Trans-nasal flexible fibre-optic laryngoscopy (TFFL) is an essential skill for otolaryngologists. There is evidence to suggest that simulators help residents acquire procedural skills. The objective of this study was to examine the effect of simulation on endoscopy skill acquistion.A randomized controlled trial was conducted utilizing medical students and junior residents with limited experience in TFFL. Learners all performed a baseline endoscopy and were then randomized to receive either 45 minutes of simulation training or not. Following this, a second endoscopy was performed. Time to adequate visualization of the glottis, the percentage of time adequate visualization of the airway was maintained, and the number of collisions with mucosa were analyzed. Qualitative assessments were also obtained from the learner, patient, and staff laryngologist.Time to adequate visualization of the glottis and the number of mucosal collisions were significantly less during the second endoscopy, irrespective of the use of simulation . Analysis using a two-way ANOVA with interaction established that none of the quantitative measures analyzed in this study improved with the addition of simulation.Improvements in time to visualization of the glottis and number of mucosal contacts were seen between the first and second endoscopy irrespective of simulator use. No additional benefit was conferred with the use of a low-fidelity simulator. Trans-nasal fibre-optic flexible laryngoscopy (TFFL) is routinely performed by otolaryngologists, both at the bedside and in the out-patient setting. This procedure allows for detailed evaluation of the nasal, pharyngeal, and laryngeal anatomy. Prior to learning how to correctly recognize pathology, trainees must learn to manipulate the endoscope and pass it through the upper aerodigestive tract.Surgical education has traditionally been based on the apprentice model, wherein skills are fostered and developed in the clinical setting . TraininOur objective was to examine the effect of low-fidelity simulation training on TFFL skills in the clinical setting. To that end, we developed a low-fidelity TFFL simulator that allowed learners to practice endoscopic manipulation skills outside of the clinical setting. We subsequently designed an experiment to examine the effect of low-fidelity simulator training on clinical endoscopy skills. Our primary outcome measure was the time taken to insert the endoscope, traverse the nasal cavity and upper aerodigestive tract and visual the glottis.Ethical approval was obtained from the University of Calgary Conjoint Health Research Ethics Board. The study was conducted through the Voice Disorders Clinic at the Rockyview General Hospital in Calgary, Alberta, a tertiary referral center for laryngeal disorders. A fellowship-trained laryngologist (JDB) supervised and evaluated all endoscopies. Learners were recruited from volunteer medical students and junior residents on otolaryngology rotations through the University of Calgary. All learners completed a questionnaire assessing prior endoscopy experience. All patients who presented to the Voice Disorders Clinic routinely require laryngeal examination and were solicited for their participation. All patients who underwent TFFL in this study were volunteers, with informed consent being obtained prior to endoscopy.All learners observed one TFFL being performed by the senior author and received instruction on how to hold and manipulate the endoscope prior to performing an endoscopy. The learner then performed a TFFL on a clinic patient. During this endoscopy the senior author only provided guidance if required to ensure patient safety.Once the first endoscopy was complete, the learner was assigned to either the simulation-group or the control-group by random draw. Learners randomized to the simulation group were given 45 minutes to practice basic endoscopy skills using the low-fidelity simulator, whereas the control group was not. To ensure that the senior author remained blinded, learners from both groups were excused from clinic for 45 minutes. After the allotted time, all learners returned to clinic and performed a second TFFL. The second TFFL was on a different patient than their initial endoscopy Figure .In total, each learner performed two separate TFFLs. The laryngologist determined which side of the nose presented the best avenue for endoscopy. Topical intra-nasal lidocaine (20 mg/mL) was used for every endoscopy . Nasal decongestant was not used. Lubricant was applied to the endoscope prior to insertion . A distal chip endoscope  was used for all endoscopies. All endoscopies were recorded for subsequent analysis.The patient was blinded to which endoscopy number and group the learner was randomized to. The senior author was blinded to which study group the learner belonged to, but was not blinded to whether this was the learner’s first or second endoscopy. The endoscopy videos were later analyzed by one of the study investigators (MWD) who was blinded to randomization group, learner name, and whether it was the learner’s first or second endoscopy.The variables assessed included: (1) time to adequate visualization of the glottis; (2) percentage of time adequate visualization of the airway was maintained during passage of the endoscope; and (3) total number of endoscope collisions with mucosa. Time to adequate visualization was defined as the amount of time required for the learner to pass the endoscope from the nasal alae until adequate visualization of the glottis was achieved. The percentage of time adequate visualization was maintained was defined as the percentage of time an adequate view of the airway was maintained, such that a structure within the nasal cavity or larynx could be readily identified rather than obscured by mucosa.During both endoscopies the learner was also subjectively evaluated by the senior author (JDB) as well as the patient. Patient comfort, ease of manipulating the endoscope, and learner comfort were assessed using a 10-point scale, with 1 being strongly agree and 10 being strongly disagree. The learners also rated their own ability using the same scoring system.The simulator consisted