These changes were followed by death or distress, necessitating euthanasia within 48 hours. Fifty percent of KO mice died within the first 6 days of initiating the 5% ethanol diet, whereas none died in the WT/ethanol group (Fig. 1A). Food intake was similar in the two EtOH groups, except for just before death

in the KO group (Fig. 1B). To avoid confounding results from animals in GDC-0068 nmr extremis, we sacrificed the remaining mice after day 6 on 5% ethanol, and the experiments described below were performed on these mice. PF KO and WT mice appeared healthy and gained weight (data not shown). EtOH KO mice were hypoglycemic with 2-fold lower blood-glucose levels than WT mice (Fig. 1C) and had 10% lower body weight (Fig. 1D). EtOH KO mice had cachexia and severely depleted intra-abdominal fat, compared with the WT/ethanol group, likely representing a baseline defect in energy homeostasis and ethanol-induced acute illness and decreased food intake http://www.selleckchem.com/products/PD-0332991.html in KO mice (Fig. 1E; Supporting Fig. 1 24). There was no difference

in body temperature between the groups. We conclude from these results that KO mice are highly susceptible to systemic toxicity and death after short exposure to ethanol ingestion. Both groups of KO mice had lower liver weight (Supporting Fig. 2). However, only PF KO mice had a lower liver:body weight ratio, compared with the corresponding WT group (Supporting Fig. 3). On microscopic examination of the liver, EtOH KO mice exhibited severe micro- and

macrovesicular steatosis in all three zones of the liver lobule. In contrast, WT mice developed only mild (predominantly zone 2) microvesicular steatosis (Fig. 2A, upper panel). Similarly, Oil red O staining for neutral lipids confirmed the presence of increased hepatic steatosis in the KO/ethanol group (Fig. 2A, bottom panel). KO mice had approximately 5-fold higher alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels than WT mice on the ethanol diet (Fig. 2B,C). Biochemical assays revealed higher liver triglyceride and cholesterol levels in the KO/ethanol group, compared with WT mice (Fig. 2D,E). Serum triglyceride and total cholesterol levels were similar in WT and KO mice (data not shown). Thus, these results show that KO mice develop severe medchemexpress liver steatosis and moderate transaminase elevation on ethanol ingestion in a time period that causes only mild lipid accumulation and no change in liver injury tests in WT mice. Increased hepatic oxidative stress is an important mechanism of ethanol-mediated liver injury, and lipid peroxidation (LPO) is used as an indicator of oxidative stress in tissues. Therefore, we performed an assay for malondialdehyde (MDA) levels as an indicator of LPO in the liver. KO mice had higher hepatic MDA levels than WT mice on the ethanol diet (Fig. 3A).

These changes were followed by death or distress, necessitating euthanasia within 48 hours. Fifty percent of KO mice died within the first 6 days of initiating the 5% ethanol diet, whereas none died in the WT/ethanol group (Fig. 1A). Food intake was similar in the two EtOH groups, except for just before death

in the KO group (Fig. 1B). To avoid confounding results from animals in AZD2014 nmr extremis, we sacrificed the remaining mice after day 6 on 5% ethanol, and the experiments described below were performed on these mice. PF KO and WT mice appeared healthy and gained weight (data not shown). EtOH KO mice were hypoglycemic with 2-fold lower blood-glucose levels than WT mice (Fig. 1C) and had 10% lower body weight (Fig. 1D). EtOH KO mice had cachexia and severely depleted intra-abdominal fat, compared with the WT/ethanol group, likely representing a baseline defect in energy homeostasis and ethanol-induced acute illness and decreased food intake Carfilzomib concentration in KO mice (Fig. 1E; Supporting Fig. 1 24). There was no difference

