Next, coculture experiments indicated that HCC cell-derived exoso

Next, coculture experiments indicated that HCC cell-derived exosomes promoted the cell growth, migration and invasion of HCC cells and had the ability to shuttle miRNAs to recipient cells. Further, our data showed that Vps4A, a key regulator of exosomes biogenesis, was frequently down-regulated in HCC tissues. The reduction of Vps4A in HCC tissues was associated with tumor progression and metastasis. In vitro studies revealed that Vps4A repressed the growth, colony formation, migration and invasion of HCC cells. We further investigated the role and involvement of Vps4A in suppressing the bioactivity of

exosomes and characterized its ability to weaken the cell response to exosomes. By small RNA sequencing, we demonstrated that Vps4A facilitated the secretion of oncogenic miRNAs in HER2 inhibitor exosomes, as well as accumulation and uptake of

tumor suppressor miRNAs in cells. A subset of Vps4A-associated miRNAs was identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the phosphatidylinositol-3-kinase (PI3K) /Akt signaling pathway was the most likely candidate pathway for modulation by these miRNAs. Indeed, we proved that the PI3K/Akt pathway was inactivated by Vps4A-overexpression. Conclusion: Exosome-mediated miRNA transfer is an important mechanism of self-modulation of the miRNA expression profiles in HCC cells. Vps4A may function as a tumor suppressor, which utilizes exosomes as mediators to regulate the secretion and uptake of miRNAs in hepatoma cells. These observations provide new insights into the development of HCC. This RGFP966 research buy article is protected by copyright. All rights reserved. “
“Death receptors, a subset of the tumor necrosis factor (TNF) receptor (TNFR) superfamily that includes TNFR1, CD95 (Fas, Apo-1),

and the TRAIL (TNF-related apoptosis-inducing ligand) receptors, transduce signals capable of engaging apoptosis or necrosis,1 depending on the status of signaling molecules in the cells. Ligation of one such receptor, cluster of differentiation 95 (CD95), has catastrophic consequences in vivo, because this results in lethal, fulminant liver destruction.2 In this issue click here of HEPATOLOGY, Hikita et al.3 employ a conditional gene deletion model to explore the molecular mechanisms of this liver failure. CD95, cluster of differentiation 95; FADD, Fas-associated protein with death domain; TNF, tumor necrosis factor; XIAP, X-linked inhibitor of apoptosis protein. Upon ligation, CD95 rapidly recruits an intracellular adapter molecule, Fas-associated protein with death domain (FADD), which in turn binds to and activates the initiator caspase, caspase-8.1 The activation of caspase-8 requires two steps: dimerization of the inactive “pro-form” of the molecule, followed by autocleavage, which generates a stable, active protease.

In this multivariate analysis, Child-Pugh score, PVT, BCLC classi

In this multivariate analysis, Child-Pugh score, PVT, BCLC classification, and use of secondary prophylaxis remained independent predictors of death (Table 5B). When the independent predictors of failure of secondary prophylaxis were evaluated, only BCLC classification

(hazard ratio [HR]: 1.78; 95% confidence interval [CI]: 1.23-2.59), presence of PVT (benign HR: 1.70; 95% CI: 0.61-4.74; malignant HR: 4.62; 95% CI: 1.96-10.90), and use of secondary prophylaxis (HR, 0.33; 95% CI: 0.14-0.75) were independently associated with outcome. Taking into account that the differences in the use of secondary prophylaxis were mainly in patients with BCLC C and D, further analysis Caspase inhibitor was performed to compare these patients with and without prophylaxis (see Supporting Table 1). Patients who received no prophylaxis had more-severe liver disease, as shown by greater Child-Pugh score and MELD score, although there were no differences in FDA approved Drug Library manufacturer severity of the HCC, as shown by the proportion of patients with BCLC C or D, PVT, or metastasis. In this study, a significantly lower survival rate was observed in patients who had HCC at the time of bleeding

than patients who did not have HCC, despite the fact that patients were matched for Child-Pugh class and age. This issue is of utmost interest because many studies that evaluated the treatment of acute bleeding episode and prophylaxis of rebleeding had excluded patients with HCC.[12-25] Furthermore, given the increasing incidence of HCC, as a result of rising hepatitis C virus (HCV)-associated advanced liver disease, click here which is expected to peak in 2020, HCC and VB are an increasingly common clinical problem that clinicians have to deal with. On the other hand, with further improvement in the management of patients

