Publications by authors named "Shing-Jyh Chang"

54 Publications

Rapid β-human chorionic gonadotropin detection in urine with electric-double-layer gated field-effect transistor biosensors and a handheld device.

Biomicrofluidics 2021 Mar 5;15(2):024106. Epub 2021 Apr 5.

Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan.

In this experimental study, a portable biosensor was developed to detect β-human chorionic gonadotropin (β-hCG), which is extensively used in pregnancy tests and serves as a biomarker for ectopic pregnancy. The sensor used is an electric-double-layer field-effect transistor biosensor with the extended-gate design. Bias voltage is applied on the sensor to measure the resulting drain current signals. Gold electrode surface is functionally activated with an anti-β-hCG antibody to capture β-hCG protein. Fluorescence imaging technique is utilized to confirm the surface functionalization. The biosensor demonstrates a dynamically wide range of molecules as detection targets at very low sample concentrations, which shows the potential to detect ectopic pregnancy in very early stages and easily keep track of its periodic changes. It can be produced en masse and does not use additional labels/reagents or pre-processing techniques for the sample. This biosensor can significantly reduce the manufacturing costs and is comparable with the currently available commercial ß-hCG assays. It is suitable for early diagnosis of ectopic pregnancy with low cost and easy operation at home with urine samples.
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http://dx.doi.org/10.1063/5.0042522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8043248PMC
March 2021

Role of IGFBP-2 in oral cancer metastasis.

Biochim Biophys Acta Mol Basis Dis 2021 Apr 14;1867(7):166143. Epub 2021 Apr 14.

Department of Medical Science, Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

Cancer metastasis is one of most main causes of failure in cancer treatment. Nonetheless, more than half of oral cancer patients were diagnosed as advanced oral cancer with dramatically decreased 5-year survival rate to lower than 20%, while the stages become more advanced. In order to improve oral cancer treatment, the identification of cancer metastatic biomarkers and mechanisms is critical. In the current study, two pairs of oral squamous cell carcinoma lines, OC3/C9, and invasive OC3-I5/C9-I5were used as model systems to investigate invasive mechanism as well as to identify potential therapy-associated targets. Based on our previous proteomic analysis, insulin-like growth factor-binding protein 2 (IGFBP-2) was reported participating in oral cancer metastasis. Subsequent studies have applied interference RNA as well as recombinant protein techniques to confirm the roles of IGFBP-2 in oral cancer metastasis and examine their potency in regulating invasion as well as the mechanism IGFBP-2 involved. The results demonstrated that expression of epithelial-mesenchymal transition (EMT) markers including Twist, Snail1, SIP1, profilin, vimentin, uPA and MMP9 were increased in both OC3-I5 and C9-I5 compared to OC3 and C9 cells, while E-cadherin expression was down-regulated in the OC3-I5 and C9-I5 cells. Moreover, IGFBP-2 is shown to affect not only migration and invasion but also wound healing ability and cell proliferation. Our results also revealed that uPA is a downstream target of IGFBP-2 to intermediate oral cancer metastasis. To sum up, the current studies indicated that elevated IGFBP-2 is strongly correlated with oral cancer metastasis and progression, and that it could potentially serve as a prognostic biomarker as well as an innovative target for the treatment of oral cancer invasion.
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http://dx.doi.org/10.1016/j.bbadis.2021.166143DOI Listing
April 2021

Characterization of TGF-β by Induced Oxidative Stress in Human Trabecular Meshwork Cells.

Antioxidants (Basel) 2021 Jan 13;10(1). Epub 2021 Jan 13.

Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 300, Taiwan.

Oxidative stress generated by reactive oxygen species (ROS) plays a critical role in the pathomechanism of glaucoma, which is a multifactorial blinding disease that may cause irreversible damage within human trabecular meshwork cells (HTMCs). It is known that the transforming growth factor-β (TGF-β) signaling pathway is an important component of oxidative stress-induced damage related to extracellular matrix (ECM) fibrosis and activates cell antioxidative mechanisms. To elucidate the dual potential roles and regulatory mechanisms of TGF-β in effects on HTMCs, we established an in vitro oxidative model using hydrogen peroxide (HO) and further focused on TGF-β-related oxidative stress pathways and the related signal transduction. Via a series of cell functional qualitative analyses to detect related protein level alterations and cell fibrosis status, we illustrated the role of TGF-β1 and TGF-β2 in oxidative stress-induced injury by shTGF-β1 and shTGF-β2 knockdown or added recombinant human TGF-β1 protein (rhTGF-β1). The results of protein level showed that p38 MAPK, TGF-β, and its related SMAD family were activated after HO stimulation. Cell functional assays showed that HTMCs with HO exposure duration had a more irregular actin architecture compared to normal TM cells. Data with rhTGF-β1 (1 ng/mL) pretreatment reduced the cell apoptosis rate and amount of reactive oxygen species (ROS), while it also enhanced survival. Furthermore, TGF-β1 and TGF-β2 in terms of antioxidant signaling were related to the activation of collagen I and laminin, which are fibrosis-response proteins. Succinctly, our study demonstrated that low concentrations of TGF-β1 (1 ng/mL) preserves HTMCs from free radical-mediated injury by p-p38 MAPK level and p-AKT signaling balance, presenting a signaling transduction mechanism of TGF-β1 in HTMC oxidative stress-related therapies.
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http://dx.doi.org/10.3390/antiox10010107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828702PMC
January 2021

Biomarker discovery in highly invasive lung cancer cell through proteomics approaches.

Cell Biochem Funct 2021 Apr 1;39(3):367-379. Epub 2020 Nov 1.

Department of Medical Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.

