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1.  Xist Deficiency and Disorders of X-Inactivation in Rabbit Embryonic Stem Cells Can Be Rescued by Transcription-Factor-Mediated Conversion 
Stem Cells and Development  2014;23(19):2283-2296.
The deficiency of X-inactive specific transcript (XIST) on the inactive X chromosome affects the behavior of female human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), and further chromosomal erosion can occur with continued passaging of these cells. However, X chromosome instability has not been identified in other species. In the present study, we investigated three female rabbit ESC (rbESC) lines and found that two of them expressed Xist normally and obtained both Xist RNA coating and H3K27me3 foci, thus defined as XiXistXa. Interestingly, the third female rbESC line lacked Xist expression during ESC maintenance and differentiation. This line showed H3K27me3 foci but no Xist RNA coating in the early passages and was thus defined as Xiw/oXistXa. Similar to Xiw/oXistXa hESCs or hiPSCs, Xiw/oXistXa rbESCs lose H3K27me3 and undergo Xi erosion (Xe) with passaging. Moreover, Xist-deficient rbESCs also exhibit impaired differentiation ability and upregulation of cancer-related genes. By overexpressing OCT4, SOX2, KLF4, and c-MYC in Xist-deficient rbESCs under optimized culture conditions, we successfully obtained mouse ESC-like (mESC-like) cells. The mESC-like rbESCs displayed dome-shaped colony morphology, activation of the LIF/STAT3-dependent pathway, and conversion of disordered X chromosome. Importantly, the defective differentiation potential was also greatly improved. Our data demonstrate that variations in X chromosome inactivation occur in early passage of rbESCs; thus, Xi disorders are conserved across species and are reversible using the proper epigenetic reprogramming and culture conditions. These findings may be very useful for future efforts toward deriving fully pluripotent rbESCs or rabbit iPSCs (rbiPSCs).
PMCID: PMC4172558  PMID: 24805295
3.  Docosahexaenoic Acid Induces Cell Death in Human Non-Small Cell Lung Cancer Cells by Repressing mTOR via AMPK Activation and PI3K/Akt Inhibition 
BioMed Research International  2015;2015:239764.
The anticancer properties and mechanism of action of omega-3 polyunsaturated fatty acids (ω3-PUFAs) have been demonstrated in several cancers; however, the mechanism in lung cancer remains unclear. Here, we show that docosahexaenoic acid (DHA), a ω3-PUFA, induced apoptosis and autophagy in non-small cell lung cancer (NSCLC) cells. DHA-induced cell death was accompanied by AMP-activated protein kinase (AMPK) activation and inactivated phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling. Knocking down AMPK and overexpressing Akt increased mTOR activity and attenuated DHA-induced cell death, suggesting that DHA induces cell death via AMPK- and Akt-regulated mTOR inactivation. This was confirmed in Fat-1 transgenic mice, which produce ω3-PUFAs. Lewis lung cancer (LLC) tumor cells implanted into Fat-1 mice showed slower growth, lower phospho-Akt levels, and higher levels of apoptosis and autophagy than cells implanted into wild-type mice. Taken together, these data suggest that DHA-induced apoptosis and autophagy in NSCLC cells are associated with AMPK activation and PI3K/Akt inhibition, which in turn lead to suppression of mTOR; thus ω3-PUFAs may be utilized as potential therapeutic agents for NSCLC treatment.
PMCID: PMC4538321  PMID: 26339598
4.  miR-150 Deficiency Protects against FAS-Induced Acute Liver Injury in Mice through Regulation of AKT 
PLoS ONE  2015;10(7):e0132734.
Although miR-150 is implicated in the regulation of immune cell differentiation and activation, it remains unknown whether miR-150 is involved in liver biology and disease. This study was performed to explore the potential role of miR-150 in LPS/D-GalN and Fas-induced liver injuries by using wild type and miR-150 knockout (KO) mice. Whereas knockout of miR-150 did not significantly alter LPS/D-GalN-induced animal death and liver injury, it protected against Fas-induced liver injury and mortality. The Jo2-induced increase in serum transaminases, apoptotic hepatocytes, PARP cleavage, as well as caspase-3/7, caspase-8, and caspase-9 activities were significantly attenuated in miR-150 KO mice. The liver tissues from Jo2-treated miR-150 KO mice expressed higher levels of Akt1, Akt2, total Akt, as well as p-Akt(Ser473) compared to the wild type livers. Pretreatment with the Akt inhibitor V reversed Jo2-induced liver injury in miR-150 KO mice. The primary hepatocytes isolated from miR-150 KO mice also showed protection against Fas-induced apoptosis in vitro (characterized by less prominent PARP cleavage, less nuclear fragmentation and less caspase activation) in comparison to hepatocytes from wild type mice. Luciferase reporter assays in hepatocytes transfected with the Akt1 or Akt2 3’-UTR reporter constructs (with or without mutation of miR-150 binding site) established Akt1 and Akt2 as direct targets of miR-150. Tail vein injection of lentiviral particles containing pre-miR-150 enhanced Jo2-induced liver injury in miR-150 KO mice. These findings demonstrate that miR-150 deficiency prevents Fas-induced hepatocyte apoptosis and liver injury through regulation of the Akt pathway.