with HCC with survival benefit,[33-37] these patients have more probabilities to present with complications of ESLD. A previous study based on ICD-9 diagnostic codes suggested similar results, although as a result of the design of the study, no in-depth analysis could be performed.[9] Interestingly, patients with HCC were less likely to have secondary prophylaxis than patients without HCC, and there was a trend for a less-frequent use of standard secondary prophylaxis with combination of beta-blockers and endoscopic band ligation in those patients with HCC. The reason why HCC patients were not offered standard therapy is unclear from this study. It is likely that this was because of the assumption, by the attending physician, that this would not result in a clinical benefit. This is also suggested by the fact that patients with HCC without secondary prophylaxis seemed to have more-severe liver disease.

5%, 29/40) (χ2 = 4933, p < 005) There was not significant diff

5%, 29/40) (χ2 = 4.933, p < 0.05). There was not significant difference of the positive rate of Sox2 among the other clinical parameter's groups (such as tumor location, size, Lauren's type, invasion depth and clinical stage). Conclusion: The low-expression of Sox2 maybe play a role in the gastric carcinogenesis and tumor cell differentiation, metastasis.

Key Word(s): 1. Stomach neoplasms; 2. SOX 2 protein; 3. expression; Presenting Author: DONGXU WANG Corresponding Author: DONGXU WANG Affiliations: PLA 254th hospital Objective: The purpose of the study is to investigate the expression of high mobility group box 1 (HMGB1) in gastric cancer and precancerous lesions, and explore its relationship with Dabrafenib in vivo the carcinogenesis and progression see more of gastric cancer. Methods: 125 cases of surgical resected gastric specimens were collected from PLA 254th hospital

between 2003–2011 Immunohistochemical S-P method was used to detect the expression of HMGB1 in 30 cases of normal gastric mucosa, 20 cases of intestinal metaplasia mucosa, 24 dysplasia mucosa, 51 cases of gastric cancer. χ2test was used to statistically analysis the difference of expression rate of HMGB1 between the normal gastric mucosa, intestinal metaplasia, dysplasia and gastric cancer lesion. The relationship between the expression rate of HMGB1 and clinical pathological parameter of gastric cancer (such as tumor location, size, differentiation, Lauren’s type, invasion depth, lymph node metastasis and clinical stage) was statistically analyzed by means of χ2test. Results: No positive expression of HMGB1 was found in normal gastric tissues. The positive expression

of HMGB1 was 35%, 41.7%, 80.4%in intestinal metaplasia, dysplasia and gastric carcinoma respectively. The positive rate of HMGB1 gradually increased along with the progression from the normal gastric tissue to intestinal metaplasia, dysplasia and gastric carcinoma (P < 0.05). It was found that the positive expression of HMGB1 in intestinal metaplasia, dysplasia and gastric carcinoma was all significantly higher than that in normal gastric mucosa.(χ2 value was 12.209, 15.341, 48.838 respectively and all p values <0.05). There was not significant difference selleck kinase inhibitor of the positive expression of HMGB1 between the dysplasia and intestinal metaplasia; The positive expression of HMGB1 in gastric cancer was statistically higher than that in intestinal metaplasia (χ2 = 13.516 P < 0.05) and dysplasia (χ2 = 11.247; P < 0.05) respectively; The positive rate of HMGB1 in gastric carcinoma with lymph node metastasis (90.6%) was higher than that in the group without lymph node metastasis (63.2%) (χ2 = 5.706, p < 0.05); it was found that the positive rate of HMGB1 in TNM III/IV stage (95.7%) was higher than that in TNM I/II stage (67.9%) (χ2 = 6.189, P < 0.05).

0 Results:  Ninety-four patients received sorafenib until August

0. Results:  Ninety-four patients received sorafenib until August 2010. The overall incidence of treatment-related adverse events was 98% of patients. Skin toxicities, including palmar-plantar erythrodysesthesia syndrome, rash, pruritus and alopecia, were the most common adverse events and were observed in 58 patients (62%). Hypertension was observed in 23 patients (24%). The median survival time was 12.5 months among the total patients. The patients with skin toxicities showed significantly longer survival than the patients without these toxicities (hazard ratio, 0.449; 95% confidence interval, 0.256–0.786; P = 0.005).