in body temperature between the groups. We conclude from these results that KO mice are highly susceptible to systemic toxicity and death after short exposure to ethanol ingestion. Both groups of KO mice had lower liver weight (Supporting Fig. 2). However, only PF KO mice had a lower liver:body weight ratio, compared with the corresponding WT group (Supporting Fig. 3). On microscopic examination of the liver, EtOH KO mice exhibited severe micro- and

macrovesicular steatosis in all three zones of the liver lobule. In contrast, WT mice developed only mild (predominantly zone 2) microvesicular steatosis (Fig. 2A, upper panel). Similarly, Oil red O staining for neutral lipids confirmed the presence of increased hepatic steatosis in the KO/ethanol group (Fig. 2A, bottom panel). KO mice had approximately 5-fold higher alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels than WT mice on the ethanol diet (Fig. 2B,C). Biochemical assays revealed higher liver triglyceride and cholesterol levels in the KO/ethanol group, compared with WT mice (Fig. 2D,E). Serum triglyceride and total cholesterol levels were similar in WT and KO mice (data not shown). Thus, these results show that KO mice develop severe MCE公司 liver steatosis and moderate transaminase elevation on ethanol ingestion in a time period that causes only mild lipid accumulation and no change in liver injury tests in WT mice. Increased hepatic oxidative stress is an important mechanism of ethanol-mediated liver injury, and lipid peroxidation (LPO) is used as an indicator of oxidative stress in tissues. Therefore, we performed an assay for malondialdehyde (MDA) levels as an indicator of LPO in the liver. KO mice had higher hepatic MDA levels than WT mice on the ethanol diet (Fig. 3A).

However, if we can predict those patients LDK378 solubility dmso who are prone to disabling outcomes, more effective and tailored treatment may be possible for patients

with CD. Recent studies have reported that early and aggressive treatment of CD with immunosuppressants and anti-tumor necrosis factor α provides improved clinical outcomes compared with standard therapy.[11-13] Considering that these agents are linked to increased risks of serious infections[14] and cancers,[15, 16] prediction of disease course could be useful to select more appropriate candidates for these treatments and to reduce overtreatment. Thus, assessment of risks and identification of predictive factors has become important to determine therapeutic strategies for CD patients. To date, there have been several studies identifying the clinical predictors of CD prognosis in Caucasians that have demonstrated that

Selleckchem NVP-AUY922 younger age at diagnosis, perianal disease, stricturing, penetrating disease behavior, ileal involvement, and upper gastrointestinal (UGI) lesions were predictive of an unfavorable course.[17-21] However, there have been no large-scale studies focusing on clinical predictors in Asian patients, and no prior studies in Korean CD patients. Therefore, this study aimed to assess the clinical characteristics at the time of CD diagnosis and investigate predictive factors of a first CD-related surgery or requirement of immunosuppressive and biological agents in a large multicenter cohort

study of Korean CD patients. This retrospective multicenter cohort study included patients diagnosed with CD between July 1987 and January 上海皓元医药股份有限公司 2012 from 13 university hospitals (Kangbuk Samsung Hospital, Samsung Medical Center, Kyung Hee University Hospital, Soonchunhyang University Hospital, Dongguk University Ilsan Hospital, Konyang University Hospital, Ewha Womans University Hospital, Chungbuk National University Hospital, Jeju National University Hospital, Hangang Sacred Heart Hospital, Seoul Paik Hospital, St. Vincent’s Hospital, and Dankook University Hospital, Republic of Korea). All patients were diagnosed and treated by inflammatory bowel disease (IBD) specialists who are the members of the Korean Association for the Study of Intestinal Diseases. The diagnosis of CD was based on clinical, radiological, endoscopic, and histopathological features according to the criteria of Lennard-Jones.[22] Patients with the following conditions were excluded: those diagnosed or suspected to have indeterminate colitis, intestinal Behçet’s disease, intestinal tuberculosis, or infectious colitis; those with a follow-up period of less than 6 months or incomplete medical records; or those who underwent any intestinal resection not related to CD.