PMCID: PMC4510058  PMID: 26196694
5.  Persistent Hepatitis C Virus Infection Impairs Ribavirin Antiviral Activity through Clathrin-Mediated Trafficking of Equilibrative Nucleoside Transporter 1 
Journal of Virology  2014;89(1):626-642.
Ribavirin (RBV) continues to be an important component of interferon-free hepatitis C treatment regimens, as RBV alone does not inhibit hepatitis C virus (HCV) replication effectively; the reason for this ineffectiveness has not been established. In this study, we investigated the RBV resistance mechanism using a persistently HCV-infected cell culture system. The antiviral activity of RBV against HCV was progressively impaired in the persistently infected culture, whereas interferon lambda 1 (IFN-λ1), a type III IFN, showed a strong antiviral response and induced viral clearance. We found that HCV replication in persistently infected cultures induces an autophagy response that impairs RBV uptake by preventing the expression of equilibrative nucleoside transporter 1 (ENT1). The Huh-7.5 cell line treated with an autophagy inducer, Torin 1, downregulated membrane expression of ENT1 and terminated RBV uptake. In contrast, the autophagy inhibitors hydroxychloroquine (HCQ), 3-methyladenine (3-MA), and bafilomycin A1 (BafA1) prevented ENT1 degradation and enhanced RBV antiviral activity. The HCV-induced autophagy response, as well as treatment with Torin 1, degrades clathrin heavy chain expression in a hepatoma cell line. Reduced expression of the clathrin heavy chain by HCV prevents ENT1 recycling to the plasma membrane and forces ENT1 to the lysosome for degradation. This study provides a potential mechanism for the impairment of RBV antiviral activity in persistently HCV-infected cell cultures and suggests that inhibition of the HCV-induced autophagy response could be used as a strategy for improving RBV antiviral activity against HCV infection.
IMPORTANCE The results from this work will allow a review of the competing theories of antiviral therapy development in the field of HCV virology. Ribavirin (RBV) remains an important component of interferon-free hepatitis C treatment regimens. The reason why RBV alone does not inhibit HCV replication effectively has not been established. This study provides a potential mechanism for why RBV antiviral activity is impaired in persistently HCV-infected cell cultures and suggests that inhibition of the HCV-induced autophagy response could be used as a strategy to increase RBV antiviral activity against HCV infection. Therefore, it is anticipated that this work would generate a great deal of interest, not only among virologists but also among the general public.
PMCID: PMC4301154  PMID: 25339775
6.  Identification of Personalized Chemoresistance Genes in Subtypes of Basal-Like Breast Cancer Based on Functional Differences Using Pathway Analysis 
PLoS ONE  2015;10(6):e0131183.
Breast cancer is a highly heterogeneous disease that is clinically classified into several subtypes. Among these subtypes, basal-like breast cancer largely overlaps with triple-negative breast cancer (TNBC), and these two groups are generally studied together as a single entity. Differences in the molecular makeup of breast cancers can result in different treatment strategies and prognoses for patients with different breast cancer subtypes. Compared with other subtypes, basal-like and other ER+ breast cancer subtypes exhibit marked differences in etiologic factors, clinical characteristics and therapeutic potential. Anthracycline drugs are typically used as the first-line clinical treatment for basal-like breast cancer subtypes. However, certain patients develop drug resistance following chemotherapy, which can lead to disease relapse and death. Even among patients with basal-like breast cancer, there can be significant molecular differences, and it is difficult to identify specific drug resistance proteins in any given patient using conventional variance testing methods. Therefore, we designed a new method for identifying drug resistance genes. Subgroups, personalized biomarkers, and therapy targets were identified using cluster analysis of differentially expressed genes. We found that basal-like breast cancer could be further divided into at least four distinct subgroups, including two groups at risk for drug resistance and two groups characterized by sensitivity to pharmacotherapy. Based on functional differences among these subgroups, we identified nine biomarkers related to drug resistance: SYK, LCK, GAB2, PAWR, PPARG, MDFI, ZAP70, CIITA and ACTA1. Finally, based on the deviation scores of the examined pathways, 16 pathways were shown to exhibit varying degrees of abnormality in the various subgroups, indicating that patients with different subtypes of basal-like breast cancer can be characterized by differences in the functional status of these pathways. Therefore, these nine differentially expressed genes and their associated functional pathways should provide the basis for novel personalized clinical treatments of basal-like breast cancer.
PMCID: PMC4488356  PMID: 26126114
7.  Identifying ultrasound and clinical features of breast cancer molecular subtypes by ensemble decision 
Scientific Reports  2015;5:11085.