Lung cancer is one of the leading causes of cancer-related death worldwide. The most common type of lung cancer is non-small cell lung cancer (NSCLC). When NSCLC is detected, patients are typically already in a metastatic stage. Metastasized cancer is a major obstacle of effective treatment and understanding the mechanisms underlying metastasis is critical to treat cancer. Herein, we selected an invasive subpopulation from the human lung cancer cell line A549 using the transwell system and named it as A549-I5. Invasive and migratory activities of this cell line were analysed using wound healing, invasion, and migration assays. In addition, epithelial-mesenchymal transition (EMT) markers, such as Snail 1, Twist, Vimentin, N-cadherin and E-cadherin, were assessed through immunoblotting. In comparison to A549 cells, the invasive A549-I5 lung cancer cells had enhanced invasiveness, motility and EMT marker expression. Proteomic analysis identified 83 significantly differentially expressed proteins in A549-I5 cells. These identified proteins were classified according to their cellular functions and most were involved in cytoskeleton, redox regulation, protein degradation and protein folding. In summary, our results provide potential diagnostic markers and therapeutic candidates for the treatment of NSCLC metastasis. SIGNIFICANCE OF THE STUDY: When NSCLC is detected, most patients are already in a metastatic stage. Herein, we selected an invasive subpopulation from a human lung cancer cell line which had increased EMT markers as well as high wound healing, invasion and migration abilities. Proteomic analysis identified numerous proteins associated with functions in cytoskeleton, redox regulation, protein degradation and protein folding that were differentially expressed in these cells. These results may provide potential diagnostic markers and therapeutic candidates for the treatment of NSCLC metastasis.
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http://dx.doi.org/10.1002/cbf.3599DOI Listing
April 2021

Establishment of three human induced pluripotent stem cell lines from a type 1 diabetic family harboring sequence variants associated with autoimmunity.

Stem Cell Res 2020 12 8;49:102029. Epub 2020 Oct 8.

Genomics Research Center, Academia Sinica, Taipei, Taiwan; Departiment of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan. Electronic address:

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-producing β cells. Genetic studies have identified > 60 T1D risk loci that harbor genes with disease-causative alleles. However, determining the biological effects of such loci is often difficult due to limited tissue availability. Disease-specific human induced pluripotent stem cells (hiPSCs) are a valuable resource for modeling T1D pathogenesis. In particular, families with complete disease penetrance offer an opportunity to further dissect T1D risk loci. Here, we describe the generation of three hiPSC lines from a T1D family with sequence variants associated with autoimmunity.
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http://dx.doi.org/10.1016/j.scr.2020.102029DOI Listing
December 2020

Targeting UDP-glucose dehydrogenase inhibits ovarian cancer growth and metastasis.

J Cell Mol Med 2020 10 7;24(20):11883-11902. Epub 2020 Sep 7.

Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.

More than 70% of patients with ovarian cancer are diagnosed in advanced stages. Therefore, it is urgent to identify a promising prognostic marker and understand the mechanism of ovarian cancer metastasis development. By using proteomics approaches, we found that UDP-glucose dehydrogenase (UGDH) was up-regulated in highly metastatic ovarian cancer TOV21G cells, characterized by high invasiveness (TOV21G ), in comparison to its parental control. Previous reports demonstrated that UGDH is involved in cell migration, but its specific role in cancer metastasis remains unclear. By performing immunohistochemical staining with tissue microarray, we found overexpression of UGDH in ovarian cancer tissue, but not in normal adjacent tissue. Silencing using RNA interference (RNAi) was utilized to knockdown UGDH, which resulted in a significant decrease in metastatic ability in transwell migration, transwell invasion and wound healing assays. The knockdown of UGDH caused cell cycle arrest in the G /G phase and induced a massive decrease of tumour formation rate in vivo. Our data showed that UGDH-depletion led to the down-regulation of epithelial-mesenchymal transition (EMT)-related markers as well as MMP2, and inactivation of the ERK/MAPK pathway. In conclusion, we found that the up-regulation of UGDH is related to ovarian cancer metastasis and the deficiency of UGDH leads to the decrease of cell migration, cell invasion, wound healing and cell proliferation ability. Our findings reveal that UGDH can serve as a prognostic marker and that the inhibition of UGDH is a promising strategy for ovarian cancer treatment.
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http://dx.doi.org/10.1111/jcmm.15808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578908PMC
October 2020

Progesterone receptor membrane component 1 is involved in oral cancer cell metastasis.

J Cell Mol Med 2020 09 16;24(17):9737-9751. Epub 2020 Jul 16.

Department of Medical Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.

Cancer metastasis is a common cause of failure in cancer therapy. However, over 60% of oral cancer patients present with advanced stage disease, and the five-year survival rates of these patients decrease from 72.6% to 20% as the stage becomes more advanced. In order to manage oral cancer, identification of metastasis biomarker and mechanism is critical. In this study, we use a pair of oral squamous cell carcinoma lines, OC3, and invasive OC3-I5 as a model system to examine invasive mechanism and to identify potential therapeutic targets. We used two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to examine the global protein expression changes between OC3 and invasive OC3-I5. A proteomic study reveals that invasive properties alter the expression of 101 proteins in OC3-I5 cells comparing to OC3 cells. Further studies have used RNA interference technique to monitor the influence of progesterone receptor membrane component 1 (PGRMC1) protein in invasion and evaluate their potency in regulating invasion and the mechanism it involved. The results demonstrated that expression of epithelial-mesenchymal transition (EMT) markers including Twist, p-Src, Snail1, SIP1, JAM-A, vimentin and vinculin was increased in OC3-I5 compared to OC3 cells, whereas E-cadherin expression was decreased in the OC3-I5 cells. Moreover, in mouse model, PGRMC1 is shown to affect not only migration and invasion but also metastasis in vivo. Taken together, the proteomic approach allows us to identify numerous proteins, including PGRMC1, involved in invasion mechanism. Our results provide useful diagnostic markers and therapeutic candidates for the treatment of oral cancer invasion.
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http://dx.doi.org/10.1111/jcmm.15535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7520311PMC
September 2020

Proteomic Analysis of Metastasis-Specific Biomarkers in Pancreatic Cancer: Galectin-1 Plays an Important Metastatic Role in Pancreatic Cancer.

J Pharm Biomed Anal 2020 Jul 25;186:113300. Epub 2020 Apr 25.

Department of Medical Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

Cancer metastasis is the major cause of death in pancreatic cancer. We have established a pair of pancreatic ductal adenocarcinoma cell line, PANC1 and invasive PANC1-I5, as a model system toinvestigate the metastatic mechanism as well as potential therapeutic targets in pancreatic cancer. We used proteomic analysis based on two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to examine the global protein expression alterations between PANC1 and PANC1-I5. Proteomic study revealed that 88 proteins are differentially expressed between PANC1-I5 and PANC1 cells, and further functional evaluations through protein expression validation, gene knockout, migration and invasion analysis revealed that galectin-1 is one of the potential players in modulating pancreatic cancer metastasis. To conclude, we have identified numerous proteins might be associated with pancreatic cancer invasiveness in the pancreatic cancer model.
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http://dx.doi.org/10.1016/j.jpba.2020.113300DOI Listing
July 2020

Rabies virus glycoprotein-amplified hierarchical targeted hybrids capable of magneto-electric penetration delivery to orthotopic brain tumor.