Loss of HNF6 results in normal apical-lateral localization of ZO-1, whereas loss of HNF1β results in very low levels of ZO-1 on the parenchymal side of forming bile duct lumen and improper apical localization of ZO-1 on the portal side. The postnatal consequences of these embryonic phenotypes that suggest loss of a cholangiocyte apical pole also differ between these two mouse models. Partial restoration of apical-basal polarity is observed when HNF6 is absent, but is not the case with HNF1β deficiency. Indeed, in patients with HNF1β mutations, ZO-1 was irregularly expressed in dysplastic ducts, and the observed DPM did not express ZO-1.

Absence of cystin-1 did not influence the apical marker osteopontin, but ZO-1 expanded to the apical surface of cholangiocytes, indicating that the basal and lateral poles were not established correctly. These phenotypes correspond Epigenetics Compound Library chemical structure to liver samples examined from ARPKD see more fetuses. Because of the polarity defects observed during biliary tubulogenesis, the authors investigated whether cholangiocyte ciliogenesis was disrupted in these mouse models. Previously, HNF6 and HNF1β were implicated in either control of cilia formation or regulation of genes involved in cilia function in the pancreas and kidney, respectively.10, 11 Because of the

random distribution of centrioles MCE observed in the absence of HNF6 or HNF1β, it is not surprising that embryonic cilia formation on cholangiocytes was significantly disrupted. The postnatal partial restoration of the apical-basal polarity in deficient HNF6 mice correlates with a few cilia present on cholangiocytes. However, the lack of cilia present on cholangiocytes remains as a postnatal defect in liver deficient for HNF1β. To determine if either HNF6 or HNF1β are involved in regulating the formation or function of cholangiocyte cilia, expression levels of candidate genes were examined in these two mouse models. Cystin-1 was the

only gene with reduced expression in both mouse models. Interestingly, in the cystin-1–deficient (cpk−/−) mouse model, the presence of a cilium is observed on some cholangiocytes. Therefore, reduced expression of cystin-1 in liver deficient in HNF6 and HNF1β is not the explanation for the reduced or absence of cilia in these two mouse models. Notably, and an avenue for further research, is the observation that the HNF1β targets in the kidney (Pkhd1, polycystic kidney and hepatic disease 1; Pkd2, polycystic kidney disease 2; Nphp1, nephronophthisis 1; IFT88, intraflagellar transport 88 homolog; and Kif12, kinesin family member 12) were not changed in HNF1β deficient liver, which indicates that HNF1β regulates a divergent transcriptional landscape for cilia in cholangiocytes versus kidney.

Loss of HNF6 results in normal apical-lateral localization of ZO-1, whereas loss of HNF1β results in very low levels of ZO-1 on the parenchymal side of forming bile duct lumen and improper apical localization of ZO-1 on the portal side. The postnatal consequences of these embryonic phenotypes that suggest loss of a cholangiocyte apical pole also differ between these two mouse models. Partial restoration of apical-basal polarity is observed when HNF6 is absent, but is not the case with HNF1β deficiency. Indeed, in patients with HNF1β mutations, ZO-1 was irregularly expressed in dysplastic ducts, and the observed DPM did not express ZO-1.

Absence of cystin-1 did not influence the apical marker osteopontin, but ZO-1 expanded to the apical surface of cholangiocytes, indicating that the basal and lateral poles were not established correctly. These phenotypes correspond this website to liver samples examined from ARPKD find more fetuses. Because of the polarity defects observed during biliary tubulogenesis, the authors investigated whether cholangiocyte ciliogenesis was disrupted in these mouse models. Previously, HNF6 and HNF1β were implicated in either control of cilia formation or regulation of genes involved in cilia function in the pancreas and kidney, respectively.10, 11 Because of the