Breast cancer is molecularly heterogeneous and categorized into four molecular subtypes: Luminal-A, Luminal-B, HER2-amplified and Triple-negative. In this study, we aimed to apply an ensemble decision approach to identify the ultrasound and clinical features related to the molecular subtypes. We collected ultrasound and clinical features from 1,000 breast cancer patients and performed immunohistochemistry on these samples. We used the ensemble decision approach to select unique features and to construct decision models. The decision model for Luminal-A subtype was constructed based on the presence of an echogenic halo and post-acoustic shadowing or indifference. The decision model for Luminal-B subtype was constructed based on the absence of an echogenic halo and vascularity. The decision model for HER2-amplified subtype was constructed based on the presence of post-acoustic enhancement, calcification, vascularity and advanced age. The model for Triple-negative subtype followed two rules. One was based on irregular shape, lobulate margin contour, the absence of calcification and hypovascularity, whereas the other was based on oval shape, hypovascularity and micro-lobulate margin contour. The accuracies of the models were 83.8%, 77.4%, 87.9% and 92.7%, respectively. We identified specific features of each molecular subtype and expanded the scope of ultrasound for making diagnoses using these decision models.
PMCID: PMC4457139  PMID: 26046791
8.  MiR-21 Targets 15-PGDH and Promotes Cholangiocarcinoma Growth 
Molecular cancer research : MCR  2014;12(6):890-900.
MicroRNAs (miRs) are a group of small, non-coding RNAs that modulate the translation of genes by binding to specific target sites in the target mRNA. This study investigated the biological function and molecular mechanism of microRNA-21 (miR-21) in human cholangiocarcinoma. In situ hybridization analysis of human cholangiocarcinoma specimens showed increased miR-21 in cholangiocarcinoma tissue compared to the non-cancerous biliary epithelium. Lentiviral transduction of miR-21 enhanced human cholangiocarcinoma cell growth and clonogenic efficiency in vitro, whereas inhibition of miR-21 decreased these parameters. Over-expression of miR-21 also promoted cholangiocarcinoma growth using an in vivo xenograft model system. The NAD+-linked 15-hydroxyprostaglandin dehydrogenase (15-PGDH/HPGD), a key enzyme that converts the protumorigenic prostaglandin E2 (PGE2) to its biologically inactive metabolite, was identified as a direct target of miR-21 in cholangiocarcinoma cells. In parallel, cyclooxygenase-2 (COX2) over-expression and PGE2 treatment increased miR-21 levels and enhanced miR-21 promoter activity in human cholangiocarcinoma cells.
PMCID: PMC4058387  PMID: 24699315
Cholangiocarcinoma; miR-21; 15-PGDH; COX-2; PGE2
9.  Chaperone-Mediated Autophagy Targets IFNAR1 for Lysosomal Degradation in Free Fatty Acid Treated HCV Cell Culture 
PLoS ONE  2015;10(5):e0125962.
Hepatic steatosis is a risk factor for both liver disease progression and an impaired response to interferon alpha (IFN-α)-based combination therapy in chronic hepatitis C virus (HCV) infection. Previously, we reported that free fatty acid (FFA)-treated HCV cell culture induces hepatocellular steatosis and impairs the expression of interferon alpha receptor-1 (IFNAR1), which is why the antiviral activity of IFN-α against HCV is impaired.
To investigate the molecular mechanism by which IFNAR1 expression is impaired in HCV cell culture with or without free fatty acid-treatment.
HCV-infected Huh 7.5 cells were cultured with or without a mixture of saturated (palmitate) and unsaturated (oleate) long-chain free fatty acids (FFA). Intracytoplasmic fat accumulation in HCV-infected culture was visualized by oil red staining. Clearance of HCV in FFA cell culture treated with type I IFN (IFN-α) and Type III IFN (IFN-λ) was determined by Renilla luciferase activity, and the expression of HCV core was determined by immunostaining. Activation of Jak-Stat signaling in the FFA-treated HCV culture by IFN-α alone and IFN-λ alone was examined by Western blot analysis and confocal microscopy. Lysosomal degradation of IFNAR1 by chaperone-mediated autophagy (CMA) in the FFA-treated HCV cell culture model was investigated.
FFA treatment induced dose-dependent hepatocellular steatosis and lipid droplet accumulation in HCV-infected Huh-7.5 cells. FFA treatment of infected culture increased HCV replication in a concentration-dependent manner. Intracellular lipid accumulation led to reduced Stat phosphorylation and nuclear translocation, causing an impaired IFN-α antiviral response and HCV clearance. Type III IFN (IFN-λ), which binds to a separate receptor, induces Stat phosphorylation, and nuclear translocation as well as antiviral clearance in FFA-treated HCV cell culture. We show here that the HCV-induced autophagy response is increased in FFA-treated cell culture. Pharmacological inhibitors of lysosomal degradation, such as ammonium chloride and bafilomycin, prevented IFNAR1 degradation in FFA-treated HCV cell culture. Activators of chaperone-mediated autophagy, including 6-aminonicotinamide and nutrient starvation, decreased IFNAR1 levels in Huh-7.5 cells. Co-immunoprecipitation, colocalization and siRNA knockdown experiments revealed that IFNAR1 but not IFNLR1 interacts with HSC70 and LAMP2A, which are core components of chaperone-mediated autophagy (CMA).
Our study presents evidence indicating that chaperone-mediated autophagy targets IFNAR1 degradation in the lysosome in FFA-treated HCV cell culture. These results provide a mechanism for why HCV induced autophagy response selectively degrades type I but not the type III IFNAR1.