Hypertension had no correlation with survival. Skin toxicities check details were also significant

prognostic factors in a multivariate analysis (hazard ratio, 0.522; 95% confidence interval, 0.274–0.997; P = 0.049), along with Child–Pugh class and α-fetoprotein level. The median development time for skin toxicities was 21 days. Conclusion:  Skin toxicities occur commonly at the early phase in patients treated with sorafenib, and could be a promising surrogate marker for the treatment outcome. “
“The association between sarcopenia and nutritional status is thought to be an important problem in patients with cirrhosis. In this study, we investigated whether nutritional factors were related to sarcopenia in patients with liver cirrhosis. The subjects were www.selleckchem.com/products/EX-527.html 50 patients with cirrhosis aged 41 years or older. In this study, the subjects were interviewed about their dietary habits, and their daily physical activity was surveyed using a pedometer. The skeletal muscle mass index (SMI) was calculated using the appendicular skeletal muscle mass (ASM) measured by bioelectric impedance analysis. The handgrip strength was measured using a hand dynamometer. Sarcopenia was defined by find more SMI and handgrip strength. The patients with cirrhosis were categorized as normal group or sarcopenia group, and the two groups were compared. Univariate

and multivariate logistic regression modeling were used to identify the relevance for sarcopenia in patients with cirrhosis. Height (odds ratio (OR), 5.336; 95% confidence interval [CI], 1.063–26.784; P = 0.042), energy intake per ideal bodyweight (IBW) (OR, 5.882; 95% CI, 1.063–32.554; P = 0.042) and number of steps (OR, 4.767; 95% CI, 1.066–21.321; P = 0.041) were independent relevant factors for sarcopenia. Moreover, a significantly greater number of the patients in the sarcopenia group had low values for both parameters’ energy intake per IBW and number of steps. Our results suggest that walking 5000 or more steps per day and maintaining a total energy intake of 30 kcal/IBW may serve as a reference for lifestyle guidelines for compensated cirrhotic patients. “
“Hepatitis C virus (HCV) infection is a major cause of hepatocellular carcinoma (HCC) and chronic liver disease worldwide.

Six hydrogen bonds were established between hydroxyl groups of EG

Six hydrogen bonds were established between hydroxyl groups of EGCG and hydrogen-bond acceptors (nitrogen or oxygen) in CBR1. The polyphenol structure of EGCG appeared to be crucial for its binding to CBR1. Importantly, the phenolic hydroxyl group in the gallate moiety of EGCG reached deeply into the active site and interacted with Ser139 and Tyr193 of the catalytic triad. The phenolic oxygen was positioned 3.43 Å from Oγ of Ser139 and 3.48 Å from Oη of Try193, and this suggested the existence of strong hydrogen-bond interactions

(Fig. 2B). EGCG is positioned differently from hydroxy-PP, which binds selleck to the substrate-binding site of CBR1.21 The structure of the substrate isatin is similar to that of hydroxy-PP and has the same pyrazolopyrimidine core, and it is thus not surprising that they compete against each other for the same site of CBR1. This suggests that EGCG does not bind to the substrate-binding

site as hydroxy-PP does. EGCG is also positioned differently from NADPH. This model is in agreement with the results of check details an enzyme assay, which showed that EGCG is a noncompetitive inhibitor against both isatin and NADPH. The model was further verified by an examination of the inhibitory activity of EGCG on CBR1 mutants. The R95A and K231A mutants, which were as active as the wild-type enzyme, were significantly less sensitive to EGCG with IC50 values 8.3-fold and 9.2-fold higher than that of the wild-type enzyme, respectively (Supporting Information Table 2). As the metabolism of DNR by CBR1 in tumor cells has been shown to contribute to drug resistance, it was expected that EGCG would enhance the antitumor effect of DNR by inhibition of the CBR1-mediated metabolism. To test this possibility, we measured the ability of EGCG to block CBR1-mediated metabolism of DNR in hepatoma cells with a cell viability assay. We carried out a protein western blot analysis to determine endogenous protein levels of CBR1 in different hepatoma cells (Fig. 3A). The expression levels of CBR1 in most of the HCC cells were comparable to those in human hepatocytes (L02). Only in Hep3B was the CBR1 expression significantly reduced for

some reason. We selected HepG2 and SMMC7721 as CBR1 high-expression cells and Hep3B as CBR1 low-expression cells in the ensuing studies. The concentration selleck chemicals llc of EGCG that exhibited minimal cytotoxicity in hepatoma cell lines when used alone was selected for the treatment in combination with DNR (Supporting Information Fig. 4). In HepG2 cells, EGCG induced a 16.2% enhancement of DNR-mediated growth inhibition (Fig. 3B, left panel), and the enhancement was 20.5% in SMMC7721 cells (Fig. 3B, middle panel). The enhancement effect of EGCG was dose-dependent. In contrast, EGCG did not affect the sensitivity of DNR in Hep3B cells (Fig. 3B, right panel), and this further supports the idea that the enhancement effect of EGCG is CBR1-dependent.