random distribution of centrioles MCE observed in the absence of HNF6 or HNF1β, it is not surprising that embryonic cilia formation on cholangiocytes was significantly disrupted. The postnatal partial restoration of the apical-basal polarity in deficient HNF6 mice correlates with a few cilia present on cholangiocytes. However, the lack of cilia present on cholangiocytes remains as a postnatal defect in liver deficient for HNF1β. To determine if either HNF6 or HNF1β are involved in regulating the formation or function of cholangiocyte cilia, expression levels of candidate genes were examined in these two mouse models. Cystin-1 was the

only gene with reduced expression in both mouse models. Interestingly, in the cystin-1–deficient (cpk−/−) mouse model, the presence of a cilium is observed on some cholangiocytes. Therefore, reduced expression of cystin-1 in liver deficient in HNF6 and HNF1β is not the explanation for the reduced or absence of cilia in these two mouse models. Notably, and an avenue for further research, is the observation that the HNF1β targets in the kidney (Pkhd1, polycystic kidney and hepatic disease 1; Pkd2, polycystic kidney disease 2; Nphp1, nephronophthisis 1; IFT88, intraflagellar transport 88 homolog; and Kif12, kinesin family member 12) were not changed in HNF1β deficient liver, which indicates that HNF1β regulates a divergent transcriptional landscape for cilia in cholangiocytes versus kidney.

LPS from Escherichia coli O14 was prepared by phenol-chloroform-petroleum ether extraction. [3H/14C]LPS was prepared using Salmonella typhimurium PR122 as described.17 Clodronate-liposomes and phosphate-buffered saline (PBS)-liposomes were prepared by J. Niederkorn (University of Texas Southwestern Mitomycin C Medical Center) using clodronate provided by Roche. Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and Nω-Nitro-D-arginine methyl ester hydrochloride (D-NAME) were from Sigma-Aldrich (St. Louis, MO). 5-Bromo-2′-deoxy-uridine (BrdU) was from Roche Diagnostics. PEGsTNF-R1, a pegylated form of the TNF neutralizing domain of Etanercept, was provided by Amgen. Anakinra and Actemra were

purchased from Amgen and Genentech, respectively. Aoah−/− C57Bl/6 mice and μMT, Aoah−/− (double knockout) mice were produced as described.6, 18 The mice were maintained in specific pathogen-free conditions in the UT Southwestern Animal Resources Center and used for experiments when they were 5-12 weeks of age. All protocols

were approved Ku-0059436 manufacturer by the UT Southwestern Institutional Animal Care and Use Committee. PE- or Alexa Fluor 647-labeled rat antimouse F4/80 (BM8), Alexa Fluor 555 conjugated goat antirat immunoglobulin G (IgG) and Qdot 565 conjugated goat anti-fluorescein isothiocyanate (FITC) antibody were from Invitrogen. Biotin-conjugated rat antimouse CD11b (M1/70), rat antimouse CD144 (11D4.1), and FITC-labeled anti-BrdU antibody were from BD Biosciences. An agonistic monoclonal antibody (UT12) to the Toll-like receptor 4 (TLR4)/MD-2 complex was produced by S. Ohta19 and prepared as described.20 Rat antimouse interleukin (IL)-10R antibody (Ab) (YL03.1b1.3a-34ABS) and isotype control Ab (MB819.7D7.180) were generously provided by Schering-Plough MCE公司 Biopharma (Palo Alta, CA). Antibodies for flow cytometry were from BD Biosciences. Groups of three

Aoah−/− and Aoah+/+ mice were injected intravenously with UT12 IgG20 (0.0125, 0.05, 0.1, or 0.25 μg/g body weight). Livers were harvested 7 days postinjection. In another experiment, Aoah−/− and Aoah+/+ mice were injected intravenously with 0.1 μg/g body weight and studied on days 7, 14, or 21 postinjection. Aoah−/− and Aoah+/+ mice were injected intravenously with 0.5 μg E. coli O14 LPS/g body weight or an equal volume of PBS. Seven days later, animals were deeply anesthetized with isoflurane and perfused with 15 mL of PBS followed by 20 mL of fixative (4% paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodylate buffer) through the left ventricle. The liver was then removed, cut into small pieces, and immersed in fixative for 1 hour at room temperature. SEM and TEM liver samples were prepared by Tom Januszewski (Molecular and Cellular Imaging Facility, UT Southwestern). The samples were examined with an XL30 ESEM SEM and a JEOL 1200 EX TEM at voltages of 30 and 120.