PMCID: PMC4427131  PMID: 25961570
10.  Predictors for Physical Activity in Adolescent Girls Using Statistical Shrinkage Techniques for Hierarchical Longitudinal Mixed Effects Models 
PLoS ONE  2015;10(4):e0125431.
We examined associations among longitudinal, multilevel variables and girls’ physical activity to determine the important predictors for physical activity change at different adolescent ages. The Trial of Activity for Adolescent Girls 2 study (Maryland) contributed participants from 8th (2009) to 11th grade (2011) (n=561). Questionnaires were used to obtain demographic, and psychosocial information (individual- and social-level variables); height, weight, and triceps skinfold to assess body composition; interviews and surveys for school-level data; and self-report for neighborhood-level variables. Moderate to vigorous physical activity minutes were assessed from accelerometers. A doubly regularized linear mixed effects model was used for the longitudinal multilevel data to identify the most important covariates for physical activity. Three fixed effects at the individual level and one random effect at the school level were chosen from an initial total of 66 variables, consisting of 47 fixed effects and 19 random effects variables, in additional to the time effect. Self-management strategies, perceived barriers, and social support from friends were the three selected fixed effects, and whether intramural or interscholastic programs were offered in middle school was the selected random effect. Psychosocial factors and friend support, plus a school’s physical activity environment, affect adolescent girl’s moderate to vigorous physical activity longitudinally.
PMCID: PMC4416015  PMID: 25928064
11.  Impaired Autophagy Response in Human Hepatocellular Carcinoma 
Autophagy is a cellular lysosomal degradation mechanism has been implicated in chronic liver diseases and hepatocellular carcinoma (HCC). Association of autophagy defect with the development of human HCC has been shown in transgenic mouse model.
We performed this study to verify whether a defect in autophagy would play a role in human hepatocellular carcinoma (HCC).
Archival tissue sections of 20 patients with HCC with or without hepatitis C virus (HCV) infection were studied. All slides were immunostained using monoclonal antibodies to p62 and glypican-3 with appropriate positive and negative controls. The expression of p62 and glycican-3 in the HCC and the surrounding non-tumor were semiquantitated. The cytoplasmic staining was graded as negative, weak or strong.
Positive p62 staining was found in 20 out of 20 (100%) HCCs and negative staining was observed in 20 out of 20 non-tumor areas and cirrhotic nodules. Positive glypican-3 staining was found in 70% of HCCs and negative staining was seen in all non-tumor areas. An autophagy defect leading to increased expression of p62 and glypican-3 was also seen in the HCC cell line (Huh-7.5), but not in the primary human hepatocytes. Activation of cellular autophagy in Huh-7.5 cells efficiently cleared p62 and glypican-3 expression and inhibition of autophagy induced the expression of p62 and glypican-3.
This study shows that p62 is increased in HCC compared to the surrounding non-tumorous liver tissue suggesting that human HCCs are autophagy defective. We provide further evidence that glypican-3 expression in HCC may be also related to defective autophagy. Our study indicates that p62 immunostain may represent a novel marker for HCC.
PMCID: PMC4364514  PMID: 24369267
Hepatocellular Carcinoma; Autophagy; Glypican-3; p62; Immunostaining
12.  MiR-122 in hepatitis B virus and hepatitis C virus dual infection 
World Journal of Hepatology  2015;7(3):498-506.
Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are the most common causes of chronic liver diseases and hepatocelluar carcinomas. Over the past few years, the liver-enriched microRNA-122 (miR-122) has been shown to differentially regulate viral replication of HBV and HCV. It is notable that the level of miR-122 is positively and negatively regulated by HCV and HBV, respectively. Consistent with the well-documented phenomenon that miR-122 promotes HCV accumulation, inhibition of miR-122 has been shown as an effective therapy for the treatment of HCV infection in both chimpanzees and humans. On the other hand, miR-122 is also known to block HBV replication, and HBV has recently been shown to inhibit miR-122 expression; such a reciprocal inhibition between miR-122 and HBV suggests an intriguing possibility that miR-122 replacement may represent a potential therapy for treatment of HBV infection. As HBV and HCV have shared transmission routes, dual infection is not an uncommon scenario, which is associated with more advanced liver disease than either HBV or HCV mono-infection. Thus, there is a clear need to further understand the interaction between HBV and HCV and to delineate the role of miR-122 in HBV/HCV dual infection in order to devise effective therapy. This review summarizes the current understanding of HBV/HCV dual infection, focusing on the pathobiological role and therapeutic potential of miR-122.
PMCID: PMC4381172  PMID: 25848473
MiR-122; Hepatitis B virus; Hepatitis C virus; Hepatitis B virus/hepatitis C virus dual infection
13.  Phosphorylation of Atg31 is required for autophagy 
Protein & Cell  2015;6(4):288-296.
Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17-Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.
Electronic supplementary material
The online version of this article (doi:10.1007/s13238-015-0138-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4383750  PMID: 25773276
autophagy; Atg31; phosphorylation; autophagosome; pre-autophagosomal structure (PAS)
14.  Phosphorylation of Atg31 is required for autophagy 
Protein & Cell  2015;6(4):288-296.
Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17-Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.
Electronic supplementary material
The online version of this article (doi:10.1007/s13238-015-0138-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4383750  PMID: 25773276
autophagy; Atg31; phosphorylation; autophagosome; pre-autophagosomal structure (PAS)
15.  The Differences in Ultrasound and Clinicopathological Features between Basal-Like and Normal-Like Subtypes of Triple Negative Breast Cancer 
PLoS ONE  2015;10(3):e0114820.
The aim of this study was to identify the ultrasound features and clinicopathological characteristics of basal-like subtype of triple negative breast cancers (TNBCs).
Materials and Methods
This study was approved by the ethical board of the Second Affiliated Hospital of Harbin Medical University. The patients’ clinicopathological information was available. The ultrasound features of 62 tumors from 62 TNBC patients were interpreted. The immunohistochemical results of cytokertain5/6 (CK5/6) and Epidermal Growth Factor Receptor (EGFR) were used to classify the tumor into basal-like and normal-like groups. The association of the ultrasound features interpreted by experienced ultrasound doctors with the immunohistochemical classification was studied.
Of the 62 TNBC cases, 42 (67.7%) exhibited the basal-like phenotype and 20 (32.3%) exhibited the normal-like phenotype based on the immunohistochemical CK5/6 and EGFR markers. Of all the tumors, 90.3% were invasive carcinomas. The basal-like tumors were significantly associated with a maximum diameter on ultrasound of more than 20 mm (36, 85.7%) (P = 0.0014). The normal-like tumors usually exhibited lateral shadows (15, 75%) (P = 0.0115) as well as microlobulated margins (12, 60%) (P = 0.0204) compared to the basal-like subtype. Other ultrasound features showed no significant differences between the two groups.
Although ultrasound cannot yet be used to differentiate between the basal-like subtype and normal-like subtype of TNBC, ultrasound can be used to provide some useful information to the clinicians.
PMCID: PMC4348341  PMID: 25734578
16.  Near-Infrared Spectroscopy as a Diagnostic Tool for Distinguishing between Normal and Malignant Colorectal Tissues 
BioMed Research International  2015;2015:472197.
Cancer diagnosis is one of the most important tasks of biomedical research and has become the main objective of medical investigations. The present paper proposed an analytical strategy for distinguishing between normal and malignant colorectal tissues by combining the use of near-infrared (NIR) spectroscopy with chemometrics. The successive projection algorithm-linear discriminant analysis (SPA-LDA) was used to seek a reduced subset of variables/wavenumbers and build a diagnostic model of LDA. For comparison, the partial least squares-discriminant analysis (PLS-DA) based on full-spectrum classification was also used as the reference. Principal component analysis (PCA) was used for a preliminary analysis. A total of 186 spectra from 20 patients with partial colorectal resection were collected and divided into three subsets for training, optimizing, and testing the model. The results showed that, compared to PLS-DA, SPA-LDA provided more parsimonious model using only three wavenumbers/variables (4065, 4173, and 5758 cm−1) to achieve the sensitivity of 84.6%, 92.3%, and 92.3% for the training, validation, and test sets, respectively, and the specificity of 100% for each subset. It indicated that the combination of NIR spectroscopy and SPA-LDA algorithm can serve as a potential tool for distinguishing between normal and malignant colorectal tissues.
PMCID: PMC4309295  PMID: 25654106
17.  Statistical Learning Methods for Longitudinal High-dimensional Data 
Recent studies have collected high-dimensional data longitudinally. Examples include brain images collected during different scanning sessions and time-course gene expression data. Because of the additional information learned from the temporal changes of the selected features, such longitudinal high-dimensional data, when incorporated with appropriate statistical learning techniques, are able to more accurately predict disease status or responses to a therapeutic treatment. In this article, we review recently proposed statistical learning methods dealing with longitudinal high-dimensional data.
PMCID: PMC4181610  PMID: 25285184
High-dimensionality; Multiple times points; Prediction; Support vector machines; Shrinkage; Temporal effects
18.  Physical Activity Trajectories and Multi-Level Factors among Adolescent Girls 
Although the decline of physical activity in adolescent girls is well-documented, there are girls whose physical activity does not follow this pattern. This study examined the relationships between physical activity trajectories and personal, psychosocial and environmental factors among adolescent girls.
Participants were from the University of Maryland field site of the Trial of Activity for Adolescent Girls. Of 730 girls measured in 8th grade, 589 were re-measured in 11th grade. Moderate to vigorous physical activity was assessed by accelererometers; participants were categorized as active maintainers (n=31), inactive maintainers (n=410), adopters (n=64), or relapsers (n=56). Height and weight were measured, personal and psychosocial information was collected from surveys, and distance from home to school and parks was assessed from Geographical Information Systems. Multivariable logistic regression was used for data analysis.
