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PDF - 119K, Legends for Figures 1-8.

PDF File - 97K, Homo sapiens microRNA pri-miR-21, complete sequence.

PDF - 655K, The effects of T3, miR-21 and TIAM1 in SK-Hep1 cells.

PDF - 57K, Basic clinical characterization of 40 patients with hepatoma.

PDF File - 47K, Effect of T3 on primary miR-21 in HepG2-TR�1 cells.

PDF - 179K, MSH2 is a miR-21 target downregulated by T3.

PDF - 55K, miRNAs differentially expressed following T3 treatment in HepG2-TR�1.

PDF File - 46K, Endogenous protein expression of TR�1 in various types of hepatoma cell lines.

PDF - 144K, Schematic effects of miR-21 regulated by T3 on cell migration and invasion.

PDF File - 116K, Full-length 3'UTR sequence of TIAM1 and MSH2.

PDF - 38K, Sequence of primary miR-21 promoter region (-3000/+1).

PDF - 55K, Expression of TR�1, miR-21 and TIAM1 from 40 patients with Hepatoma.

Background Hepatocellular carcinoma (HCC) is one of the most common primary liver malignancies worldwide. The long-term prognosis for HCC remains extremely poor, with drug resistance being the major underlying cause of recurrence and mortality. The lncRNA colorectal neoplasia differentially expressed (CRNDE) is an epigenetic mediator and plays an important role to drive proliferation and drug resistance in HCC. However, CRNDE as an epigenetic regulator with influences sorafenib resistance in HCC is unclear. Thus, we explore the potential of targeting the CRNDE/p300/YY1 axis as a novel therapeutic strategy to overcome sorafenib resistance of HCC. Method Detection of the expression level of CRNDE and EGFR in clinical specimens of HCC. CRNDE, EGFR, p300, and YY1expression were altered in HCC cells through transfection with different plasmids, and cell proliferation, migration, invasion, and sorafenib resistance were subsequently observed. Immunoprecipitation, chromatin immunoprecipitation, re-chromatin immunoprecipitation, site-directed mutagenesis, RNA Immunoprecipitation, immune fluorescence, qRT-PCR, and western blotting were performed to uncover the mechanisms of CRNDE regulation. The xenograft nude mice model was used to investigate the tumor growth and sorafenib resistance. Results In this study, we showed that CRNDE expression is significantly positively correlated with that of epidermal growth factor receptor (EGFR) in clinical specimens of HCC and induces proliferation and sorafenib resistance of HCC via EGFR-mediated signaling. Mechanistically, CRNDE stabilized the p300/YY1 complex at the EGFR promoter and simultaneously enhanced histone H3K9 and H3K27 acetylation, which serve as markers of relaxed chromatin. EGFR was positively upregulated by the epigenetic complex, p300/YY1, in a manner dependent on CRNDE expression, leading to enhanced tumor cell proliferation and sorafenib resistance. Furthermore, C646, a p300 inhibitor, suppressed EGFR transcriptional activity by decreasing chromatin relaxation and YY1 binding, which effectively reduced proliferation/sorafenib resistance and prolonged overall survival. Conclusion Our collective findings support the potential of targeting the CRNDE/p300/YY1 axis as a novel therapeutic strategy to overcome sorafenib resistance of HCC.

Background: Decisions regarding the staging, prognosis, and treatment of patients with head and neck squamous cell carcinomas (HNSCCs) are made after determining their p16 expression levels and human papillomavirus (HPV) infection status. Methods: We investigated the prognostic roles of p16-positive and p16-negative circulating tumor cells (CTCs) and their cell counts in HNSCC patients. We enrolled patients with locally advanced HNSCCs who received definitive concurrent chemoradiotherapy for final analysis. We performed CTC testing and p16 expression analysis before chemoradiotherapy. We analyzed the correlation between p16-positive and p16-negative CTCs and HPV genotyping, tissue p16 expression status, response to chemoradiotherapy, disease-free survival, and overall survival. Results: Forty-one patients who fulfilled the study criteria were prospectively enrolled for final analysis. The detection rates of p16-positive (&gt, 0 cells/mL blood) and p16-negative (≥3 cells/mL blood) CTCs were 51.2% (n = 21/41) and 70.7%, respectively. The best responses of chemoradiotherapy and the p16 positivity of CTCs are independent prognostic factors of disease progression, with hazard ratios of 1.738 (95% confidence interval (CI): 1.031–2.927), 5.497 (95% CI: 1.818–16.615), and 0.176 (95% CI: 0.056–0.554), respectively. The p16 positivity of CTCs was a prognostic factor for cancer death, with a hazard ratio of 0.294 (95% CI: 0.102–0.852). Conclusions: The p16-positive and p16-negative CTCs could predict outcomes in HNSCC patients receiving definitive chemoradiotherapy. This non-invasive CTC test could help stratify the risk and prognosis before chemoradiotherapy in clinical practice and enable us to perform de-intensifying therapies.