LPS from Escherichia coli O14 was prepared by phenol-chloroform-petroleum ether extraction. [3H/14C]LPS was prepared using Salmonella typhimurium PR122 as described.17 Clodronate-liposomes and phosphate-buffered saline (PBS)-liposomes were prepared by J. Niederkorn (University of Texas Southwestern this website Medical Center) using clodronate provided by Roche. Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and Nω-Nitro-D-arginine methyl ester hydrochloride (D-NAME) were from Sigma-Aldrich (St. Louis, MO). 5-Bromo-2′-deoxy-uridine (BrdU) was from Roche Diagnostics. PEGsTNF-R1, a pegylated form of the TNF neutralizing domain of Etanercept, was provided by Amgen. Anakinra and Actemra were

purchased from Amgen and Genentech, respectively. Aoah−/− C57Bl/6 mice and μMT, Aoah−/− (double knockout) mice were produced as described.6, 18 The mice were maintained in specific pathogen-free conditions in the UT Southwestern Animal Resources Center and used for experiments when they were 5-12 weeks of age. All protocols

were approved SB203580 by the UT Southwestern Institutional Animal Care and Use Committee. PE- or Alexa Fluor 647-labeled rat antimouse F4/80 (BM8), Alexa Fluor 555 conjugated goat antirat immunoglobulin G (IgG) and Qdot 565 conjugated goat anti-fluorescein isothiocyanate (FITC) antibody were from Invitrogen. Biotin-conjugated rat antimouse CD11b (M1/70), rat antimouse CD144 (11D4.1), and FITC-labeled anti-BrdU antibody were from BD Biosciences. An agonistic monoclonal antibody (UT12) to the Toll-like receptor 4 (TLR4)/MD-2 complex was produced by S. Ohta19 and prepared as described.20 Rat antimouse interleukin (IL)-10R antibody (Ab) (YL03.1b1.3a-34ABS) and isotype control Ab (MB819.7D7.180) were generously provided by Schering-Plough 上海皓元医药股份有限公司 Biopharma (Palo Alta, CA). Antibodies for flow cytometry were from BD Biosciences. Groups of three

Aoah−/− and Aoah+/+ mice were injected intravenously with UT12 IgG20 (0.0125, 0.05, 0.1, or 0.25 μg/g body weight). Livers were harvested 7 days postinjection. In another experiment, Aoah−/− and Aoah+/+ mice were injected intravenously with 0.1 μg/g body weight and studied on days 7, 14, or 21 postinjection. Aoah−/− and Aoah+/+ mice were injected intravenously with 0.5 μg E. coli O14 LPS/g body weight or an equal volume of PBS. Seven days later, animals were deeply anesthetized with isoflurane and perfused with 15 mL of PBS followed by 20 mL of fixative (4% paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodylate buffer) through the left ventricle. The liver was then removed, cut into small pieces, and immersed in fixative for 1 hour at room temperature. SEM and TEM liver samples were prepared by Tom Januszewski (Molecular and Cellular Imaging Facility, UT Southwestern). The samples were examined with an XL30 ESEM SEM and a JEOL 1200 EX TEM at voltages of 30 and 120.