Variables at individual, social, and environmental levels predicted active maintainers and inactive maintainers, while only individual-level variables predicted adoption. None predicted relapse. Higher (favorable) scores for physical self-concept, perceived body fat, friend and family physical activity support, frequency of physical activity with friends, and shorter distance from home to a park predicted active maintainers. Overweight/obese status, earlier age at menses, and lower scores for physical self-concept, perceived body fat, friend physical activity support, and frequency of physical activity with friends, and further distance from home to school predicted inactive maintainers. High physical self-concept and not being overweight/obese predicted adopters.
Multi-level factors appear to predict behavior maintenance rather than actual change.
Implications and Contribution
Although physical activity declines among girls during adolescence, some maintain and others increase their physical activity. Our results identified factors across individual, social, and environmental levels that predicted physical activity maintenance over 3 years. These may guide future interventions to enhance adolescent girls’ physical activity and combat the overall decline.
PMCID: PMC3872172  PMID: 23992761
19.  Epigenetic regulation of miR-122 by PPARγ and hepatitis B virus X protein in hepatocellular carcinoma cells 
Hepatology (Baltimore, Md.)  2013;58(5):1681-1692.
MiR-122, a pivotal liver specific miRNA, has been implicated in several liver diseases including hepatocellular carcinoma (HCC) and hepatitis C and B viral infection. This study aimed to explore epigenetic regulation of miR-122 in human hepatocellular carcinoma (HCC) cells and to examine the effect of hepatitis C virus (HCV) and hepatitis B virus (HBV). We performed microRNA microarray analysis and identified miR-122 as the most up-regulated miRNA (6-fold) in human hepatocellular cancer cells treated with 5′aza-2′deoxycytidine (5-Aza-CdR, DNA methylation inhibitor) and 4-phenylbutyric acid (PBA, histone deacetylation inhibitor). Real-time PCR analysis verified significant upregulation of miR-122 by 5′aza and PBA in HCC cells, and to a lesser extent in primary hepatocytes. Peroxisome proliferator activated receptor-gamma (PPARγ) and retinoid X receptor alpha (RXRα) complex was found to be associated with the DR1 and DR2 consensus site in the miR-122 gene promoter which enhanced miR-122 gene transcription. 5-Aza-CdR and PBA treatment increased the association of PPARγ/RXRα, but decreased the association of its co-repressors (N-CoR and SMRT), with the miR-122 DR1 and DR2 motifs. The aforementioned DNA-protein complex also contains SUV39H1, a H3K9 histone methyl transferase, which downregulates miR-122 expression. Our findings establish a novel role of the PPARγ binding complex for epigenetic regulation of miR-122 in human HCC cells. Moreover, we show that hepatitis B virus X protein (HBX) binds PPARγ and inhibits the transcription of miR-122, whereas hepatitis C viral particles exhibited no significant effect; these findings provide mechanistic insight into reduction of miR-122 in patients with HBV but not with HCV infection.
PMCID: PMC3773012  PMID: 23703729
miR-122; PPARγ; HCC; epigenetic regulation; Hepatitis B virus X protein; HCV; liver; hepatocytes
20.  Impaired Expression of Type I and Type II Interferon Receptors in HCV-Associated Chronic Liver Disease and Liver Cirrhosis 
PLoS ONE  2014;9(9):e108616.
Chronic Hepatitis C Virus (HCV)-infected patients with liver cirrhosis (LC) respond poorly to interferon-alpha (IFN-α) and ribavirin (RBV) combination therapy, but the reason for this is unclear. We previously reported that HCV-infection induces endoplasmic reticulum (ER) stress and autophagy response that selectively down regulates the type I IFN-α receptor-1 (IFNAR1) and RBV transporters (CNT1 and ENT1), leading to IFN-α/RBV resistance. The goal of this study is to verify whether an increase in ER stress and autophagy response is also associated with the reduced expression of IFNAR1 and RBV transporters in chronic HCV-infected patients.
Primary human hepatocytes (PHH) were infected with cell culture grown HCV particles (JFH-ΔV3-Rluc). HCV replication was confirmed by the detection of viral RNA by RT-qPCR and HCV-core protein by Western blotting. The ER stress and autophagy response and expression of IFN receptors and RBV transporters in HCV infected PHH and liver tissues derived from patients were measured by Western blotting.
HCV infection of PHH showed impaired expression of IFNAR1, IFNγR1 (Type II IFN receptor) and RBV transporters but not IL10Rβ (Type III IFN-λ receptor). ER stress markers (BiP, IRE1α and peIF2α) and autophagy response (LC3II, Beclin 1 and ATG5) were induced in HCV infected chronic liver disease (CLD) and LC patients. Liver biopsies (CLD) show a 50% reduced expression of IFNAR1 and RBV transporters. Furthermore, the expression of IFNAR1 and RBV transporters was impaired in almost all LC patients.
HCV infection induces ER stress and autophagy response in infected PHH and chronically infected liver tissues. The expression of IFNAR1, IFNγR1 and RBV transporters were significantly impaired in CLD and cirrhotic livers. Our study provides a potential explanation for the reduced response rate of IFN-α and RBV combination therapy in HCV infected patients with liver cirrhosis.
PMCID: PMC4180933  PMID: 25265476
21.  Hedgehog Signaling Pathway Regulates Autophagy in Human Hepatocellular Carcinoma Cells 
Hepatology (Baltimore, Md.)  2013;58(3):995-1010.