The heterogeneity of the drug resistance of cancer cells in a tumor is regarded as an important cause of therapeutic resistance and thus treatment failure. An understanding of the heterogeneity of cancer cells in a tumor in terms of their anti-cancer drug resistance is of great value for clinical applications or fundamental studies. To achieve this goal, a combination of cell-based drug testing and optically induced dielectrophoresis (ODEP)-based cell manipulation for sorting, separation, and isolation of drug-treated cells with various degrees of cell viability is proposed. For the latter, the key working principle is based on the difference in the ODEP force generated on the cells with various degrees of viability. To test the proposed idea, an ODEP microfluidic system was designed and fabricated in which two types of ODEP-based cell manipulation schemes were tested. The results successfully demonstrated that the proof-of-concept and practical application schemes were capable of effectively sorting, separating, and isolating doxorubicin-treated Dx5 (i.e., cells with drug resistance) and MES-SA (i.e., cells without drug resistance) cells that exhibited various degrees of viability and levels of anti-cancer drug resistance gene (i.e., ABCB1) expression. Moreover, the results also demonstrated that the proposed technique was capable of sorting and separating cell of the same type (i.e., drug-treated Dx5 cells) but different degrees of viability and anti-cancer drug resistance gene expression levels. Overall, this study presents a technique that is able to effectively sort, separate, and isolate drug-treated cancer cells with phenotypic heterogeneity for subsequent clinical applications or fundamental studies.

Circulating tumor cell (CTC) clusters play a more critical role in cancer metastasis than single CTCs. The study of CTC clusters might therefore provide more biologically meaningful information for a clear understanding of cancer metastasis. To achieve this goal, the isolation of high-purity, and integral CTC clusters from the blood samples of cancer patients is required. We herein propose to integrate optically-induced-dielectrophoresis (ODEP)-based cell manipulation with a microfluidic system for isolating cancer cell clusters based on their size characteristics. In this work, a two-step ODEP-based cell manipulation was designed. A dynamic square light image array was designed for the preliminary isolation of cancer cell clusters, followed by the utilization of another ODEP-based cell manipulation to refine the purity of the cancer cell clusters harvested in the first step. In this study, the optimal ODEP operating conditions for cell isolation process were experimentally determined. Moreover, the performance of cell isolation was experimentally evaluated for the isolation of CTC clusters with a cell purity and recovery rate of 91.5 ± 5.6%, and 70.5 ± 5.2%, respectively, without compromising the integrity of cancer cell clusters. Overall, this study presents a method and device capable of isolating high-purity and integral cancer cell clusters.

Simple Summary Liver cancer has a high mortality rate. Here, we retrospectively discuss the current progress and dilemmas in the clinical research and treatment of liver cancer. We primarily focus on microRNAs because of their extremely high value in applications and research. We discuss whether microRNAs can be used for the development of better biomarkers and/or therapeutic drugs, and address the difficulties, requirements for improved diagnostic technologies, and side effects related to microRNA-based drugs. Abstract Liver cancer is the leading cause of cancer-related mortality in the world. This mainly reflects the lack of early diagnosis tools and effective treatment methods. MicroRNAs (miRNAs) are a class of non-transcribed RNAs, some of which play important regulatory roles in liver cancer. Here, we discuss microRNAs with key impacts on liver cancer, such as miR-122, miR-21, miR-214, and miR-199. These microRNAs participate in various physiological regulatory pathways of liver cancer cells, and their modulation can have non-negligible effects in the treatment of liver cancer. We discuss whether these microRNAs can be used for better clinical diagnosis and/or drug development. With the advent of novel technologies, fast, inexpensive, and non-invasive RNA-based biomarker research has become a new mainstream approach. However, the clinical application of microRNA-based markers has been limited by the high sequence similarity among them and the potential for off-target problems. Therefore, researchers particularly value microRNAs that are specific to or have special functions in liver cancer. These include miR-122, which is specifically expressed in the liver, and miR-34, which is necessary for the replication of the hepatitis C virus in liver cancer. Clinical treatment drugs have been developed based on miR-34 and miR-122 (MRX34 and Miravirsen, respectively), but their side effects have not yet been overcome. Future research is needed to address these weaknesses and establish a feasible microRNA-based treatment strategy for liver cancer.

The current approaches for the characterization of cellular electrical properties normally require a tested sample with high cell quantity, restricting their application in the cases in which the cell number in a sample is limited. To address this issue, this study presented a low-sample-loss microfluidic system capable of characterizing the size-independent electrical properties (e.g., specific membrane capacitance and cytoplasm conductivity) of single cells. In the design, a capillary tube was used to transfer cells directly into the entrance of a microfluidic constriction microchannel to possibly minimize cell sample loss. Results revealed that the microfluidic system was able to significantly reduce the sample loss phenomenon, by which the cell processing ratio could be raised from the original 0.2% to 49.3–60.0%. As a demonstration, moreover, the feasibility of using the proposed method for the identification of the EpCAM-positive CTCs after a CTC isolation process, and for the differentiation of the EpCAM-positive CTCs of three different (hepatic, oral and lung) cancer types were experimentally evaluated. Within the experimental conditions explored, our findings indicated that the proposed method was able to significantly identify the EpCAM-positive CTCs from the surrounding EpCAM-negative cells, as well as to differentiate the EpCAM-positive CTCs of the three different cancer types tested, based on their unique electrical properties of cells. As a whole, the proposed microfluidic system was found useful for the identification of CTCs after a conventional CTC isolation process. In addition to CTC identification, the proposed method might hold potential for the detection of a specific cell species in a cell suspension sample with limited cell quantity.