J Control Release 2020 05 8;321:159-173. Epub 2020 Feb 8.

Department of Biomedical Engineering and Environmental Science, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

Compact nanohybrids can potentially unite various therapeutic features and reduce side effects for precise cancer therapy. However, the poor accumulation and limited tumor penetration of drugs at the tumor impede the manifestation of nanomedicine. We developed a rabies virus glycoprotein (RVG)-amplified hierarchical targeted hybrid that acts as a stealthy and magnetolytic carrier that transports dual tumor-penetrating agents incorporating two drugs (boron-doped graphene quantum dots (B-GQDs)/doxorubicin and pH-responsive dendrimers (pH-Den)/palbociclib). The developed RVG-decorated hybrids (RVG-hybrids) enhance the accumulation of drugs at tumor by partially bypassing the BBB via spinal cord transportation and pH-induced aggregation of hierarchical targeting. The penetrated delivery of dual pH-Den and B-GQD drugs to deep tumors is actuated by magnetoelectric effect, which are able to generate electrons to achieve electrostatic repulsion and disassemble the hybrids into components of a few nanometers in size. The synergy of magnetoelectric drug penetration and chemotherapy was achieved by delivery of the B-GQDs and pH-Den to orthotopic tumors, which prolonged the host survival time. This RVG-amplified dual hierarchical delivery integrated with controlled and penetrated release from this hybrid improve the distribution of the therapeutic agents at the brain tumor for synergistic therapy, exhibiting potential for clinic use.
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http://dx.doi.org/10.1016/j.jconrel.2020.02.018DOI Listing
May 2020

Identification of hyperglycemia-associated microbiota alterations in saliva and gingival sulcus.

Arch Biochem Biophys 2020 03 23;682:108278. Epub 2020 Jan 23.

Institute of Bioinformatics and Structural Biology and Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

Oral microbes are a contributing factor to hyperglycemia by inducing an increase in insulin resistance resulting in uncontrolled blood glucose levels. However, the relationship between the distribution of oral flora and hyperglycemia is still controversial. Combining the power of MALDI-Biotyper with anaerobic bacterial culture, this study explores the correlation between anaerobic bacteria in the oral cavity and blood glucose levels. The results demonstrated that altered blood glucose levels contributed to a varied bacterial distribution in the oral cavity. Specifically, Veillonella spp. and Prevotella spp. were identified in a higher proportion in people with elevated blood glucose levels. Six bacterial species identified in this study (Prevotella melaninogenica, Campylobacter rectus, Streptococcus gordonii, Streptococcus mitis, Streptococcus salivarius, and Veillonella parvula) not only demonstrated a positive association with higher blood glucose levels, but also likely contribute to the development of the condition. The data demonstrated MALDI-TOF MS to be a simpler, faster, and more economical clinical identification tool that provides clarity and depth to the research on blood glucose and oral microbiota.
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http://dx.doi.org/10.1016/j.abb.2020.108278DOI Listing
March 2020

Role of PGRMC1 in cell physiology of cervical cancer.

Life Sci 2019 Aug 16;231:116541. Epub 2019 Jun 16.

Dept. of Medical Sciences & Inst. of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

Aims: The most frequent cancers among women worldwide. The mortality of cervical cancer has declined significantly primarily due to the widespread use of Pap smear tests as a screening test and therapeutic vaccination. However, cervical cancer still remains a severe disease among the female population, as the prognosis of metastatic cervical cancer is very poor.

Key Methods: In this study, we performed 2D-DIGE and MALDI-TOF/TOF MS to analyze differentially expressed proteins between HeLa and invasive HeLa-I5 cells..

Key Findings: According to our proteomics data, 68 differentially expressed proteins between the HeLa and HeLa-I5 cells were identified. One of these differentially expressed proteins, Progesterone receptor membrane component 1 (PGRMC1), was selected as a candidate for further studies. To correlate the role of PGRMC1 with cellular migration and cancer progression, small interfering RNA (siRNA) was used to knockdown the expression of PGRMC1. Similar function of PGRMC1 was also observed in two other cervical cancer lines, CaSki and ME-180.

Significance: PGRMC1 plays an essential role in regulating cancer progression and metastasis of cervical cancer cells, thus serving as a potential therapeutic target for cervical cancer.
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http://dx.doi.org/10.1016/j.lfs.2019.06.016DOI Listing
August 2019

Remote Magnetic Control of Autophagy in Mouse B-Lymphoma Cells with Iron Oxide Nanoparticles.

Nanomaterials (Basel) 2019 Apr 4;9(4). Epub 2019 Apr 4.

Institute of Biomedical Engineering, College of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 30010, Taiwan.

Autophagy is the spontaneous degradation of intracellular proteins and organelles in response to nutrient deprivation. The phagocytosis of iron oxide nanoparticles (IONPs) results in intracellular degradation that can be exploited for use in cancer treatment. Non-invasive magnetic control has emerged as an important technology, with breakthroughs achieved in areas such as magneto-thermal therapy and drug delivery. This study aimed to regulate autophagy in mouse B-lymphoma cells (A20) through the incorporation of IONPs-quantum dots (QDs). We hypothesized that with the application of an external magnetic field after phagocytosis of IONPs-QDs, autophagy of intracellular IONPs-QDs could be regulated in a non-invasive manner and subsequently modulate the regulation of inflammatory responses. The potential of this approach as a cancer treatment method was explored. The application of IONPs and an external magnetic force enabled the non-invasive regulation of cell autophagy and modulation of the self-regulatory function of cells. The combination of non-invasive magnetic fields and nanotechnology could provide a new approach to cancer treatment.
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http://dx.doi.org/10.3390/nano9040551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524120PMC
April 2019

Proteomic investigating the cooperative lethal effect of EGFR and MDM2 inhibitors on ovarian carcinoma.

Arch Biochem Biophys 2018 Jun 12;647:10-32. Epub 2018 Apr 12.