of two parts: (1) a flexible endoscope attached to a digital camera and light source; and 2) a hollow plastic ball with multiple fenestrations located inside a glove . An relevant ,5. CombiIn total 69 learners were enrolled in the study. Four learners’ data sets were removed because they were incomplete. In three cases, the video files were corrupt, and in one instance the learner was unable to complete the endoscopy and, for the sake of patient comfort and safety, the principle investigator completed the endoscopy. The result was 32 learners in the simulation group and 33 in the control group . Similarly, the senior author’s assessment of patient comfort during the examination did not differ between the first endoscopy of the simulation and control groups . In addition, there was also no difference in learners’ comfort and their ability to manipulate the endoscope as assessed by the senior author . There was also no difference in patient comfort and willingness to repeat the examination, as assessed by the patients . In summary, there was no qualitative or quantitative difference between the simulation and control groups’ first endoscopy.Both groups had an equivalent amount of endoscopy experience prior to the study Table . The firThirteen endoscopies  were randomly selected and re-analyzed by the same study investigator (MWD). The intraclass correlation coefficient (ICC) was used to calculate intra-rater reliability of the 3 main objective variables. A ICC of 0.7-0.8 indicates strong agreement and a ICC of > 0.8 indicates almost perfect agreement. The ICCs for time to adequate visualization of the glottis, percentage of time adequate visualization of the airway was maintained, and the total number of endoscope collisions with mucosa were 0.99, 0.76, and 0.81 respectively.Overall, there was a significant improvement both in the time to adequate visualization of the glottis and number of mucosal contacts between the first and second endoscopies, irrespective of simulator use (84.8 ± 38.0 sec vs. 68.0 ± 33.9 sec, p < 0.01; 5.0 ± 4.6 vs. 3.2 ± 3.0, p < 0.01; Table Data from the simulation group and control group, irrespective of the first and second endoscopy were compared. No significant differences in any of the quantitative or qualitative measures described were identified Table .A two-way ANOVA with interaction was used to explore the effect of both simulation and endoscopy. No interaction between simulation and repeat endoscopy was detected. Time to adequate visualization of the glottis, the number of mucosal contacts, and the percentage of time adequate visualization of the airway were maintained were not affected Table .In this study we examined the effect of low-fidelity simulation training, as well as the interaction of repeat clinical endoscopy and simulation training, on both clinical endoscopy skills and patient comfort. Overall, there was a significant reduction in time to adequate visualization and number of mucosal contacts between first and second endoscopy. This suggests that the benefits of repetition quickly become appreciable. A recent study has shown that learners become competent after 6 endoscopies [Interestingly, when data were stratified by both endoscopy number and simulation, there was no significant interaction seen. This suggests that, in this study, there was no additional benefit, above and beyond repeat endoscopy, conferred by the use of the simulator.Learners performed TFFL on different patients for each endoscopy. While patients may have mild variations in their anatomy, none had had previous endoscopic sinus surgery, major head and neck surgery or head and neck external beam radiotherapy. Using different patients for the first and second endoscopy was important to prevent any improvement on the second endoscopy being attributable to familiarity with the patient’s anatomy.In our study, we compared the second endoscopy following randomization with the baseline endoscopy. We did not analyze the subsequent endoscopies beyond this. It is possible, and would be interesting to examine, if the rate of skill acquisition may have been faster in the simulation group compared to the control group if subsequent endoscopies had been analyzed.Based on our observations, the most challenging aspect of the endoscopy was navigating the nasal cavity. This anatomy was not reflected in the design of the simulator. It may be that, in this case, a simulator that better reflected nasal anatomy would be more beneficial. However, a randomized controlled study looking at rigid and flexible nasal endoscopy utilizing an accurate nasal model also did not show any quantitative or qualitative difference among simulator and control groups when they subsequently performed endoscopies on standardized patients . This isThe aim of this study was to assess the effect of a low-fidelity simulator on endoscopy skills and patient comfort. Overall, we demonstrated that repeat endoscopy in the clinical setting had a significant impact on time to adequate visualization of the glottis and the number of mucosal contacts, but that no additional benefit was conferred with the use of a low-fidelity simulator. In the modern medical education environment, there is considerable focus on the use of simulation as a means of acquiring technical skills. While this can most certainly be a valuable education tool, not all skills require simulation training. We believe that this study demonstrates this fact and establishes that flexible trans-nasal endoscopy is such a skill.The authors declare that they have no competing interests.MD was responsible for data collection and analysis, as well as drafting the manuscript. WY was responsible for the design of the study, performing the statistical analysis, and editing the manuscript. JC was responsible for data analysis and drafting the manuscript. MA was responsible for the design of the study, acquisition of data, and editing the manuscript. BS was also responsible for the design of the study, acquisition of data, and editing the manuscript. JB conceived the study, participated in its design and coordination and edited the manuscript. All authors read and approved the final manuscript.