Hypoglycemia is one of the most common issues encountered in daily emergency practice. In addition to the treatment of hypoglycemia, certain other situations concomitant with hypoglycemia require further treatment. The aim of the present study was to compare demographic and clinical characteristics of infected [urinary tract infection (UTI), pneumonia or biliary tract infection (BTI)] vs. non-infected hypoglycemic patients to establish which hypoglycemic patients required further observation or hospitalization. This was a retrospective cross-sectional study of hypoglycemic (

High purity isolation of circulating tumor cells (CTCs) is important for their subsequent gene-related analysis. Nevertheless, the conventional CTC isolation schemes might not be able to avoid the contamination of leukocytes in a treated sample. To address this issue, we proposed to integrate the technique of optically induced dielectrophoresis (ODEP)-based cell manipulation, and flow velocity control in a microfluidic system for further isolating CTCs after a conventional CTC isolation process. The working principle was based on the cell size difference between the CTCs and leukocytes. In the ODEP microfluidic system, a four-cascade cell isolation using four optical light-based virtual cell filters was designed. Based on this, four different selection conditions were simultaneously implemented for a higher-resolution cell separation, and thus higher-purity cell isolation. In this work, the ODEP microfluidic system was designed and fabricated. Moreover, the optimal ODEP operating conditions such as light bar width (40 μm), gap (80 μm), and number (4), as well as the sample flow rate (0.4 μl min −1 ) were experimentally determined. Results revealed the presented method was able to isolate cancer cells with cell purity as high as 94.9 ± 0.3% (cancer cell recovery rate: 54 ± 7%). Overall, this study has presented an ODEP microfluidic system capable of refining CTC purity after a conventional CTC isolation process.

Objective Hypoglycaemia is a common complication of diabetes mellitus. Previous studies suggest that hypoglycaemic episodes may occur with other comorbidities, influencing the outcome of recovery. Recognising the predisposing factors for hypoglycaemic episodes in the emergency department is important. Therefore, we investigated the characteristics of and predisposing factors for hypoglycaemia in the emergency department. Methods Data from 186 patients were retrospectively collected from a medical centre in northern Taiwan. We divided the patients into the advanced-age group (132 patients) and the younger group (54 patients). Associated data collected for statistical analysis included vital signs on arrival, first measured blood glucose level, laboratory results, related comorbidities, length of hospital stay, and survival to discharge. Results Hypoglycaemia was more frequently observed in women in the advanced-age group than in the younger group. Tachycardia and elevated systolic blood pressure were less predominant in the advanced-age group than younger group. More patients in the advanced-age group had concurrent infection, and more patients in the younger group had liver dysfunction, elevated liver enzymes, liver cirrhosis, and concurrent stroke. Conclusions Closer attention should be paid to the possibility of infection in patients of advanced age. Liver disease and stroke need to be ruled out in younger patients.

NEK2 (NIMA-related expressed kinase 2) is a serine/threonine centrosomal kinase that acts as a critical regulator of centrosome structure and function. Aberrant NEK2 activities lead to failure in regulating centrosome duplication. NEK2 overexpression promotes tumorigenesis and is associated with poor prognosis in several cancers. Increased NEK2 expression during the late pathological stage has been detected in the Oncomine liver dataset and hepatocellular carcinoma (HCC) specimens. Elevated NEK2 protein is associated with poor overall survival in patients with HCC. However, the precise roles and mechanisms of NEK2 in liver cancer progression remain largely unknown. An earlier functional study revealed that NEK2 mediates drug resistance (cisplatin or lipo-doxorubicin) via expression of an ABCC10 transporter. Active angiogenesis and metastasis underlie the rapid recurrence and poor survival of HCC. Results from the current study showed that NEK2 mediates tumor growth, metastasis and angiogenesis in vivo. NEK2-mediated drug resistance was blocked by a specific PI3K or AKT inhibitor. Moreover, NEK2 mediated liver cancer cell migration via pAKT/NF-κB signaling and matrix metalloproteinase (MMP) activation. Angiogenesis was induced via the same signaling pathway and IL-8 stimulation. Our findings collectively indicate that NEK2 modulates hepatoma cell functions, including growth, drug resistance, metastasis and angiogenesis via downstream genes activation.