Hedgehog (Hh) signaling plays an important role in embryonic development and in the regulation of a variety of cellular functions. Aberrant activation of Hh signaling has been implicated in several human cancers including hepatocellular carcinoma (HCC). In this study we examined the pathobiological functions and molecular mechanisms of Hh signaling pathway in HCC cells. Treatment of cultured human HCC cells (Huh7, Hep3B and HepG2) with the Hh signaling ligand (recombinant Shh) or agonist, SAG and purmorphamine, prevented the induction of autophagy. In contrast, GANT61 (a small molecule inhibitor of Gli1 and Gli2) induced autophagy, as determined by immunobloting for microtubule-associated protein light chain 3 (LC3) and p62, GFP-LC3 puncta, monodansylcadaverine (MDC) staining and transmission electron microscopy. Hh inhibition-induced autophagy was associated with upregulation of Bnip3, as determined by immunoblotting and real-time PCR assay. Knockdown of Bnip3 by RNAi impaired GANT61-induced autophagy. Additionally, Hh inhibition-induced autophagy was associated with Bnip3-mediated displacement of Bcl-2 from Beclin-1, as determined by immunoblotting and immunoprecipitation assays. Furthermore, inhibition of Hh signaling increased HCC cell apoptosis and decreased cell viability, as determined by caspase and WST-1 assays. Pharmacological or genetic inhibition of autophagy by 3-methyladenine (3-MA) or Beclin-1 siRNA partially suppressed GANT61-induced cell apoptosis and cytotoxicity. In a tumor xenograft model using SCID mice inoculated with Huh7 cells, administration of GANT61 inhibited tumor formation and decreased tumor volume; this effect was partially blocked by the autophagy inhibitor, 3-MA. These findings provide novel evidence that hedgehog inhibition induces autophagy through upregulation of Bnip3 and that this mechanism contributes to apoptosis. Therefore, the status of autophagy is a key factor that determines the therapeutic response to Hh-targeted therapies.
PMCID: PMC3706478  PMID: 23504944
Hedgehog signaling; GANT61; autophagy; hepatocellular carcinoma; apoptosis
22.  High TFAP2C/low CD44 expression is associated with an increased rate of pathologic complete response following neoadjuvant chemotherapy in breast cancer 
The Journal of surgical research  2013;184(1):10.1016/j.jss.2013.04.042.
In luminal breast cancer cell lines, TFAP2C regulates expression of key genes in the estrogen receptor–associated cluster and represses basal-associated genes including CD44. We examined the effect of TFAP2C overexpression in a basal cell line and characterized the expression of TFAP2C and CD44 in breast cancer specimens to determine if expression was associated with clinical response.
MDA-MB-231 breast cancer cells were treated with a TFAP2C-containing plasmid and evaluated for effects on CD44 expression. Pretreatment biopsy cores from patients receiving neoadjuvant chemotherapy for breast cancer were evaluated for TFAP2A, p53, TFAP2C, and CD44 expression by immunohistochemistry.
Overexpression of TFAP2C in MDA-MB-231 cells resulted in decreased expression of CD44 mRNA and protein, P< 0.05. A pathologic complete response (pCR) following neoadjuvant chemotherapy was achieved in 17% of patients (4/23). Average expression for TFAP2C by immunohistochemistry in patients with a pCR was 93%, compared with 46% in patients with residual disease, P= 0.016; and in tumors that stained at ≥80% for TFAP2C, 4 of 9 (44%) achieved pCR, compared with 0 of 14 below 80%, P= 0.01. Additionally, in tumors that stained ≤80% for CD44, 4 of 10 (40%) achieved pCR, compared with 0 of 13 >80%, P = 0.02. In tumors that stained high for TFAP2C (≥80%) and low for CD44 (≥80%), 4 of 7 (57%) achieved pCR, compared with 0 of 16 in all other groups (P= 0.004).
TFAP2C repressed CD44 expression in basal-derived breast cancer. In primary breast cancer specimens, high TFAP2C and low CD44 expression were associated with pCR after neoadjuvant chemotherapy and could be predictive of tumors that have improved response to neoadjuvant chemotherapy.
PMCID: PMC3820425  PMID: 23764310
TFAP2C; Breast cancer; Pathologic complete response; Neoadjuvant chemotherapy; CD44
23.  15-PGDH inhibits hepatocellular carcinoma growth through 15-keto-PGE2/PPARγ-mediated activation of p21WAF1/Cip1 
Oncogene  2013;33(9):1101-1112.