Expression of sialylated Lex, involved in SabA-mediated adherence

Expression of sialylated Lex, involved in SabA-mediated adherence of H. pylori, was mainly observed in the body. Of known canine non-H. pylori Helicobacter

species, H. heilmannii sensu stricto presented the highest adherence scores to the antral mucosa in canine paraffin-embedded sections. The relationship between pet ownership or frequent exposure to dogs and infection with different gastric Helicobacter species was assessed [35]. A significant correlation was found between human and canine infection for H. felis and to a lesser extent for H. bizzozeronii. The poultry gut microbiota was little studied, while chickens are a major meat source worldwide and are considered as important reservoirs for foodborne pathogens. High abundance of Campylobacter species H. pullorum and Megamonas species

was found in the buy MLN0128 cecal microbiome of Ross broiler chickens housed indoors under standard commercial conditions [36]. The gastrointestinal tract microbiota was characterized in king, gentoo, macaroni, and little penguin species [37]. 16S rRNA gene pyrosequencing revealed that Helicobacteriaceae was the third dominant family in king penguins (8%) in contrast to other penguin species. In the Proteobacteria phylum, Helicobacter INCB024360 in vitro species ranged from 1 to 11% in these four marine seabird species. Of 3889 16S rRNA sequences analyzed from the feces of migrating birds (migratory stopover, Delaware Bay, USA), 6.5% corresponded to Epsilonproteobacteria, that is, Campylobacter (82.3%) and Helicobacter (17.7%) species.

Most Helicobacter-like sequences were closely related to H. pametensis and H. anseris, while the low percentage of sequence identity (92%) with H. anseris suggests a different Helicobacter species [38]. Helicobacters were detected at low frequence in feces and intestinal tissues of tropical terrestrial wild birds (Venezuela) by molecular methods [39], suggesting that these bacteria may be uncommon in the populations studied. PCR selleck compound arrays for commonly reported rodent infectious agents were used in naturally infected index mice and sentinel mice exposed by contact and soiled-bedding transfer [40]. Helicobacters and pinworms were detected in fewer than half of the soiled-bedding sentinels. Of the four Helicobacter species identified in index mice, only H. ganmani was found in soiled-bedding and contact sentinels. The prevalence of enterohepatic Helicobacter (EHH) infection was determined in a study on old rhesus monkeys [41]. Helicobacter infection (PCR, culture) was present in 97% of the monkeys; 13 of 14 monkeys diagnosed with intestinal adenocarcinoma were infected. H. macacae and “Helicobacter sp. rhesus monkey” taxons 2 and 4 were detected on the epithelial colonic surface. In vitro experiments showed bacterial adherence to epithelia, invasion as well as induction of proinflammatory gene expression, while genes involved in the inflammasome were downregulated.


“Several recent studies have shown that in migraine patien


“Several recent studies have shown that in migraine patients, the prevalence of an incomplete Circle of Willis is higher than in controls.1-3 This might suggest that such an anatomic anomaly is a risk factor for developing migraine. As an explanation, it has been commonly proposed that an incomplete Circle of Willis could prevent regional cerebral KU-57788 molecular weight blood flow adaptation in the face of increased metabolic demand. A resulting local ischemia could then, in hyperexcitable persons,

lead to a cortical spreading depression that is by many investigators believed to be the cause of migraine attacks.[3] Unfortunately, these studies did not measure regional cerebral blood flow during increased brain activity or, eg, a one-sided carotid artery occlusion. We doubt whether in subjects with an incomplete see more Circle of Willis such conditions will importantly diminish cerebral perfusion. In this paper, we argue that an incomplete Circle of Willis might increase the risk for migraine by elevated wall shear stress in small-diameter anastomotic vessels. Aberrations in the Circle of Willis do not automatically cause a dramatic reduction of anastomotic flow to the brain. Only a simultaneous absence of both the anterior communicating artery (or an A1 segment) and