Isolation of high-purity, label-free, and viable circulating tumour cells (CTCs) from cancer patients is crucial for subsequent analyses. To address this issue, a two-step CTC isolation scheme was proposed, wherein a spheroid cell culture was used to further purify viable CTCs after conventional negative selection-based CTC isolation methods. Our results from a cancer cell line model revealed that the survival of leukocytes in spheroid cell cultures was significantly decreased with time, whereas OECM-1 cells maintained viability and proliferated. Therefore, such a cell culture operation was expected to increase cancer cell purity in the cell spheroids. This assumption was confirmed by our results, which showed that cancer cell purities were 10.6 to 80.3-fold increased after spheroid cell culture for 8 days. In the following clinical tests, CTC-related cells were observed in 6 of 13 blood samples. Furthermore, the average purity of CTC-related cells was 34.8 ± 14.0%. By utilizing a second-step spheroid cell culture operation, the purity of CTC-related cells was greatly improved when compared with that (less than 10%) achievable by conventional negative selection-based CTC isolation. Overall, this study proposed a two-step process for the isolation of high-purity, label-free, and viable CTCs.

Circulating tumour cells (CTCs) in blood circulation play an important role in cancer metastasis. CTCs are generally defined as the cells in circulating blood expressing the surface antigen EpCAM (epithelial cell adhesion molecule). Nevertheless, CTCs with a highly metastatic nature might undergo an epithelial-to-mesenchymal transition (EMT), after which their EpCAM expression is downregulated. In current CTC-related studies, however, these clinically important CTCs with high relevance to cancer metastasis could be missed due to the use of the conventional CTC isolation methodologies. To precisely explore the clinical significance of these cells (i.e., CD45neg/EpCAMneg cells), the high-purity isolation of these cells from blood samples is required. To achieve this isolation, the integration of fluorescence microscopic imaging and optically induced dielectrophoresis (ODEP)-based cell manipulation in a microfluidic system was proposed. In this study, an ODEP microfluidic system was developed. The optimal ODEP operating conditions and the performance of live CD45neg/EpCAMneg cell isolation were evaluated. The results demonstrated that the proposed system was capable of isolating live CD45neg/EpCAMneg cells with a purity as high as 100%, which is greater than the purity attainable using the existing techniques for similar tasks. As a demonstration case, the cancer-related gene expression of CD45neg/EpCAMneg cells isolated from the blood samples of healthy donors and cancer patients was successfully compared. The initial results indicate that the CD45neg/EpCAMneg nucleated cell population in the blood samples of cancer patients might contain cancer-related cells, particularly EMT-transformed CTCs, as suggested by the high detection rate of vimentin gene expression. Overall, this study presents an ODEP microfluidic system capable of simply and effectively isolating a specific, rare cell species from a cell mixture.

Background & Aims Thyroid hormone (T 3 ) and its receptor (TR) are involved in cell growth and cancer progression. Although deregulation of microRNA (miRNA) expression has been detected in many tumor types, the mechanisms underlying functional impairment and specific involvement of miRNAs in tumor metastasis remain unclear. In the current study, we aimed to elucidate the involvement of deregulated miRNA-130b (miR-130b) and its target genes mediated by T 3 /TR in cancer progression. Methods Quantitative reverse transcription-PCR, luciferase and chromatin immunoprecipitation assays were performed to identify the miR-130b transcript and the mechanisms implicated in its regulation. The effects of miR-130b on hepatocellular carcinoma (HCC) invasion were further examined in vitro and in vivo . Clinical correlations among miR-130b, TRs and interferon regulatory factor 1 (IRF1) were examined in HCC samples using Spearman correlation analysis. Results Our experiments disclosed negative regulation of miR-130b expression by T 3 /TR. Overexpression of miR-130b led to marked inhibition of cell migration and invasion, which was mediated via suppression of IRF1. Cell migration ability was promoted by T 3 , but partially suppressed upon miR-130b overexpression. Furthermore, miR-130b suppressed expression of epithelial-mesenchymal transition (EMT)-related genes, matrix metalloproteinase-9, phosphorylated mammalian target of rapamycin (mTOR), p-ERK1/2, p-AKT and p-signal transducer and activator of transcription (STAT)-3. Notably, miR-130b was downregulated in hepatoma samples and its expression patterns were inversely correlated with those of TRα1 and IRF1. Conclusions Our data collectively highlight a novel pathway interlinking T 3 /TR, miR-130b, IRF1, the EMT-related genes, p-mTOR, p-STAT3 and the p-AKT cascade, which regulates the motility and invasion of hepatoma cells.