Adults recognize that if event A predicts event B, intervening on A might generate B. Research suggests that young children have difficulty making this inference unless the events are initiated by goal-directed actions  Causal representations are central to human cognition. They support prediction, explanation, and intervention and underlie folk theories across domains Consistent with this hypothesis, research has shown that infants' causal reasoning is closely related to their reasoning about intentional agents and their goal-directed actions. Leslie, for instance, showed that 6-month-old infants are sensitive to contact causality between entities in a causal event when the event is agent-initiated, but are insensitive to contact causality when the event is object-initiated The influence of representations of agency on causal reasoning continues beyond early infancy These findings raise questions concerning the nature of the relationship between representations of agency and causality early in development. One possibility is that although toddlers can learn predictive relationships among events Alternatively, toddlers might have an adult-like concept of causation but fail to understand some physical mechanisms of as means of causal transmission. Research suggests, for instance, that infants expect causation on contact This suggests the possibility that toddlers may be more likely to use predictive relations as a basis for establishing a causal representation of an event in domains involving less restrictive transmission relations. In particular, toddlers might more successfully integrate prediction and intervention for psychological causal events, which can occur either through direct contact or  at a distance. By circumventing constraints on toddlers' expectations about mechanisms of causal transmission, toddlers might have no difficulty with the basic task of expecting a predictive relation to be representative of a causal relation.Consistent with this possibility, previous research suggests that young infants represent many aspects of psychological causal relations. Schlottmann and colleagues, for example, have shown that infants as young as 6 months of age seem to perceive causality in simple social outcomes such as one object chasing another object and causing it to flee In the current study, we replicate Bonawitz and colleagues' study All toddlers viewed a three-part predictive event in which (1) a block spontaneously began to slide across a stage from rest towards a base block and (2) contacted the base block, after which (3) an effect occurred see . In the The first set of analyses assessed whether toddlers had learned the predictive relationship between the block's motion and the outcome. We assessed toddlers' learning of the predictive event by coding whether toddlers looked up towards the toy in the first 3 seconds following an Off trial during the familiarization phase. During the Off trial, the block contacted the base, but the effect did not occur. If in the experimenter's online judgment (see coding below) toddlers did not look up towards the toy within 3 seconds of the block contacting the base, we presented an additional On trial in which the effect occurred, followed by an additional Off trial. Following each toddler's final Off trial, they viewed one final On trial, to show that the effect had not spontaneously stopped occurring.p = n.s.). Of those toddlers that learned the predictive relationship, no toddlers in the Psychological outcome condition and 3 toddlers in the Physical outcome condition needed an additional Off trial . Although there were no significant differences between conditions, children were only included in further analyses if they learned the predictive relationship. This ensured that any differences that emerged during subsequent phases were not due to differential initial encoding of the predictive relationship.All results used for analyses were coded from videotape by two coders blind to conditions. (See Methods.) In all cases where the experimenter introduced a second Off trial the blind coders' judgment agreed with the experimenter's online judgment. p = .08). If toddlers failed to act spontaneously, the experimenter prompted them by moving the block part way towards the base. All of the remaining 4 toddlers in the Psychological outcome condition and 8 of the 11 remaining toddlers in the Physical outcome condition completed the action. Three toddlers in the Physical outcome condition failed to perform the action either spontaneously or following a prompt and were thus removed from further analysis.The next set of analyses explored toddlers' ability to perform the target action following the predictive events. Toddlers acted spontaneously on the block in both conditions, although there was a trend for more toddlers to act on the block spontaneously in the Psychological outcome condition. Seven of the 18 toddlers (38.89%) in the Physical outcome condition spontaneously placed the block in contact with the base during the test phase. By contrast, 11 of the 15 toddlers (73.33%) in the Psychological outcome condition spontaneously performed the action  and their tendency to act on the block might reflect only different levels of overall motivation and engagement.Although toddlers were marginally more likely to act on the block in the Psychological condition than in the Physical condition, the action by itself is hard to interpret. Toddlers may have been slightly more likely to act spontaneously in the Psychological condition than the Physical condition because they were more likely to treat the events as causal in the Psychological than Physical condition. Alternatively, toddlers might have understood the causal relationship equivalently in both conditions (either failing to infer that