Institute of Bioinformatics and Structural Biology and Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

With the concept of precision medicine, combining multiple molecular-targeting therapies has brought new approaches to current cancer treatments. Malfunction of the tumor suppressor protein, p53 is a universal hallmark in human cancers. Under normal conditions, p53 is degraded through an ubiquitin-proteosome pathway regulated by its negative regulator, MDM2. In contrast, cellular stress such as DNA damage will activate p53 to carry out DNA repair, cell cycle arrest, and apoptosis. In this study, we focused on ovarian carcinoma with high EGFR and MDM2 overexpression rate. We assessed the effects of combined inhibition by MDM2 (JNJ-26854165) and EGFR (gefitinib) inhibitors on various ovarian cell lines to determine the importance of these two molecular targets on cell proliferation. We then used a proteomic strategy to investigate the relationship between MDM2 and EGFR inhibition to explore the underlying mechanisms of how their combined signaling blockades work together to exert cooperative inhibition. Our results demonstrated that all four cell lines were sensitive to both individual and combined, MDM2 and EGFR inhibition. The proteomic analysis also showed that gefitinib/JNJ-treated CAOV3 cells exhibited downregulation of proteins involved in nucleotide biosynthesis such as nucleoside diphosphate kinase B (NME2). In conclusion, our study showed that the combined treatment with JNJ and gefitinib exerted synergistic inhibition on cell proliferation, thereby suggesting the potential application of combining MDM2 inhibitors with EGFR inhibitors for enhancing efficacy in ovarian cancer treatment.
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http://dx.doi.org/10.1016/j.abb.2018.04.004DOI Listing
June 2018

Endothelial angiogenesis is directed by RUNX1T1-regulated VEGFA, BMP4 and TGF-β2 expression.

PLoS One 2017 22;12(6):e0179758. Epub 2017 Jun 22.

Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan.

Tissue angiogenesis is intimately regulated during embryogenesis and postnatal development. Defected angiogenesis contributes to aberrant development and is the main complication associated with ischemia-related diseases. We previously identified the increased expression of RUNX1T1 in umbilical cord blood-derived endothelial colony-forming cells (ECFCs) by gene expression microarray. However, the biological relevance of RUNX1T1 in endothelial lineage is not defined clearly. Here, we demonstrate RUNX1T1 regulates the survival, motility and tube forming capability of ECFCs and EA.hy926 endothelial cells by loss-and gain-of function assays, respectively. Second, embryonic vasculatures and quantity of bone marrow-derived angiogenic progenitors are found to be reduced in the established Runx1t1 heterozygous knockout mice. Finally, a central RUNX1T1-regulated signature is uncovered and VEGFA, BMP4 as well as TGF-β2 are demonstrated to mediate RUNX1T1-orchested angiogenic activities. Taken together, our results reveal that RUNX1T1 serves as a common angiogenic driver for vaculogenesis and functionality of endothelial lineage cells. Therefore, the discovery and application of pharmaceutical activators for RUNX1T1 will improve therapeutic efficacy toward ischemia by promoting neovascularization.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179758PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481149PMC
September 2017

Proteomic analysis of prognostic plasma biomarkers in peripheral arterial occlusive disease.

Mol Biosyst 2017 Jun;13(7):1297-1303

Division of Plastic Surgery, Department of Surgery, Ditmanson Medical Foundation Chia-Yi Christian Hospital, Chiayi, Taiwan.

Peripheral arterial occlusive disease (PAOD), one of the major manifestations of systemic atherosclerosis, causes intermittent claudication and rest pain. Patients with PAOD not only have reduced quality of life, but also have a substantial risk of cardiovascular morbidity and death. In this study, we adopted a proteomics-based approach using 2D-DIGE and MALDI-TOF MS to compare the differential plasma proteome between good and poor prognosis of PAOD. We identified 196 plasma proteins, which represent 42 unique gene products. These proteins mainly have roles in the inflammatory response and coagulation. This approach identified several potential prognostic plasma markers in PAOD, including transthyretin and complement factor B, which may be associated with the evaluation of good/poor prognosis of PAOD. In conclusion, we report a comprehensive patient-based plasma proteomic approach for the identification of potential plasma biomarkers for the screening and detection of good/poor prognosis of PAOD. Among these, transthyretin and complement factor B are potential markers for monitoring the PAOD disease in the plasma.
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http://dx.doi.org/10.1039/c7mb00229gDOI Listing
June 2017

PI3K inhibitor enhances the cytotoxic response to etoposide and cisplatin in a newly established neuroendocrine cervical carcinoma cell line.

Oncotarget 2017 Jul;8(28):45323-45334

Department of Obstetrics and Gynecology, Hsinchu MacKay Memorial Hospital, Hsinchu, 30071, Taiwan (R.O.C.).

Background: Neuroendocrine cervical carcinoma (NECC) is a rare and aggressive subtype of cervical cancer. To date, no NECC cell-based model is available, which hinders the development of new therapeutic strategies for NECC. In this study, we derived a new NECC cell line from an ex vivo biopsy and used it to explore novel drug combination approach for NECC.

Results: The stable HM-1 cell line displayed high expression levels of the neuroendocrine marker, synaptophysin. HM-1 cell transplantation could induce tumor growth in nude mice. As expected, the combination of etoposide and cisplatin synergistically inhibited HM-1 cell proliferation. Strikingly, when etoposide and cisplatin were combined with PI3K inhibitor BEZ235, the growth of HM-1 cells was significantly reduced. Taken together, the data implied the combination of etoposide and cisplatin with BEZ235 not only inhibited HM-1 cell proliferation but also increased cell apoptosis.

Materials And Methods: A NECC tissue sample from a 75-year-old female patient was processed to derive a primary cell line annotated as HM-1. The features of HM-1 were analyzed to establish its characteristic profile. Next, HM-1 was treated with PI3K inhibitors, BKM120 and/or BEZ235, in combination with two well-known genotoxic drugs, etoposide and/or cisplatin, to evaluate which combination could serve as a more effective treatment approach. Their inhibiting effects on HM-1 were evaluated by cell viability, apoptosis, and target kinase expression.

Conclusions: The newly established NECC cell line HM-1 could serve as a cell-based model for NECC research. The synergistic drug combination of PI3K inhibitor with genotoxic drugs might become a potential new treatment strategy against NECC.
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http://dx.doi.org/10.18632/oncotarget.17335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542189PMC
July 2017

Proteomic Analysis of Various Rat Ocular Tissues after Ischemia-Reperfusion Injury and Possible Relevance to Acute Glaucoma.

Int J Mol Sci 2017 Feb 5;18(2). Epub 2017 Feb 5.