Objective To study the epidemiology, etiologies and prognostic factors of hypoglycemia. Methods A retrospective chart review of hypoglycemic cases from December, 2009 to February, 2012 was conducted to gather the following patient data: age, gender, vital signs at triage, white blood cell count, serum glucose, C-reactive protein, glutamic oxaloacetic transaminase, creatinine, sodium, potassium, past history of liver cirrhosis, uremia, concomitant infection, concomitant cancer/malignancy, length of stay, lack of recent meal, status of acute renal failure and concomitant stroke. A total of 186 cases were enrolled in our study. We analyzed the data using commercial statistics software (SPSS for Windows, version 11.0, SPSS Inc., Chicago, IL). We used the Student's t-test and χ2 test for the statistical analyses, and significance was set at a P value less than 0.05. Results Hypoglycemia is related to several co-morbidities. In total, 10.2% of the patients had liver cirrhosis and 7.0% had uremia. More than half (55.4%) were bacterial infection during hospitalization. Acute renal failure accounted for 26.3% of the hypoglycemic episodes. In addition to the etiology of infection, the lack of a recent meal accounted for 44.6% hypoglycemic episodes. A total of 2.2% of the cases resulted from an acute cerebrovascular accident. Approximately 8.6% were concomitant with malignancy. Conclusions When hypoglycemic patients present in the emergency department, physicians should pay attention to the presence of infection, malignancy, liver diseases (liver cirrhosis and biliary tract infection), and acute renal failure.

The thyroid hormone, 3,3',5-triiodo-l-thyronine (T3 ), mediates several physiological processes, including embryonic development, cellular differentiation, metabolism and regulation of cell proliferation. Thyroid hormone (T3 ) and its receptor (TR) are involved in metabolism and growth. In addition to their developmental and metabolic functions, TRs play a tumor suppressor role, and therefore, their aberrant expression can lead to tumor transformation. Aberrant epigenetic silencing of tumor suppressor genes promotes cancer progression. The epigenetic regulator, Ubiquitin-like with PHD and ring finger domains 1 (UHRF1), is overexpressed in various cancers. In our study, we demonstrated that T3 negatively regulates UHRF1 expression, both in vitro and in vivo. Our results further indicate that UHRF1 regulation by T3 is indirect and mediated by Sp1. Sp1-binding elements of UHRF1 were identified at positions -664/-505 of the promoter region using the luciferase and chromatin immunoprecipitation assays. Notably, UHRF1 and Sp1 levels were elevated in subgroups of hepatocellular carcinoma patients and inversely correlated with TRα1 expression. Knockdown of UHRF1 expression should therefore provide a means to inhibit hepatoma cell proliferation. Expression of UHRF1 was downregulated by TRs, in turn, relieving silencing of the UHRF1 target gene, p21. Based on the collective findings, we propose that T3 /TR signaling induces hepatoma cell growth inhibition via UHRF1 repression.

Magnetic microbeads are widely utilized in microfluidic systems for various applications. For these tasks, the effective and efficient manipulation of magnetic microbeads is important. Among the techniques for microparticle manipulation, the optically induced electrokinetic (OEK) [e.g., optically induced dielectrophoresis (ODEP) or light-actuated AC electroosmosis (LACE)]-based technique is promising. However, its utilization for magnetic microbead manipulation in a microfluidic system has not yet been fundamentally studied. To address this issue, the effect of operating conditions on the OEK-based magnetic microbead manipulation investigated. The results showed that the maximum terminal velocity (Vterminal) of a light image that can manipulate microbeads decreased significantly with increasing AC frequency. In addition, the results revealed that the Vterminal increased with increasing intrinsic a-Si:H layer thickness (Ta-Si) when the AC frequency was higher than 30 kHz, whereas the results completely reversed when the frequency was lower than 30 kHz. Additionally, the Vterminal of larger magnetic microbeads was higher than that of smaller microbeads when the AC frequency was higher than 50 kHz; conversely, the results significantly reversed when the frequency was lower than 30 kHz. Moreover, microbead manipulation under high conductivity conditions could significantly affect the magnitude of the Vterminal. Based on the fast manipulation velocity in LACE-based microbead manipulation, its application for basic unit operations in a microfluidic system was demonstrated. Overall, this study has provided some fundamental information for the selection of optimum operating conditions for OEK-based magnetic microbead manipulation in a microfluidic system.

Conventional positive and negative selection-based circulating tumor cell (CTC) isolation methods might generally ignore metastasis-relevant CTCs that underwent epithelial-to- mesenchymal transition and suffer from a low CTC purity problem, respectively. To address these issues, we previously proposed a 2-step CTC isolation method integrating a negative selection CTC isolation and subsequent spheroid cell culture. In addition to its ability to isolate CTCs, more importantly, the spheroid cell culture used could serve as a cell culture model mimicking the process of new tumor tissue formation during cancer metastasis. Therefore, it is promising not only to selectively isolate metastasis-relevant CTCs but also to test the potential of cancer metastasis and thus the prognosis of disease. To explore these issues, experiments were performed. The key findings of this study demonstrated that the method was able to harvest both epithelial (E)- and mesenchymal (M)-type CTCs without selection bias. Moreover, both the M-type CTC count and the information obtained from the multidrug resistance-associated protein 2 (MRP2) and MRP5 gene expression analysis of the CTCs isolated via the 2-step CTC isolation method might be able to serve as prognostic factors for progression-free survival in head and neck squamous cell carcinoma.