a posterior communicating artery (or P1 segment) would definitely prohibit flow compensation via the Circle of Willis (see Figure). The presence of this combination seems, however, rare because it was not observed in a study on 360 normal fixed brains.[4] Furthermore, even a moderate decrease of cerebral blood flow (from 47 to 37 mL/100 g/min) is sufficient to attenuate attention and motor reactions.[5] It is hard to believe that in half of the population, having similar morphologic variants, such signs of diminished brain function, will be elicited by increased brain activity or by head rotation. Indeed, occlusion of a carotid artery in patients with incomplete Circle of Willis, anesthetized for open arch surgery or kept conscious for carotid endarterectomy, did not elicit signs of transient cerebral

ischemia, even not in the presence of severe bilateral carotid artery stenosis.[6, 7] In addition, secondary collateral pathways are formed by the external carotid artery via the ophthalmic selleck artery and via leptomeningeal anastomoses at the brain surface.[8] An imminent shortage of regional cerebral blood supply will obviously be corrected by a rise of blood flow velocity in the patent portions of the circuit, as a result of an increased pressure gradient. An example of this is observed in a study with carotid occlusion in healthy volunteers, showing increased flow velocity, as measured by transcranial color-coded duplex sonography, in a posterior communicating artery.[9] An increased flow velocity may enhance wall shear stress.

Table 5 presents the results from adjusted logistic regression mo

Table 5 presents the results from adjusted logistic regression models for the associations of childhood trauma categories with obesity, smoking status, substance abuse, depression, and anxiety. All models were adjusted for age, gender, race, education, household CH5424802 income levels, obesity (BMI ≥ 30 kg/m2), smoking status, and substance abuse. The models were additionally adjusted for current depression and anxiety. Odds ratios for the relationships between particular childhood abuse and neglect (compared with those without exposure to any trauma category) and the variables of interest

are reported in Table 5. Obesity, current smoking, and current substance abuse were not associated with any of the childhood trauma categories. Prior substance abuse (which included medication overuse) was, however, associated with physical, sexual abuse (P = .0004 for both), and physical (P = .007), emotional neglect (P = .005). Current depression was associated with physical (P = .003), sexual (P = .007), and emotional abuse (P < .001), and physical

and emotional neglects (P = .001 MK-2206 cell line for both). Current anxiety was associated with all childhood abuse and neglect categories (P < .001 for all). A graded relationship of childhood maltreatment was observed with current depression and anxiety (Table 6). Eighteen percent of the study population reported 1, 15% reported 2, and 25% reported 3 or more categories of childhood trauma. With an increase in the number of maltreatment types, the likelihood of current depression, anxiety, or both, also increased significantly. For migraineurs reporting 3 or more types of maltreatment in childhood there check details was a 4-fold prevalence of depression and anxiety compared with those not reporting maltreatment. Prevalence of self-reported physician diagnosis of depression and anxiety was also higher in persons reporting childhood maltreatment.

In this study, 41% (n = 538) had been diagnosed with depression and 31% (n = 410) with anxiety. Diagnosis of both depression and anxiety were significantly higher in migraineurs reporting childhood abuse and neglect (P < .001 for all categories of abuse and neglect). In adjusted logistic regression analysis, migraineurs reporting 3 or more types of maltreatment were more likely to have had a physician-diagnosis of both depression and anxiety in the past (OR = 6.91, 95% CI: 3.97-12.03, P < .001), or either depression or anxiety (OR = 3.66, 95% CI: 2.28-5.88). This is the largest study to date of abuse in a migraine clinic population.

(Table 1)10-13 TARE compared to TACE has been reported to be sup

(Table 1).10-13 TARE compared to TACE has been reported to be superior in the ability to downstage T3 to T2, shorter median time to radiographic response and associated with significantly prolonged TTP. The potential

implications for patients listed for orthotopic liver transplantation (i.e., enabling patients to wait longer without drop out) INCB024360 cell line are merely speculative. Moreover, data supports the prognostic role of the response to liver directed therapy acting as a biological stress test to provide insight into a tumor’s aggressiveness.14 Any differences exerted in selection pressure by different forms of LDT remains to be seen and can only be addressed in well developed randomized controlled trials. Data comparing sorafenib to TARE in patients with PVT is even sparser, currently existing only across studies and therefore less clinically meaningful. To this end, RCTs comparing standard of care (TACE, sorafenib) to TARE are warranted. Logistic concerns include the number of patients required; a power