Institute of Bioinformatics and Structural Biology & Department of Medical Sciences, National Tsing Hua University, Hsinchu 300, Taiwan.

Glaucoma is a group of eye diseases that can cause vision loss and optical nerve damage. To investigate the protein expression alterations in various intraocular tissues (i.e., the cornea, conjunctiva, uvea, retina, and sclera) during ischemia-reperfusion (IR) injury, this study performed a proteomic analysis to qualitatively investigate such alterations resulting from acute glaucoma. The IR injury model combined with the proteomic analysis approach of two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to monitor the protein expression alterations in two groups of specimens (an IR injury group and a control group). The analysis results revealed 221 unique differentially expressed proteins of a total of 1481 proteins in the cornea between the two groups. In addition, 97 of 1206 conjunctival proteins, 90 of 1354 uveal proteins, 61 of 1180 scleral proteins, and 37 of 1204 retinal proteins were differentially expressed. These findings imply that different ocular tissues have different tolerances against IR injury. To sum up, this study utilized the acute glaucoma model combined with 2D-DIGE and MALDI-TOF MS to investigate the IR injury affected protein expression on various ocular tissues, and based on the ratio of protein expression alterations, the alterations in the ocular tissues were in the following order: the cornea, conjunctiva, uvea, sclera, and retina.
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http://dx.doi.org/10.3390/ijms18020334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343869PMC
February 2017

Dysregulation of Vascular Endothelial Growth Factor Receptor-2 by Multiple miRNAs in Endothelial Colony-Forming Cells of Coronary Artery Disease.

J Vasc Res 2017 26;54(1):22-32. Epub 2017 Jan 26.

Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan, ROC.

Background/aims: Endothelial colony-forming cells (ECFCs) have the potential to be used in regenerative medicine. Dysfunction of ECFCs is correlated with the onset of cardiovascular disorders, especially coronary artery disease (CAD). Binding of vascular endothelial growth factor A (VEGFA) to vascular endothelial growth factor receptor-2 (VEGFR2) triggers cell motility and angiogenesis of ECFCs, which are crucial to vascular repair.

Methods: To identify the miRNA-VEGFR2-dependent regulation of ECFC functions, ECFCs isolated from peripheral blood of disease-free and CAD individuals were subjected to small RNA sequencing for identification of anti-VEGFR2 miRNAs. The angiogenic activities of the miRNAs were determined in both in vitro and in vivo mice models.

Results: Three miRNAs, namely miR-410-3p, miR-497-5p, and miR-2355-5p, were identified to be upregulated in CAD-ECFCs, and VEGFR2 was their common target gene. Knockdown of these miRNAs not only restored the expression of VEGFR2 and increased angiogenic activities of CAD-ECFCs in vitro, but also promoted blood flow recovery in ischemic limbs in vivo. miR-410-3p, miR-497-5p, and miR-2355-5p could serve as potential biomarkers for CAD detection as they are highly expressed in the plasma of CAD patients.

Conclusions: This modulation could help develop new therapeutic modalities for cardiovascular diseases and other vascular dysregulated diseases, especially tumor angiogenesis.
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http://dx.doi.org/10.1159/000449202DOI Listing
May 2017

Interactome Analysis Reveals a Novel Role for RAD6 in the Regulation of Proteasome Activity and Localization in Response to DNA Damage.

Mol Cell Biol 2017 03 1;37(6). Epub 2017 Mar 1.

Research Center for Translational Medicine at East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, Shanghai, People's Republic of China

RAD6, an E2 ubiquitin-conjugating enzyme, is a key node for determining different DNA damage repair pathways, controlling both the error-prone and the error-free DNA damage repair pathways through differential regulation of the ubiquitination of the proliferating cell nuclear antigen (PCNA) protein. However, whether other pathways are involved in the RAD6-mediated regulation of DNA damage repair is still unclear. To deeply understand the molecular mechanisms of RAD6 in DNA damage repair, we performed a proteomic analysis and identified the changes of the protein-protein interaction (PPI) networks of RAD6 before and after X-ray irradiation. Furthermore, our study indicated that a proteasome-related event is likely involved in the DNA damage repair process. Moreover, we found that RAD6 promotes proteasome activity and nuclear translocation by enhancing the degradation of PSMF1 and the lamin B receptor (LBR). Therefore, we provide a novel pathway that is employed by RAD6 in response to DNA damage.
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http://dx.doi.org/10.1128/MCB.00419-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5335506PMC
March 2017

YM500v3: a database for small RNA sequencing in human cancer research.

Nucleic Acids Res 2017 01 29;45(D1):D925-D931. Epub 2016 Nov 29.

Graduate Institute of Biomedical Sciences, China Medical University, Taichung, 40402, Taiwan

We previously presented the YM500 database, which contains >8000 small RNA sequencing (smRNA-seq) data sets and integrated analysis results for various cancer miRNome studies. In the updated YM500v3 database (http://ngs.ym.edu.tw/ym500/) presented herein, we not only focus on miRNAs but also on other functional small non-coding RNAs (sncRNAs), such as PIWI-interacting RNAs (piRNAs), tRNA-derived fragments (tRFs), small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). There is growing knowledge of the role of sncRNAs in gene regulation and tumorigenesis. We have also incorporated >10 000 cancer-related RNA-seq and >3000 more smRNA-seq data sets into the YM500v3 database. Furthermore, there are two main new sections, 'Survival' and 'Cancer', in this updated version. The 'Survival' section provides the survival analysis results in all cancer types or in a user-defined group of samples for a specific sncRNA. The 'Cancer' section provides the results of differential expression analyses, miRNA-gene interactions and cancer miRNA-related pathways. In the 'Expression' section, sncRNA expression profiles across cancer and sample types are newly provided. Cancer-related sncRNAs hold potential for both biotech applications and basic research.
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http://dx.doi.org/10.1093/nar/gkw1084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210564PMC
January 2017

Oncogenic Functions of Gli1 in Pancreatic Adenocarcinoma Are Supported by Its PRMT1-Mediated Methylation.

Cancer Res 2016 12 6;76(23):7049-7058. Epub 2016 Oct 6.

Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas.