The thyroid hormone, T3, regulates cell growth, differentiation and development through binding to the nuclear thyroid hormone receptor (THR), a member of the steroid/TR superfamily of ligand-dependent transcriptional factors. T3 modulates lipid metabolism in liver, although the detailed molecular mechanisms are unclear at present. Here, by a microarray analysis, we identified a novel chromosome 19 open reading frame 80 (C19orf80) which was activated by T3. T3 stimulation led to upregulation of both mRNA and protein levels of C19orf80. Immunofluorescence analysis revealed a vesicle-like pattern of C19orf80 around lipid droplets or within the lysosome-associated compartment in cells. Furthermore, T3 treatment as well as C19orf80 overexpression specifically activated the autophagic response and lipid metabolism, as observed from lipidated LC3 (LC3-II) and levels of oxygen consumption rate, respectively. Reciprocally, knockdown of C19orf80 obstructed T3-activated autophagy and lipolysis. Moreover, treatment with autolysosome maturation inhibitors, ammonium chloride and chloroquine, not only suppressed the T3-activated autophagic process but also lipid metabolism. Our results collectively suggested that T3 regulates lipid metabolism through a C19orf80-activated autophagic process.

NEK2 (NIMA-related expressed kinase 2) is a serine/threonine centrosomal kinase that acts as a critical regulator of centrosome structure and function. Aberrant NEK2 activities lead to failure in regulating centrosome duplication. NEK2 overexpression promotes tumorigenesis and is associated with poor prognosis in several cancers. Increased NEK2 expression during the late pathological stage has been detected in the Oncomine liver dataset and hepatocellular carcinoma (HCC) specimens. Elevated NEK2 protein is associated with poor overall survival in patients with HCC. However, the precise roles and mechanisms of NEK2 in liver cancer progression remain largely unknown. An earlier functional study revealed that NEK2 mediates drug resistance (cisplatin or lipo-doxorubicin) via expression of an ABCC10 transporter. Active angiogenesis and metastasis underlie the rapid recurrence and poor survival of HCC. Results from the current study showed that NEK2 mediates tumor growth, metastasis and angiogenesis in vivo. NEK2-mediated drug resistance was blocked by a specific PI3K or AKT inhibitor. Moreover, NEK2 mediated liver cancer cell migration via pAKT/NF-κB signaling and matrix metalloproteinase (MMP) activation. Angiogenesis was induced via the same signaling pathway and IL-8 stimulation. Our findings collectively indicate that NEK2 modulates hepatoma cell functions, including growth, drug resistance, metastasis and angiogenesis via downstream genes activation.

Although accumulating evidence has confirmed the important roles of thyroid hormone (T(3)) and its receptors (TRs) in tumor progression, the specific functions of TRs in carcinogenesis remain unclear. In the present study, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) was directly upregulated by T(3) in TR-overexpressing hepatoma cell lines. TRAIL is an apoptotic inducer, but it can nonetheless trigger non-apoptotic signals favoring tumorigenesis in apoptosis-resistant cancer cells. We found that TR-overexpressing hepatoma cells treated with T(3) were apoptosis resistant, even when TRAIL was upregulated. This apoptotic resistance may be attributable to simultaneous upregulation of Bcl-xL by T(3), because (1) knockdown of T(3)-induced Bcl-xL expression suppressed T(3)-mediated protection against apoptosis, and (2) overexpression of Bcl-xL further protected hepatoma cells from TRAIL-induced apoptotic death, consequently leading to TRAIL-promoted metastasis of hepatoma cells. Moreover, T(3)-enhanced metastasis in vivo was repressed by the treatment of TRAIL-blocking antibody. Notably, TRAIL was highly expressed in a subset of hepatocellular carcinoma (HCC) patients, and this high-level expression was significantly correlated with that of TRs in these HCC tissues. Together, our findings provide evidence for the existence of a novel mechanistic link between increased TR and TRAIL levels in HCC. Thus, TRs induce TRAIL expression, and TRAIL thus synthesized acts in concert with simultaneously synthesized Bcl-xL to promote metastasis, but not apoptosis.

Thyroid hormone (T3) mediates cellular growth, development, and differentiation by binding to the nuclear thyroid hormone receptor (TR). Recent studies suggest that long-term hypothyroidism is associated with human hepatocellular carcinoma (HCC) independent from other major HCC risk factors. Dickkopf (DKK) 4, a secreted protein, antagonizes the Wnt signal pathway. In this study, we demonstrate that T3 may play a suppressor role by inducing DKK4 expression in HCC cells at both the messenger RNA (mRNA) and protein levels. DKK4 was down-regulated in 67.5% of HCC cancerous tissues. The decrease in DKK4 levels was accompanied by a concomitant decrease in TR protein levels in the matched cancerous tissues in 31% of tissues compared by immunoblotting with the adjacent noncancerous tissues. Further, TR and DKK4 expression levels were positively correlated in both normal and cancerous specimens by tissue array analysis. In function assays, stable DKK4 transfected into J7 or HepG2 cells decreased cell invasion in vitro. Conversely, knocking down DKK4 restores cell invasiveness. DKK4-expressing J7 clones showed increased degradation of β-catenin, but down-regulation of CD44, cyclin D1, and c-Jun. To investigate the effect of DKK4 and TR on tumor growth in vivo, we established a xenograft of J7 cells in nude mice. J7-DKK4 and J7-TRα1 overexpressing mice, which displayed growth arrest, lower lung colony formation index, and smaller tumor size than in control mice, supporting an inhibitory role of DKK4 in tumor progression. Conclusion: Taken together, these data suggest that the TR/DKK4/Wnt/β-catenin cascade influences the proliferation and migration of hepatoma cells during the metastasis process and support a tumor suppressor role of the TR. (Hepatology 2012)