p<.0005).If toddlers truly represent the psychological, but not the physical, outcome events causally, then toddlers should expect their action on the block to bring about the effect only in the Psychological condition. As a result, our primary measure of interest was whether toddlers appeared to expect their own action to generate the outcome. For the 15 toddlers in each condition who both learned the predictive relationship and acted (either spontaneously or after prompting) on the block, we coded whether they looked up towards to the toy within a 3 second window after they placed the block in contact with the base; the toy remained in the OFF position after the toddlers' interventions in both conditions. In the Physical outcome condition, only 5 of the 15 toddlers (33.33%) looked to the toy after intervening. By contrast, in the Psychological outcome condition, all of the toddlers did so  influenced toddlers' tendency to generalize the outcome from the spontaneously occurring event to the consequence of their own intervention. This is consistent with previous research suggesting that toddlers have difficulty representing non-agentive physical predictive relationships as potential causal relationships Note that we always verified that the toddlers had learned the predictive relationship by including a familiarization trial in which the effect did not occur. Thus all the events presented occurred probabilistically. One possibility is that toddlers are more willing to accept probabilistic relationships for psychological events than physical events. If so, toddlers might generalize the outcome in the Psychological but not Physical condition because they are more willing to accept probabilistic relationships among agents than among objects. We think this interpretation is possible but unlikely to account for the current results. Critically, Bonawitz and colleagues' study However, our results do provide some suggestive evidence that the Psychological condition might have been more motivating and engaging than the Physical condition. Although the children were equally likely to learn the predictive relationship in both conditions, there was a non-significant trend for toddlers to act on the block more often in the Psychological outcome condition than in the Physical outcome condition. To the degree that this trend is meaningful, it might (as discussed) be due to different levels of overall engagement with the task. If so, such differential engagement  might account for the condition differences.physical predictive event. The children were instructed to greet the puppet and the puppet laughed at the start of ever familiarization trial; the puppet's laughing was identical to that in the Psychological condition. If the puppet's laughing simply enhances toddlers' arousal or increases their motivation to participate in the task, then toddlers' performance in this condition should improve. However, if, as we hypothesize, toddlers have difficulty generalizing from prediction to intervention for spontaneously occurring physical events, then they should continue to fail to generalize the outcome associated with the predictive event to their own interventions in this condition.To investigate the possibility that children's interest in the puppet contributed to children's different performance in the Psychological and Physical conditions, we ran an Audience condition, intended to increase the children's engagement with the Physical outcome condition. In the Audience condition we introduced a laughing puppet before each p = n.s.). Of those toddlers who learned the predictive relationship, two toddlers needed an additional Off trial; there was no significant difference between this condition and either the Physical outcome or Psychological outcome conditions in the number toddlers needing an additional Off trial . Nonetheless, to ensure that subsequent results were not due to toddlers' failure to learn the initial predictive relationship, toddlers who failed to learn the predictive relationship (n = 5) were removed from subsequent analyses.Again, children were counted as learning a predictive relationship if the independent coders judged that the child had look to the (still and silent) puppet within 3 seconds of the block contacting the base. First, we found that the majority of toddlers learned the predictive relationship between the block and the effect (17 of 22 toddlers (77.27%)), no different from either the Psychological outcome or Physical outcome conditions . Ten of the 17 toddlers (58.82%) in the Audience condition spontaneously intervened by placing the block in contact with the base; five additional toddlers completed the intervention following the experimenter's prompted action. The remaining toddlers who never performed the intervention (n = 2) were removed from subsequent analysis.Toddlers' tendency to act spontaneously on the block in the Audience condition did not differ from either the Psychological outcome or Physical outcome conditions . Toddlers' performance in this condition was not significantly different from their performance in the Physical outcome condition .As noted however, merely acting on the block does not mean that children expected their action to cause the outcome. Thus our final measure of interest again was whether, having learned the predictive relationship and demonstrating their ability to perform the target intervention, toddlers looked towards the outcome following their intervention. Toddlers were significantly less likely to look towards the outcome in the Audience condition than in the Psychological outcome condition (6 of 15 toddlers (40.00%); Fisher's exact test, These results suggest that although toddlers might have found the psychological task (involving a laughing puppet) more interesting