The oncogenic transcription factor Gli1 is a critical effector in the Hedgehog (Hh) pathway, which is necessary for the development and progression of pancreatic ductal adenocarcinoma (PDAC). Although TGFβ and K-Ras are known regulators of Gli1 gene transcription in this setting, it is not understood how Gli1 functional activity is regulated. Here, we report the identification of Gli1 as a substrate for the protein arginine N-methyltransferase PRMT1 in PDAC. We found that PRMT1 methylates Gli1 at R597, promoting its transcriptional activity by enhancing the binding of Gli1 to its target gene promoters. Interruption of Gli1 methylation attenuates oncogenic functions of Gli1 and sensitizes PDAC cells to gemcitabine treatment. In human PDAC specimens, the levels of both total Gli1 and methylated Gli1 were correlated positively with PRMT1 protein levels. Notably, PRMT1 regulated Gli1 independently of the canonical Hh pathway as well as the TGFβ/Kras-mediated noncanonical Hh pathway, thereby signifying a novel regulatory mechanism for Gli1 transcriptional activity. Taken together, our results identified a new posttranslational modification of Gli1 that underlies its pivotal oncogenic functions in PDAC. Cancer Res; 76(23); 7049-58. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-16-0715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5135656PMC
December 2016

Rapid diagnosis of pseudomosaicism in a case of Level II mosaicism for trisomy 5 in a single colony from an in situ culture of amniocytes and a review of mosaic trisomy 5 at amniocentesis.

Taiwan J Obstet Gynecol 2016 Aug;55(4):602-3

Department of Medical Research, MacKay Memorial Hospital, Taipei, Taiwan; Department of Bioengineering, Tatung University, Taipei, Taiwan.

Objective: We present prenatal diagnosis of pseudomosaicism for trisomy 5 and a review of the literature of mosaic trisomy 5 at amniocentesis.

Case Report: A 39-year-old woman underwent amniocentesis at 17 weeks of gestation, which revealed a karyotype of 47,XY,+5[1]/46,XY[20]. The single colony with trisomy 5 had five metaphase cells, and all five cells had the karyotype of 47,XY,+5. Repeat amniocentesis performed at 20 weeks of gestation revealed a karyotype of 46,XY in 27/27 colonies. Simultaneously, interphase fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH), and quantitative fluorescent polymerase chain reaction (QF-PCR) were performed on uncultured amniocytes. Interphase FISH revealed no trisomy 5 in 100 uncultured amniocytes. aCGH revealed no genomic imbalance. QF-PCR excluded uniparental disomy 5. A healthy 3662-g male baby was delivered with a normal karyotype in cord blood and 3.75% (3/80 cells) of trisomy 5 cells in uncultured urinary cells compared with 0.95% (1/105 cells) of trisomy 5 cells in normal control examined by FISH at 1.5 months of age. A review of seven cases with mosaic trisomy 5 at amniocentesis shows that 4/7 had clinically normal outcome, 3/7 had structural defects, mainly the heart, 6/6 had normal karyotype in blood, and 2/3 had mosaic trisomy 5 in the fetal tissues.

Conclusion: Prenatal diagnosis of mosaic trisomy 5 should alert the possibility of fetal structural abnormalities, especially the heart, and culture artifacts. We suggest that the application of molecular cytogenetic techniques such as aCGH, interphase FISH, and QF-PCR on uncultured amniocytes is useful in our understanding of the mosaic status at repeat amniocentesis.
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http://dx.doi.org/10.1016/j.tjog.2016.05.005DOI Listing
August 2016

Small RNA and RNA-IP Sequencing Identifies and Validates Novel MicroRNAs in Human Mesenchymal Stem Cells.

OMICS 2016 Mar 24;20(3):191-8. Epub 2016 Feb 24.

1 Department of Obstetrics and Gynecology, Hsinchu Mackay Memorial Hospital , Hsinchu, Taiwan .

Organ regeneration therapies using multipotent mesenchymal stem cells (MSCs) are currently being investigated for a variety of common complex diseases. Understanding the molecular regulation of MSC biology will benefit regenerative medicine. MicroRNAs (miRNAs) act as regulators in MSC stemness. There are approximately 2500 currently known human miRNAs that have been recorded in the miRBase v21 database. In the present study, we identified novel microRNAs involved in MSC stemness and differentiation by obtaining the global microRNA expression profiles (miRNomes) of MSCs from two anatomical locations bone marrow (BM-MSCs) and umbilical cord Wharton's jelly (WJ-MSCs) and from osteogenically and adipogenically differentiated progenies of BM-MSCs. Small RNA sequencing (smRNA-seq) and bioinformatics analyses predicted that 49 uncharacterized miRNA candidates had high cellular expression values in MSCs. Another independent batch of Ago1/2-based RNA immunoprecipitation (RNA-IP) sequencing datasets validated the existence of 40 unreported miRNAs in cells and their associations with the RNA-induced silencing complex (RISC). Nine of these 40 new miRNAs were universally overexpressed in both MSC types; nine others were overexpressed in differentiated cells. A novel miRNA (UNI-118-3p) was specifically expressed in BM-MSCs, as verified using RT-qPCR. Taken together, this report offers comprehensive miRNome profiles for two MSC types, as well as cells differentiated from BM-MSCs. MSC transplantation has the potential to ameliorate degenerative disorders and repair damaged tissues. Interventions involving the above 40 new microRNA members in transplanted MSCs may potentially guide future clinical applications.
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http://dx.doi.org/10.1089/omi.2015.0136DOI Listing
March 2016

Discovering monotonic stemness marker genes from time-series stem cell microarray data.

BMC Genomics 2015 21;16 Suppl 2:S2. Epub 2015 Jan 21.

Background: Identification of genes with ascending or descending monotonic expression patterns over time or stages of stem cells is an important issue in time-series microarray data analysis. We propose a method named Monotonic Feature Selector (MFSelector) based on a concept of total discriminating error (DEtotal) to identify monotonic genes. MFSelector considers various time stages in stage order (i.e., Stage One vs. other stages, Stages One and Two vs. remaining stages and so on) and computes DEtotal of each gene. MFSelector can successfully identify genes with monotonic characteristics.

Results: We have demonstrated the effectiveness of MFSelector on two synthetic data sets and two stem cell differentiation data sets: embryonic stem cell neurogenesis (ESCN) and embryonic stem cell vasculogenesis (ESCV) data sets. We have also performed extensive quantitative comparisons of the three monotonic gene selection approaches. Some of the monotonic marker genes such as OCT4, NANOG, BLBP, discovered from the ESCN dataset exhibit consistent behavior with that reported in other studies. The role of monotonic genes found by MFSelector in either stemness or differentiation is validated using information obtained from Gene Ontology analysis and other literature. We justify and demonstrate that descending genes are involved in the proliferation or self-renewal activity of stem cells, while ascending genes are involved in differentiation of stem cells into variant cell lineages.