Human papilloma virus infection is critical but not sufficient to cause cervical cancer. Molecular markers of cervical carcinogenesis are essential. The aim of this study was to identify aberrantly expressed proteins in cervical cancer and determine their clinical significance. A two-dimensional polyacrylamide gel electrophoresis (2-DE) proteomic strategy was used for screening candidate proteins. Immunoblotting and immunohistochemical (IHC) analyses were performed to confirm the results of 2-DE, and the clinical significance was estimated. Glucose-regulated protein 58 (Grp58) was overexpressed in 73% of cancers. The IHC staining showed that the Grp58 histoscore was significantly higher in patients with adenocarcinoma (AD) compared with squamous cell carcinoma (P < 0.05). Grp58 staining was intense in AD with a penetration depth greater than half of the cervical stroma (P = 0.033). High Grp58 expression was associated with low overall survival and recurrence-free survival (RFS) rates (P = 0.007 and P = 0.013, respectively). In multivariate analysis, high Grp58 expression (P = 0.042) and lymph node metastasis (P = 0.026) were determined as independent prognostic factors for RFS. Patients exhibiting both high Grp58 expression and lymph node metastasis displayed poorer outcomes than the other patient groups. In functional studies, knockdown of Grp58 in HeLa cells led to decreased cell invasiveness and inhibition of lung metastasis in a xenograft mouse model. In conclusion, Grp58 serves as a potent prognostic factor of cervical AD. Estimation of the Grp58 index in conjunction with the lymph node metastasis status might aid in predicting the prognosis of cervical AD.

Cervical carcinoma is the second most common cause of death from gynecological cancers worldwide. Knowledge of the molecular mechanisms underlying the tumorigenesis of cervical cancer cell, except human papilloma virus infection, is limited.A microarray was used to study the differential expression of genes in cancerous tissues to identify new molecular markers for diagnosis and prognosis. Their differential expression was confirmed with Western blotting and immunohistochemical analyses. The clinical correlations and prognostic significance of the aberrantly expressed proteins were evaluated to identify novel biomarkers of cervical cancer.The expression of gelsolin was significantly upregulated in 78% of patients with cervical cancer, and gelsolin was selected for further study. Gelsolin expression was stronger in cervical tumor tissues than in the surrounding noncancerous tissues (P0.001). Gelsolin expression in the plasma of cervical cancer patients was increased 2.2-fold compared with that of healthy control subjects (P0.001). The levels of plasma gelsolin in the early and late stages were significantly different (P=0.006). According to immunohistochemical analysis, increased gelsolin expression was associated with histological type and FIGO stage II. The 5-year overall survival and recurrence-free survival rates for the low-expression group (cut-off=115) were significantly higher than those of the high-expression group. Cancer cells with reduced gelsolin expression exhibited reduced migration and proliferation.These results provide strong evidence that gelsolin plays an important role in cellular proliferation and migration in cervical cancer and suggest that gelsolin is a promising marker for cervical cancer screening and prognosis.

The thyroid hormone 3,3′,5-triiodo-l-thyronine (T3) regulates growth, development, and differentiation processes in animals. These activities are mediated by the nuclear thyroid hormone receptors (TRs). Microarray analyses were performed previously to study the mechanism of regulation triggered by T3 treatment in hepatoma cell lines. The results showed that spondin 2 was regulated positively by T3. However, the underlying mechanism and the physiological role of T3 in the regulation of spondin 2 are not clear. To verify the microarray results, spondin 2 was further investigated using semi-quantitative reverse transcription-PCR and western blotting. After 48 h of T3 treatment in the HepG2–TRα1#1 cell line, spondin 2 mRNA and protein levels increased by 3.9- to 5.7-fold. Similar results were observed in thyroidectomized rats. To localize the regulatory region in spondin 2, we performed serial deletions of the promoter and chromatin immunoprecipitation assays. The T3 response element on the spondin 2 promoter was localized in the −1104/−1034 or −984/−925 regions. To explore the effect of spondin 2 on cellular function, spondin 2 knockdown cell lines were established from Huh7 cells. Knockdown cells had higher migration ability and invasiveness compared with control cells. Conversely, spondin 2 overexpression in J7 cells led to lower migration ability and invasiveness compared with control cells. Furthermore, this study demonstrated that spondin 2 overexpression in some types of hepatocellular carcinomas is TR dependent. Together, these experimental findings suggest that spondin 2, which is regulated by T3, has an important role in cell invasion, cell migration, and tumor progression.