calculation performed to determine the sample size to demonstrate therapeutic equivalency between TACE and TARE in BCLC B patients showed that more than 1000 patients would be needed.13 The feasibility KU-60019 datasheet of a large trial due to cost and the number of centers with adequate expertise in both treatment modalities requires careful consideration; however, the number of centers utilizing TARE appears to be increasing making this less of a limitation for conducting such a trial. Lastly, stratification for lobar versus selective selleck chemicals treatment and standardization of TACE methodologies would be required given differences in treatment practices. In BCLC C patients, the anticipated trial design would be sorafenib ± TARE with a primary endpoint of TTP. There are several examples of accepted treatment practices

for HCC that are based on cohort analyses (not RCTs) that have been accepted into treatment guidelines including RFA (<3 cm) versus hepatic resection, transplantation versus hepatic resection, and open versus laparoscopic hepatic resection. Such trials for TARE are unlikely to come to fruition. TARE is currently not recognized by the American Association for the Study of Liver Diseases or EASL in the management of HCC due to lack of randomized data. However the National Comprehensive Cancer Networks have endorsed TARE as one of the treatment options for HCC.15 At our institution on ongoing RCT (PREMIERE Trial) is comparing TARE to various liver directed therapies (RFA, TACE, or RFA+TACE) based on tumor size and number.

Four days after AAV8 injection in mice, we administered Jo2 antib

Four days after AAV8 injection in mice, we administered Jo2 antibody intraperitoneally and observed the effect of miR-221 overexpression on Jo2-induced apoptosis. We found delayed death due to fulminant liver failure in mice overexpressing miR-221 compared

to control mice (Fig. 4A). To confirm that the observed survival effect of miR-221 was indeed due to inhibition of apoptosis in the liver, four mice in both groups were sacrificed at 9 hours after Jo2 injection. We found that miR-221 overexpressing mice had reduced Alpelisib cost pathological signs of liver injury (Fig. 4B). Consistent with liver morphology, miR-221 overexpressing mice had decreased levels of serum transaminases and less hepatocyte apoptosis as detected by lower caspase-3/7 activity (Fig. 4C,D) and a reduced number of TUNEL-positive nuclei (Fig. 4E) than their respective controls. Thus, miR-221 overexpression in mouse liver delays FAS-induced fulminant liver failure by inhibiting hepatocyte apoptosis. Next, we investigated the mechanism of protection of hepatocytes from apoptosis by miR-221. To find protein targets of miR-221 we used PICTAR,

Target Scan, and miRANDA algorithms. In silico analyses identified PUMA, a proapoptotic protein, as a target for miR-221. If Puma mRNA is a true target of miR-221, expression of PUMA protein levels should change in the liver at 12 hours after Jo2-injection in mice, because miR-221 expression increases at this timepoint (Table 1; Supporting Fig. S2c). We therefore determined the level of PUMA at 12 hours after Jo2 injection in primary hepatocytes. We found that PUMA protein expression Sirolimus research buy in hepatocytes was dramatically reduced at 12 hours after Jo2 injection compared to 0 hours control hepatocyte lysates (Fig. 5A), albeit its transiently elevated levels at earlier timepoints (Supporting Fig. S3a). click here Decrease in PUMA protein levels at 12 hours suggests regulation of PUMA at the posttranscriptional

level. However, this decrease may simply be due to transcriptional down-regulation. We therefore determined Puma mRNA levels after Jo2-induced apoptosis by qRT-PCR. We found that mRNA levels of Puma did not decrease; we rather observed a 1.8-fold increase in Puma mRNA expression levels in isolated primary hepatocytes at 12 hours after Jo2-injection (Fig. 5B). Thus, decreased protein levels of PUMA at 12 hours but not those of its mRNA strongly suggest posttranscriptional regulation of PUMA in hepatocytes. To further investigate whether Puma is posttranscriptionally regulated by miR-221 we cloned the 3′ UTR of Puma downstream of a firefly luciferase gene in a miRGLO vector (henceforth, this construct will be referred as miR-GLO-PUMA). Luciferase reporter assay using the miRGLO vector was used to demonstrate the direct binding of a mature miRNA with 3′ UTR of mRNA.