長 ......68 4.2.4 錫鬚性質比較 ......69 4.2.4.1錫鬚成長三階段下的型態比較 ......69 4.2.4.2錫鬚尺寸(直徑、長度) ......71 4.2.4.3短時間下觀察錫鬚成長 ......72 4.2.4.4細長狀錫鬚之成長性質(密度、成長速度) ......74 4.2.4.5 山丘狀錫鬚之成長性質 ......75 4.2.5 稀土添加導致錫鬚生成的成長機構 ......77 4.2.5.1 橫截面觀察CeSn3氧化 ......77 4.2.5.2 錫鬚成長之驅動力(壓應力) ......78 4.2.5.3 稀土添加導致錫鬚生成之成長機構 ......79 4.2.5.4 成份分析(EDX) ......80 4.2.5.5 錫鬚成長速度 ......81 伍、結論 ......170 陸、參考文獻 ......172 附錄、作者簡介 ......18

Correlates of Attitudes Toward Homosexuality and Intention to Care for Homosexual People Among Psychiatric Nurses in Southern Taiwan

This study examined the association between attitudes toward homosexual individuals and intention to provide care and demographic and occupational factors, sexual orientation, knowledge about homosexuality, and experiences of contact with homosexual people among psychiatric nurses in southern Taiwan. In total, 133 psychiatric nurses from a medical center, three regional teaching hospitals, and one psychiatric hospital in southern Taiwan were recruited into this study. Their attitudes toward homosexual people as recorded on the Attitudes Toward Homosexuality Questionnaire, intention to provide care to homosexual individuals, and related factors were examined. The results revealed that psychiatric nurses who had a bachelor's or master's degree, higher level of knowledge about homosexuality, and friends or relatives with a homosexual orientation had a more positive attitude toward homosexuality. These psychiatric nurses, with more positive attitudes, and who worked in the medical center or regional teaching hospitals had a higher intention to care for homosexual people. The factors related to attitudes toward homosexuality and intention to care for homosexual people identified in this study should be taken into consideration when intervening in psychiatric nurses' attitudes toward homosexuality and intention to care for homosexual people

Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

<div><p>Background</p><p>Nasopharyngeal carcinoma (NPC) is known for its high metastatic potential and locoregional recurrence, although the molecular alterations that are driving NPC metastasis remain unclear at this time. This study aimed to examine the expression of fibulin-5 in NPC, correlate the results with clinicopathological variables and survival, and to investigate the role of fibulin-5 in human NPC cell lines.</p> <p>Material and Methods</p><p>Standard semi-quantitative-RT-PCR, quantitative-RT-PCR, immunoblotting, and immunohistochemistry were used to investigate the mRNA and protein expression profiles of fibulin-5 in normal and NPC tissues. Immunohistochemistry of fibulin-5 was correlated with clinicopathological characteristics by univariate analyses. NPC cells overexpressing fibulin-5 or fibulin-5-siRNA cells were generated by stable transfection to characterize the molecular mechanisms of fibulin-5-elicited cell growth and metastasis.</p> <p>Results</p><p>Our results demonstrated that fibulin-5 overexpression in NPC specimens and significantly correlated with advanced tumor metastasis indicating a poor 5-year overall survival. Fibulin-5 was mainly expressed in the nucleus in human NPC specimens and cell lines. Functionally, fibulin-5 overexpression yielded fast growth in NPC cells. In addition, fibulin-5 promotes cell metastasis in NPC cells through increased FLJ10540 and phosphor-AKT activity. In contrast, siRNA depletion of fibulin-5 suppressed FLJ10540 expression and phosphor-AKT activity. Suppression of either fibulin-5 or FLJ10540 can cause significant inhibition with regards to cell motility in NPC cells. Finally, immunohistochemical analysis of human aggressive NPC specimens showed a significant and positive correlation between fibulin-5 and FLJ10540 expression.</p> <p>Conclusion</p><p>Higher fibulin-5 expression is not only an important indicator of poor survival, but also contributes to the development of new therapeutic strategies in the FLJ10540/AKT pathway for NPC treatment.</p> </div

NPC cells stimulated with fibulin-5 protein encourages cell growth and motility.