Conclusions: We have developed a novel system, easy to use even with no pre-existing knowledge, to identify gene sets with monotonic expression patterns in multi-stage as well as in time-series genomics matrices. The case studies on ESCN and ESCV have helped to get a better understanding of stemness and differentiation. The novel monotonic marker genes discovered from a data set are found to exhibit consistent behavior in another independent data set, demonstrating the utility of the proposed method. The MFSelector R function and data sets can be downloaded from: http://microarray.ym.edu.tw/tools/MFSelector/.
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http://dx.doi.org/10.1186/1471-2164-16-S2-S2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4331716PMC
December 2015

Mitochondrial proteomics with siRNA knockdown to reveal ACAT1 and MDH2 in the development of doxorubicin-resistant uterine cancer.

J Cell Mol Med 2015 Apr 30;19(4):744-59. Epub 2015 Jan 30.

Department of Applied Science, National Hsinchu University of Education, Hsinchu, Taiwan.

Mitochondria are key organelles in mammary cells in responsible for a number of cellular functions including cell survival and energy metabolism. Moreover, mitochondria are one of the major targets under doxorubicin treatment. In this study, low-abundant mitochondrial proteins were enriched for proteomic analysis with the state-of-the-art two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assistant laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) strategy to compare and identify the mitochondrial protein profiling changes in response to the development of doxorubicin resistance in human uterine cancer cells. The mitochondrial proteomic results demonstrate more than fifteen hundred protein features were resolved from the equal amount pooled of three purified mitochondrial proteins and 101 differentially expressed spots were identified. In which, 39 out of these 101 identified proteins belong to mitochondrial proteins. Mitochondrial proteins such as acetyl-CoA acetyltransferase (ACAT1) and malate dehydrogenase (MDH2) have not been reported with the roles on the formation of doxorubicin resistance in our knowledge. Further studies have used RNA interference and cell viability analysis to evidence the essential roles of ACAT1 and MDH2 on their potency in the formation of doxorubicin resistance through increased cell viability and decreased cell apoptosis during doxorubicin treatment. To sum up, our current mitochondrial proteomic approaches allowed us to identify numerous proteins, including ACAT1 and MDH2, involved in various drug-resistance-forming mechanisms. Our results provide potential diagnostic markers and therapeutic candidates for the treatment of doxorubicin-resistant uterine cancer.
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http://dx.doi.org/10.1111/jcmm.12388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395189PMC
April 2015

miRNome traits analysis on endothelial lineage cells discloses biomarker potential circulating microRNAs which affect progenitor activities.

BMC Genomics 2014 Sep 18;15:802. Epub 2014 Sep 18.

Division of Cardiology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.

Background: Endothelial progenitor cells (EPCs) play a fundamental role in not only blood vessel development but also post-natal vascular repair. Currently EPCs are defined as early and late EPCs based on their biological properties and their time of appearance during in vitro culture. Both EPC types assist angiogenesis and have been linked to ischemia-related disorders, including coronary artery disease (CAD).

Results: We found late EPCs are more mobile than early EPCs and matured endothelial cells (ECs). To pinpoint the mechanism, microRNA profiles of early EPCs late EPCs, and ECs were deciphered by small RNA sequencing. Obtained signatures made up of both novel and known microRNAs, in which anti-angiogenic microRNAs such as miR-221 and miR-222 are more abundant in matured ECs than in late EPCs. Overexpression of miR-221 and miR-222 resulted in the reduction of genes involved in hypoxia response, metabolism, TGF-beta signalling, and cell motion. Not only hamper late EPC activities in vitro, both microRNAs (especially miR-222) also hindered in vivo vasculogenesis in a zebrafish model. Reporter assays showed that miR-222, but not miR-221, targets the angiogenic factor ETS1. In contrast, PIK3R1 is the target of miR-221, but not miR-222 in late EPCs. Clinically, both miR-221-PIK3R1 and miR-222-ETS1 pairs are deregulated in late EPCs of CAD patients.

Conclusions: Our results illustrate EPCs and ECs exploit unique miRNA modalities to regulate angiogenic features, and explain why late EPC levels and activities are reduced in CAD patients. These data will further help to develop new plasma biomarkers and therapeutic approaches for ischemia-related diseases or tumor angiogenesis.
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http://dx.doi.org/10.1186/1471-2164-15-802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176563PMC
September 2014

Dysregulated miR-361-5p/VEGF axis in the plasma and endothelial progenitor cells of patients with coronary artery disease.

PLoS One 2014 27;9(5):e98070. Epub 2014 May 27.

Division of Cardiology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.

Dysfunction and reduction of circulating endothelial progenitor cell (EPC) is correlated with the onset of cardiovascular disorders including coronary artery disease (CAD). VEGF is a known mitogen for EPC to migrate out of bone marrow to possess angiogenic activities, and the plasma levels of VEGF are inversely correlated to the progression of CAD. Circulating microRNAs (miRNAs) in patient body fluids have recently been considered to hold the potential of being novel disease biomarkers and drug targets. However, how miRNAs and VEGF cooperate to regulate CAD progression is still unclear. Through the small RNA sequencing (smRNA-seq), we deciphered the miRNome patterns of EPCs with different angiogenic activities, hypothesizing that miRNAs targeting VEGF must be more abundant in EPCs with lower angiogenic activities. Candidates of anti-VEGF miRNAs, including miR-361-5p and miR-484, were enriched in not only diseased EPCs but also the plasma of CAD patients. However, we found out only miR-361-5p, but not miR-484, was able to suppress VEGF expression and EPC activities. Reporter assays confirmed the direct binding and repression of miR-361-5p to the 3'-UTR of VEGF mRNA. Knock down of miR-361-5p not only restored VEGF levels and angiogenic activities of diseased EPCs in vitro, but further promoted blood flow recovery in ischemic limbs of mice. Collectively, we discovered a miR-361-5p/VEGF-dependent regulation that could help to develop new therapeutic modalities not only for ischemia-related diseases but also for tumor angiogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098070PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4035317PMC
December 2015

Biomarker discovery for neuroendocrine cervical cancer.