viverrini–associated Thai intrahepatic CCA. Antigens were retrieved from deparaffinized and rehydrated tissues by pretreating the slides in citrate buffer (pH 6.0) for 10 minutes at 108°C by way of autoclave. Immunohistochemical staining was performed using purified anti–MTA-1 immunoglobulin prepared as described.24, 29 Scoring was assessed semiquantitatively as negative (no detectable staining or positive staining in <10% of tumor cells); weakly positive (positive staining in 10%-25% of Vismodegib tumor cells); positive (positive staining in 25%-75% of tumor cells), and strongly positive (>75%) by two independent investigators. Quantitative real-time polymerase

chain reaction (PCR) was performed as described.24, 25-27 Sequences of primers are available on request. Differences among groups were compared using analysis of variance and the Student t test. P ≤ 0.05 was considered statistically significant. To investigate the influence of MTA1 on infection

and the establishment of O. viverrini, we isolated liver, small intestine, and kidney tissues from infected age-matched Mta1+/+ and Mta1−/− mice. Histopathological analyses using thin hematoxylin and eosin–stained sections revealed significant changes in the inflammatory response in Mta1+/+ and age-matched Mta1−/− mice. In particular, there was a higher occurrence of periductal fibrosis and infiltrating polymorphonuclear cells in the livers of wild-type mice compared with Mta1−/− mice (Fig. 1A; top panel). An increase in inflammatory response also correlated with a higher percentage (12%) of inflammatory zones in the Mta1+/+ mice. In addition, analysis this website of hematoxylin and eosin–stained sections of the kidney supported the observation medchemexpress that O. viverrini infection resulted in a higher magnitude of inflammatory response in Mta1+/+ mice when compared with age-matched Mta1−/− mice (Fig. 1A, bottom panel).

To determine whether the presence or absence of MTA1 had a significant effect on the pathology associated with infection, levels of critical cellular markers known to be up-regulated during O. viverrini infection were evaluated using immunohistochemistry and quantitative reverse-transcription PCR (RT-PCR). We tested expression levels of CK-19, CK-18, and annexin-2. Expression of CK-19 has been widely used to study proliferation of biliary epithelium after O. viverrini infection, whereas annexin 2 appears to be a prognostic marker of O. viverrini infection-induced CCA.19, 31 There were significant increases in expression levels of CK-19, CK-18, and annexin 2, in the liver tissues from the Mta1+/+ mice when compared with age-matched Mta1 −/− mice using both immunohistochemistry (Fig. 2A-D) and quantitative real-time PCR (Fig. 2E-G). The T cell repertoire and secreted cytokines play an important role in determining the outcome of parasitic infections.

viverrini–associated Thai intrahepatic CCA. Antigens were retrieved from deparaffinized and rehydrated tissues by pretreating the slides in citrate buffer (pH 6.0) for 10 minutes at 108°C by way of autoclave. Immunohistochemical staining was performed using purified anti–MTA-1 immunoglobulin prepared as described.24, 29 Scoring was assessed semiquantitatively as negative (no detectable staining or positive staining in <10% of tumor cells); weakly positive (positive staining in 10%-25% of Selumetinib tumor cells); positive (positive staining in 25%-75% of tumor cells), and strongly positive (>75%) by two independent investigators. Quantitative real-time polymerase

chain reaction (PCR) was performed as described.24, 25-27 Sequences of primers are available on request. Differences among groups were compared using analysis of variance and the Student t test. P ≤ 0.05 was considered statistically significant. To investigate the influence of MTA1 on infection

and the establishment of O. viverrini, we isolated liver, small intestine, and kidney tissues from infected age-matched Mta1+/+ and Mta1−/− mice. Histopathological analyses using thin hematoxylin and eosin–stained sections revealed significant changes in the inflammatory response in Mta1+/+ and age-matched Mta1−/− mice. In particular, there was a higher occurrence of periductal fibrosis and infiltrating polymorphonuclear cells in the livers of wild-type mice compared with Mta1−/− mice (Fig. 1A; top panel). An increase in inflammatory response also correlated with a higher percentage (12%) of inflammatory zones in the Mta1+/+ mice. In addition, analysis KU-57788 mouse of hematoxylin and eosin–stained sections of the kidney supported the observation MCE公司 that O. viverrini infection resulted in a higher magnitude of inflammatory response in Mta1+/+ mice when compared with age-matched Mta1−/− mice (Fig. 1A, bottom panel).