<p>(A) Hone 1 cells were treated with indicated concentrations of fibulin-5, and cell growth was analyzed on days 0-3 by MTT assay. Data were normalized against the OD₅₇₀ value on day 1 of each treatment. The results represent the mean ± SD of 3 independent experiments. (B) Migration and invasion of Hone1 cells (200×). For the migration assays, Hone1 cells stimulated with indicate concentrations of fiblin-5 protein were seeded into the top of a Transwell insert. After 24 hour, the cells on top were scraped, and the cells that had migrated to the bottom were fixed and stained with Giemsa. The relative-fold migration values for the clones were normalized against the DMSO and are represented diagrammatically.</p

The activity of AKT induced cell motility is regulated by FLJ10540 in NPC cells.

<p>(A) Hone1-expressing FLJ10540 cells were serum-starved for 24 hour and treated with or without AKT inhibitor. The Total cell lysates of vehicle-Hone and FLJ10540-Hone1, transfected cells were immunoblotted for the unphosphorylated and phosphorylated forms of AKT. β-actin was used as the internal loading control. (B) A negative control and FLJ10540 siRNAs were transfected into Hone1 cells for 24 hour and western blotting was performed as in (B). (C) Vehicle-Hone1 and FLJ10540-Hone1 transfected cells were serum-starved and treated with the AKT inhibitor for 24 hour. The migration and invasion ratios of vehicle-Hone1 and fibulin-5-Hone1 transfected cells were determined as previously described.</p

The distributions of fibulin-5 in NPC and other human cancer cell lines.

<p>(A) Immunoblot analysis of total protein from normal tissues, TW01, TW04, and Hone1 cells using monoclonal anti-fibulin-5 antibody. (B) Hone1, A-431 and U2-OS cells were grown on glass cover slips and cultured overnight. Indirect immunofluorescence of endogenous fibulin-5 protein in Hone1 cells was detected by using anti-fibulin-5 antibody. (Bottom panel) Hone1 cells were transfected with DDK-fused fibulin-5 by Plus/Lipofectamine reagents and cultured overnight. Indirect immunofluorescence of highly expressed fibulin-5 protein in Hone1 cells was detected with anti-DDK antibody. The cells were double-stained with DAPI to detect DNA. (C) (Upper panel) Western blot analysis for fibulin-5 of cytoplasm extracts (left) and nuclear extracts (right) of TW01 and Hone1 cells. β-actin and LaminA/C were used as control. (Bottom panel) Immunoprecipitation analysis for fibulin-5 of cell culture supernatants and intra-cellular total cell extracts of TW01 and Hone1 cells.</p

The proliferation, migratory, and invasive abilities of NPC cells are inhibited by <i>fibulin-5</i>-specific siRNA.

<p>(A) A negative control siRNA plus <i>fibulin-5</i> siRNA was transfected into Hone1 cells for 24 hour. After transfection, western blotting was performed with anti-fibulin-5 and β-actin antibodies. The mRNA expression level of endogenous fibulin-5 was also measured by Q-RT-PCR. (B) Using the same panel, the sifibulin-5 transfectants and negative control were seeded into 96-well plates with 5.0% FBS. The cells were cultured for 0-3 days followed by MTT assay to quantitate cell growth. The data were normalized against the value on day 0 of each treatment. The growth curves of Hone1 cells are shown as the mean ± SD of 3 independent experiments. (C and D) The wound healing, migration, and invasion results of negative control-Hone1 and sifibulin-5-Hone1 stable cells are shown (200×). The relative-fold migration and invasion of sifibulin-5-Hone1 cells were normalized against the values for the negative control cells and are represented diagrammatically. The results represent the mean ± SD of 3 independent experiments. </p