Electrophoresis 2014 Jul 5;35(14):2039-45. Epub 2014 Jun 5.

Department of Medical Sciences, Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.

Neuroendocrine cervical cancer is an aggressive but rare form of cervical cancer. The majority of neuroendocrine cervical cancer patients present with advanced-stage diseases. However, the limited numbers of neuroendocrine tumor markers are insufficient for clinical purposes. Thus, we used a proteomic approach combining lysine labeling 2D-DIGE and MALDI-TOF MS to investigate the biomarkers for neuroendocrine cervical cancer. By analyzing the global proteome alteration between the neuroendocrine cervical cancer line (HM-1) and non-neuroendocrine cervical cancer lines (CaSki cells, ME-180 cells, and Hela cells), we identified 82 proteins exhibiting marked changes between HM-1 and CaSki cells, and between ME-180 and Hela cells. Several proteins involved in protein folding, cytoskeleton, transcription control, signal transduction, glycolysis, and redox regulation exhibited significant changes in abundance. Proteomic and immunoblot analyses indicated respective 49.88-fold and 25-fold increased levels of transgelin in HM-1 cells compared with that in other non-neuroendocrine cervical cancer cell lines, implying that transgelin is a biomarker for neuroendocrine cervical cancer. In summary, we used a comprehensive neuroendocrine/non-neuroendocrine cervical cancer model based proteomic approach for identifying neuroendocrine cervical cancer markers, which might contribute to the prognosis and diagnosis of neuroendocrine cervical cancer.
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http://dx.doi.org/10.1002/elps.201400014DOI Listing
July 2014

Deficiency of the microRNA-31-microRNA-720 pathway in the plasma and endothelial progenitor cells from patients with coronary artery disease.

Arterioscler Thromb Vasc Biol 2014 Apr 20;34(4):857-69. Epub 2014 Feb 20.

From the Institute of Microbiology and Immunology, School of Life Science (H.-W.W., T.-S.H., H.-H.L., K.-H.L., C.-F.T., Y.-C.C., Y.-L.C.), Cancer Research Center & Genome Research Center (H.-W.W.), School of Medicine (P.-H.H., C.-C.L.), and Cardiovascular Research Center (P.-H.H.), National Yang-Ming University, Taipei, Taiwan; Division of Cardiology, Department of Medicine (P.-H.H.) and Division of Nephrology, Department of Medicine (C.-C.L.), Taipei Veterans General Hospital, Taipei, Taiwan ; Department of Education and Research, Taipei City Hospital, Taipei, Taiwan (H.-W.W.); Department of Obstetrics and Gynecology, Hsin-Chu Mackay Memorial Hospital, Hsin Chu, Taiwan (S.-J.C., C.-H.T., C.-H.C.); and Division of Cardiology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan (T.-N.T., C.-C.C., S.-M.C.).

Objective: Defects in angiogenesis/vasculogenesis or vessel repair are major complications of coronary artery disease (CAD). Endothelial progenitor cells (EPCs) play a fundamental role in postnatal vascular repair and CAD. The role of microRNAs in CAD pathogenesis and their potential as biomarkers remain to be elucidated.

Approach And Results: MicroRNA-31 (miR-31) level in both the plasma and EPCs of patients with CAD is found lower. miR-31 regulates EPC activities by targeting FAT atypical cadherin 4 and thromboxane A2 receptor, which show increased expression in CAD EPCs. Overexpressing miR-31 in CAD EPCs rescued their angiogenic and vasculogenic abilities both in vitro and in vivo. When exploring approaches to restore endogenous miR-31, we found that far-infrared treatment enhanced the expression of not only miR-31, but also miR-720 in CAD EPCs. miR-720, which was also decreased in EPCs and the plasma of patients with CAD, stimulated EPC activity by targeting vasohibin 1. The miR720-vasohibin 1 pair was shown to be downstream of FAT atypical cadherin 4, but not of thromboxane A2 receptor. FAT atypical cadherin 4 inhibited miR-720 expression via repression of the planar cell polarity signaling gene four-jointed box 1 (FJX1), which was required for miR-720 expression through a hypoxia-inducible factor 1, α subunit-dependent mechanism. Restoring miR-720 level strengthened activity of CAD EPCs. The miR-31-miR-720 pathway is shown critical to EPC activation and that downregulation of this pathway contributes to CAD pathogenesis. Circulating levels of miR-31, miR-720, and vasohibin 1 have the potential to allow early diagnosis of CAD and to act as prognosis biomarkers for CAD and other EPC-related diseases.

Conclusions: Manipulating the expression of the miR-31-miR-720 pathway in malfunction EPCs should help develop novel therapeutic modalities.
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http://dx.doi.org/10.1161/ATVBAHA.113.303001DOI Listing
April 2014

Differential expression of distinct surface markers in early endothelial progenitor cells and monocyte-derived macrophages.

Gene Expr 2013 ;16(1):15-24

Division of Cardiology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.

Bone marrow-derived endothelial progenitor cells (EPCs) play a fundamental role in postnatal angiogenesis. Currently, EPCs are defined as early and late EPCs based on their biological properties and their time of appearance during in vitro culture. Reports have shown that early EPCs share common properties and surface markers with adherent blood cells, especially CD14+ monocytes. Distinguishing early EPCs from circulating monocytes or monocyte-derived macrophages (MDMs) is therefore crucial to obtaining pure endothelial populations before they can be applied as part of clinical therapies. We compared the gene expression profiles of early EPCs, blood cells (including peripheral blood mononuclear cells, monocytes, and MDMs), and various endothelial lineage cells (including mature endothelial cells, late EPCs, and CD133+ stem cells). We found that early EPCs expressed an mRNA profile that showed the greatest similarity to MDMs than any other cell type tested. The functional significance of this molecular profiling data was explored by Gene Ontology database search. Novel plasma membrane genes that might potentially be novel isolation biomarkers were also pinpointed. Specifically, expression of CLEC5A was high in MDMs, whereas early EPCs expressed abundant SIGLEC8 and KCNE1. These detailed mRNA expression profiles and the identified functional modules will help to develop novel cell isolation approaches that will allow EPCs to be purified; these can then be used to target cardiovascular disease, tumor angiogenesis, and various ischemia-related diseases.
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http://dx.doi.org/10.3727/105221613x13776146743307DOI Listing
January 2014