or arousing than the physical task (involving the airplane), differential attention, motivation, and arousal are unlikely to account for toddlers' different performance in the Psychological outcome and Physical outcome conditions. The presence of a laughing puppet in the Audience condition did not significantly increase toddlers' tendency to generalize the outcome of their interventions from the outcomes learned predictively.In a final study, we investigated whether constraints specific to the domain of psychological causality affect toddlers' tendency to treat predictive relations as relations that might support effective interventions. Although psychological events are not subject to a contact constraint, they are subject to the epistemic state of the participants. If the puppet cannot see or hear the block's motion and resulting contact, then he shouldn't laugh in response to the event. To test whether toddlers would (appropriately) fail to represent psychologically implausible events causally, we ran an Epistemic Status condition. In this condition, we blocked the puppet's visual access to the block's motion by blindfolding the puppet and placing a wall between the puppet and the blocks. We pointed out that the puppet could not see or hear the events behind the wall. If toddlers' differential performance in the Psychological outcome and Physical outcome conditions is due to the toddlers' understanding of psychological causality, then they should not represent the predictive relation as a causal event when the events are psychologically unlikely.p = n.s.). Of these 17 toddlers, all demonstrated learning of the predictive relationship on the first Off trial . One additional toddler was removed from subsequent analyses for never having learned the predictive relationship on either Off trial.We found that the majority of toddlers learned the predictive relationship between the block and the effect (17 of 18 toddlers (94.44%)) and that this performance was no different from either the Psychological outcome or Physical outcome conditions . Nine of the 17 toddlers (52.94%) in the Epistemic Status condition spontaneously intervened by placing the block in contact with the base, and six additional toddlers completed the intervention following the experimenter's prompted action. The one remaining toddler, who never performed the intervention, was removed from subsequent analysis.Toddlers were as likely to intervene in the Epistemic Status condition as they were in the Psychological outcome and Physical outcome conditions ; toddlers' performance was not significantly different from the Physical outcome condition .As in the previous studies, merely acting on the block is not evidence that the children expected their action to cause the outcome. Of the toddlers who learned the predictive relationship and demonstrated their ability to perform the target action, toddlers were significantly less likely to look towards the outcome following their intervention in the Epistemic Status condition than in the Psychological outcome condition (5 of 15 toddlers (33.33%); Fisher's exact test, These results suggest that toddlers' causal representations of the psychological event were subject to constraints specific to the domain of psychological causality: toddlers did not represent the predictive psychological outcome as a causal event when the puppet lacked informational access to the block's movement . Additionally, these results replicate the Audience condition in suggesting that the mere salient, arousing presence of a laughing puppet cannot account for children's different performance in the Physical and Psychological conditions. Therefore, these results suggest that toddlers can reason about causal events involving psychological outcomes even when the initiating events occur spontaneously.Previous work How then do children reason about physical causal events? One speculative possibility is that infants initially recognize agent-initiated events – events involving their own actions or those other goal-directed agents – as causal events Future work might investigate this developmental story about how children understand mechanisms of physical transmission. Even if an account like this is correct however, the question remains of why children readily accept the entire range of these transmission events as causal, as long as goal-directed agents initiate the events. Additional research might look at whether children can bootstrap from their understanding of the goal-directed causal events to their understanding of means of transmission in the absence of dispositional agency.Another outstanding question from this research concerns the extent of toddlers' understanding of psychological outcomes. The current study investigated only one type of psychological outcome . However, the class of events that encompass the domain of psychological outcomes is vast. Events can make us laugh, cry, perform an action, or inhibit a response. Moreover, philosophers have long suggested that we may perceive psychological outcomes as “uncaused” insofar as we believe they are generated by the agent's free will; thus adults often draw a distinction between reasons for actions and causes for actions The current results suggest that, although children have an adult-like abstract understanding of the concept of causation that binds prediction and action by two years of age, their ability to recognize particular events as instances of causation may depend on domain-specific constraints. Sensitivity to these constraints may develop in a piecemeal manner, in which viable means of causal transmission are learned event-by-event. This study thus adds to a growing body of research suggesting that in tandem with or even before they have an accurate understanding of the specific