The thyroid hormone receptor (TR) and human testicular orphan receptor 4 (TR4) belong to the nuclear hormone receptor superfamily. They are ligand-dependent transcription factors. TR and TR4 bind to a similar thyroid response element (TRE), known as a direct repeat with four nucleotide spacing (DR4). This study examined the possible interaction or cross-talking between those two receptors. We hypothesized that protein-protein interaction between TR4 and TR may promote TR-mediated transcriptional activity. Glutathione S-transferase pull-down and immunoprecipitation assays showed direct interaction between TR and TR4. Electrophoretic mobility-shift assay demonstrated that TR and TR4 could co-occupy the same TRE. The interaction between TR4 and TR may enhance regulation of genes targeted by TR, such as furin, fibrinogen, cdk2 and p21 expression. We found that TR4 function is similar with TR as TR4 alone could regulate expression of some TR target genes, and could increase cell migration or inhibit cell proliferation. Importantly, the TR-dependent inhibition of cell proliferation and stimulation of cell migration are more enhanced in the HepG2-TR cells stably over-expressing TR4. Overall, TR4 not only has modulation abilities similar to TR but also can cross-talk with TR and promote the TR signaling pathway.

Cervical carcinoma is the third-most common cause of cancer-related deaths in women worldwide. However, the molecular mechanisms underlying the metastasis of cervical cancer are still unclear. Oligonucleotide microarrays coupled with bioinformatics analysis show that cytoskeletal remodeling and epithelial-to- mesenchymal transition (EMT) are significant pathways in clinical specimens of cervical cancer. In accord with clinical observations demonstrating ectopic expression of lipocalin 2 (LCN2), an oncogenic protein associated with EMT, in malignant tumors, was significantly upregulated in cervical cancer and correlated with lymph node metastasis. Overexpression of LCN2 enhanced tumor cell migration and invasion both in vitro and in vivo. Conversely, knockdown or neutralization of LCN2 reduced tumor cell migration and invasion. LCN2-induced migration was stimulated by activation of the EMT-associated proteins, Snail, Twist, N-cadherin, fibronectin, and MMP-9. Our findings collectively support a potential role of LCN2 in cancer cell invasion through the EMT pathway and suggest that LCN2 could be effectively utilized as a lymph node metastasis marker in cervical cancer.

// I-Hsiao Chung 1 , Cheng-Yi Chen 2 , Yang-Hsiang Lin 1 , Hsiang-Cheng Chi 1 , Ya-Hui Huang 3 , Pei-Ju Tai 1 , Chia-Jung Liao 1 , Chung-Ying Tsai 1 , Syuan-Ling Lin 1 , Meng-Han Wu 1 , Ching-Ying Chen 1 , Kwang-Huei Lin 1 1 Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan, Taiwan 2 Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan 3 Liver Research Center, Department of Hepato-Gastroenterology, Chang-Gung Memorial Hospital, Linkou, Taoyuan, Taiwan Correspondence to: Kwang-Huei Lin, e-mail: khlin@mail.cgu.edu.tw Keywords: thyroid hormone receptor, LCN2, Met/FAK cascade Received: December 02, 2014 Accepted: March 25, 2015 Published: April 10, 2015 ABSTRACT The thyroid hormone, 3,3′,5-triiodo-L-thyronine (T 3 ), regulates cell growth, development and differentiation via interactions with thyroid hormone receptors (TR), but the mechanisms underlying T 3 -mediated modulation of cancer progression are currently unclear. Lipocalin 2 (LCN2), a tumor-associated protein, is overexpressed in a variety of cancer types. Oligonucleotide microarray, coupled with proteomic analysis, has revealed that LCN2 is positively regulated by T 3 /TR. However, the physiological role and pathway of T 3 -mediated regulation of LCN2 in hepatocellular carcinogenesis remain to be characterized. Upregulation of LCN2 after T 3 stimulation was observed in a time- and dose-dependent manner. Additionally, TRE on the LCN2 promoter was identified at positions -1444/-1427. Overexpression of LCN2 enhanced tumor cell migration and invasion, and conversely, its knockdown suppressed migration and invasion, both in vitro and in vivo . LCN2-induced migration occurred through activation of the Met/FAK cascade. LCN2 was overexpressed in clinical hepatocellular carcinoma (HCC) patients, compared with normal subjects, and positively correlated with TRα levels. Both TRα and LCN2 showed similar expression patterns in relation to survival rate, tumor grade, tumor stage and vascular invasion. Our findings collectively support a potential role of T 3 /TR in cancer progression through regulation of LCN2 via the Met/FAK cascade. LCN2 may thus be effectively utilized as a novel marker and therapeutic target in HCC.