To determine whether the presence or absence of MTA1 had a significant effect on the pathology associated with infection, levels of critical cellular markers known to be up-regulated during O. viverrini infection were evaluated using immunohistochemistry and quantitative reverse-transcription PCR (RT-PCR). We tested expression levels of CK-19, CK-18, and annexin-2. Expression of CK-19 has been widely used to study proliferation of biliary epithelium after O. viverrini infection, whereas annexin 2 appears to be a prognostic marker of O. viverrini infection-induced CCA.19, 31 There were significant increases in expression levels of CK-19, CK-18, and annexin 2, in the liver tissues from the Mta1+/+ mice when compared with age-matched Mta1 −/− mice using both immunohistochemistry (Fig. 2A-D) and quantitative real-time PCR (Fig. 2E-G). The T cell repertoire and secreted cytokines play an important role in determining the outcome of parasitic infections.

Eph-ephrin signaling mainly affects cell shape and motility by regulating cytoskeletal organization and cell adhesion and

also influences cell proliferation and cell-fate determination.38 In our research, we found that EphA4 suppressed cell migration and invasion JAK phosphorylation but promoted cell adhesion, which was the inverse of the functions of miR-10a in HCC cells. As described above, EMT is a process that plays important roles in both development and oncogenesis. During EMT, epithelial cells acquire a mesenchymal phenotype that is characterized by the loss of intercellular junctions and increased cell migration. A previous study has also indicated that EphA4 participates in the MET process,20 and the morphology of the QGY-7703 cells changed after alteration of miR-10a or EphA4 expression (Supporting Fig. 11). We speculated that miR-10a and EphA4 played roles

in the EMT process in HCC. Usually, the loss of intercellular junctions and the increased cell migration during EMT are evidenced by increasing expression of vimentin and decreasing expression of E-cadherin.19 To test our hypothesis, we examined the expression of the epithelial marker E-cadherin and the mesenchymal markers vimentin and ICAM-1. As expected, down-regulation of miR-10a or up-regulation of EphA4 suppressed the EMT phenotype. In other words, miR-10a can increase, whereas EphA4 can suppress HCC cell migration and invasion by mediating the EMT process. Furthermore, Xiang et al.39 indicated

that tumor cells with an epithelial phenotype have a growth advantage in the tissue environment when compared with Endocrinology antagonist those with a mesenchymal 上海皓元医药股份有限公司 phenotype. When miR-10a is up-regulated, the expression level of EphA4 is accordingly down-regulated, and the blockage of the EMT process is relieved. HCC cells with enhanced miR-10a expression reacquire the mesenchymal phenotype, which may impair the proliferation capacity in the liver, resulting in decreased intrahepatic metastatic nodules. Although EphA4 is the direct target of miR-10a, we further explored the pathway by which miR-10a and EphA4 affected cell adhesion. Bourgin et al.32 reported that EphA4 regulates dendritic spine remodeling by affecting β1-integrin signaling pathways. Davy and Robbins40 also suggested that Ephrin-A5 modulates cell adhesion and morphology in an integrin-dependent manner, and previous studies have indicated that EphA4 can interact with Ephrin-A5 and participate in signal transduction. Integrin is an α/β heterodimeric membrane protein that mediates the adhesion of cells to components of the ECM. The integrin β1 subunit is crucial for adhesion to fibronectin (FN),41 which is one important component of the ECM. We measured the protein level of β1-integrin and found that it was up-regulated by miR-10a inhibition or EphA4 overexpression. These observations suggest that miR-10a and EphA4 regulate cell adhesion by mediating the β1-integrin signaling pathway.