15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme in prostaglandin metabolism. This study provides important evidence for inhibition of hepatocellular carcinoma (HCC) growth by 15-PGDH through the 15-keto-PGE2/PPARγ/p21WAF1/Cip1 signaling pathway. Forced overexpression of 15-PGDH inhibited HCC cell growth in vitro, whereas knockdown of 15-PGDH enhanced tumor growth parameters. In a tumor xenograft model in SCID mice, inoculation of human HCC cells (Huh7) with overexpression of 15-PGDH led to significant inhibition of tumor growth, while knockdown of 15-PGDH enhanced tumor growth. In a separate tumor xenograft model in which mouse HCC cells (Hepa1-6) were inoculated into syngeneic C57BL/6 mice, intratumoral injection of adenovirus vector expressing 15-PGDH (pAd-15-PGDH) significantly inhibited xenograft tumor growth. The anti-tumor effect of 15-PGDH is mediated through its enzymatic product, 15-keto-PGE2, which serves as an endogenous PPARγ ligand. Activation of PPARγ by 15-PGDH-derived 15-keto-PGE2 enhanced the association of PPARγ with the p21WAF1/Cip1 promoter and increased p21 expression and association with CDK2, CDK4 and PCNA. Depletion of p21 by shRNA reversed 15-PGDH-induced inhibition of HCC cell growth; overexpression of p21 prevented 15-PGDH knockdown-induced tumor cell growth. These results demonstrate a key 15-PGDH/15-keto-PGE2-mediated activation of PPARγ and p21WAF1/Cip1 signaling cascade that regulates hepatocarcinogenesis and tumor progression.
PMCID: PMC3912217  PMID: 23542179
Hepatocellular carcinoma (HCC); 15-hydroxyprostaglandin dehydrogenase (15-PGDH); 15-keto-PGE2; p21; PPARγ; liver
24.  Docosahexaenoic acid-induced apoptosis is mediated by activation of mitogen-activated protein kinases in human cancer cells 
BMC Cancer  2014;14:481.
The role of omega-3 polyunsaturated fatty acids (ω3-PUFAs) in cancer prevention has been demonstrated; however, the exact molecular mechanisms underlying the anticancer activity of ω3-PUFAs are not fully understood. Here, we investigated the relationship between the anticancer action of a specific ω3-PUFA docosahexaenoic acid (DHA), and the conventional mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK) and p38 whose dysregulation has been implicated in human cancers.
MTT assays were carried out to determine cell viability of cancer cell lines (PA-1, H1299, D54MG and SiHa) from different origins. Apoptosis was confirmed by TUNEL staining, DNA fragmentation analysis and caspase activity assays. Activities of the conventional MAPKs were monitored by their phosphorylation levels using immunoblotting and immunocytochemistry analysis. Reactive oxygen species (ROS) production was measured by flow cytometry and microscopy using fluorescent probes for general ROS and mitochondrial superoxide.
DHA treatment decreased cell viability and induced apoptotic cell death in all four studied cell lines. DHA-induced apoptosis was coupled to the activation of the conventional MAPKs, and knockdown of ERK/JNK/p38 by small interfering RNAs reduced the apoptosis induced by DHA, indicating that the pro-apoptotic effect of DHA is mediated by MAPKs activation. Further study revealed that the DHA-induced MAPKs activation and apoptosis was associated with mitochondrial ROS overproduction and malfunction, and that ROS inhibition remarkably reversed these effects of DHA.
Together, these results indicate that DHA-induced MAPKs activation is dependent on its capacity to provoke mitochondrial ROS generation, and accounts for its cytotoxic effect in human cancer cells.
PMCID: PMC4094407  PMID: 24993937
Docosahexaenoic acid; Reactive oxygen species; Mitogen-activated protein kinases; Apoptosis; Cancer
25.  The In Vitro and In Vivo Antitumor Effects of Clotrimazole on Oral Squamous Cell Carcinoma 
PLoS ONE  2014;9(6):e98885.
Clotrimazole is an antifungal imidazole derivative showing anti- neoplastic effect in some tumors, but its anticancer potential is still unclear in oral squamous cell carcinoma (OSCC). The aim of this study was to evaluate the antitumor effect of clotrimazole, and to investigate the possible mechanism of clotrimazole-mediated antitumor activity in OSCC.
In vitro experiments, the cell viability and clonogenic ability of three human OSCC cell lines CAL27, SCC25 and UM1 were detected after clotrimazole treatment by CCK8 assay and colony formation assay. Cell cycle progression and apoptosis were assessed by flow cytometry, and the involvement of several mediators of apoptosis was examined by western blot analysis. Then, the in vivo antitumor effect of clotrimazole was investigated in CAL27 xenograft model. Immunohistochemistry and western blot analysis were performed to determine the presence of apoptotic cells and the expression of Bcl-2 and Bax in tumors from mice treated with or without clotrimazole.
Clotrimazole inhibited proliferation in all three OSCC cell lines in a dose-and time-dependent manner, and significantly reduced the colony formation of OSCC cells in vitro. Clotrimazole caused cell cycle arrest at the G0/G1 phase. In addition, clotrimazole induced apoptosis in OSCC cells, and significantly down-regulated the anti-apoptotic protein Bcl-2 and up-regulated the pro-apoptotic protein Bax. Notably, clotrimazole treatment inhibited OSCC tumor growth and cell proliferation in CAL27 xenograft model. Clotrimazole also markedly reduced Bcl-2 expression and increased the protein level of Bax in tumor tissues of xenograft model.
Our findings demonstrated a potent anticancer effect of clotrimazole by inducing cell cycle arrest and cellular apoptosis in OSCC.
PMCID: PMC4043897  PMID: 24892421

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