causal mechanisms, young children have a rich, abstract understanding of causality The Massachusetts Institute of Technology Institutional Review Board approved the procedures for all research described in this paper. We obtained written consent from the participants' parents.p = n.s.).Seventy-eight toddlers  were recruited at two children's museums. Toddlers were assigned to one of four conditions: a Psychological outcome condition (n = 16), a Physical outcome condition (n = 22), an Audience condition (n = 22), or an Epistemic Status condition (n = 18). Note that participants were recruited to match in the final sample included for analysis (n = 15/condition); different n's in condition assignment reflect non-significant differences in the number of participants failing to meet the inclusion criteria described below. An additional 10 toddlers were recruited but not included in the final sample due to: inability to complete the session , parental interference , or experimenter error . There were no age differences between the conditions (All events occurred on a white stage (30 in.×12 in.) that blocked a confederate from view See . A blue The experiment had two phases: a familiarization phase and a test phase. There were two types of familiarization trials: On trials and Off trials see . Each toAt the start of the test phase, the experimenter handed the block to the toddler and asked the child to make the effect occur. If the child did not spontaneously place the block in contact with the base, the experimenter prompted the toddler to place the block in contact with the base. The prompt involved the experimenter pushing the block across the stage towards, but stopping just short of, the base block. The experimenter then handed the block back to the toddler and encouraged them to make the effect occur.The Physical outcome condition was identical in structure  to the Psychological outcome condition except that instead of a puppet laughing after the block's movement, a toy airplane spun for three seconds at the top left corner of the stage see .The Audience condition mirrored the Physical outcome condition. Additionally, the puppet used in the Psychological outcome condition was placed on the experimenter's lap and acted as an audience member with the toddler to the familiarization events. At the start of each familiarization trial , the experimenter drew the child's attention to the puppet, who was seated next to the stage, on the experimenter's lap. The experimenter asked the child to say hello to the puppet, and the puppet then laughed and wiggled for three seconds . After the puppet laughed, the experimenter told the child that the puppet was going to watch the show with them and then turned the puppet to face the stage. The puppet laughed and turned towards the stage in an identical manner prior to the start of every familiarization trial. We had the puppet laugh before the trials rather than after so that the puppet's laughter could not be construed as an effect; we had the puppet laugh on the experimenter's lap rather than on the stage so the puppet could not be construed as a dispositional agent initiating the events. The trials then proceeded as On trials or Off trials, which mirrored the Physical outcome condition. During the test phase, the puppet did not laugh. The experimenter gave the block to the child and asked them to make the effect occur.The Epistemic Status condition was identical to the Psychological outcome condition, except as follows. Prior to the start of the familiarization phase, the experimenter placed a blindfold over the puppet's eyes. The experimenter then told the toddler that the puppet could not see or hear what was happening during the study (“I'm going to cover his eyes and his ears so that he can't see or hear what is happening during the show.”). Additionally, a wall was placed between the puppet and the blocks (prior to the child entering the room), such that the puppet had no visual access to the predicting event. The remainder of the familiarization phase was identical to that of the Psychological outcome condition. The block spontaneously moved towards and contacted the base, after which the puppet laughed for 3 s.Following data collection, two raters, blind to experimental condition, independently scored toddlers' behaviors. 75% of responses were double-coded; inter-rater agreement was high .


There are four changes that need to be made to this article. These changes affect the Introduction, References, Discussion and Figure Legends, respectively.1. In the last sentence of the second paragraph of the Introduction, "a non-endocytic mechanism" should instead read "an exosomal mechanism".2. Reference 13 is incorrect and should instead read:"Dreux M, Garaigorta U, Boyd B, Decembre E, Chung J, et al. (2012) Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity. Cell Host Microbe 12: 558-570."3. In the second sentence of the fourth paragraph of the Discussion, reference 13 should instead refer to reference 12.4. There is an error in the Figure 4 legend. The descriptions of panels A and B should be switched, so that the text instead reads as follows:"A) RIG-I was knocked down in pDC-GEN2.2 cells using siRNA and then stimulated with the pU/UC or X-region RNA. When compared to the Scrambled siRNA condition, the RIG-I knock-down condition (mean knock-down 13% by PCR) produced significantly less IFN mRNA. Combined data from 3 independent experiments. Bars represent the mean and error bars are +/- SEM. p values are the Mann-Whitney result for the scrambled siRNA condition compared to the RIG-I siRNA condition. * p<0.05 ** p<0.01 *** p<0.001 # p=0.0001. B) Gene expression graphs show that, after normalization to the reference gene GAPDH and Mock transfected condition, when compared to untreated HCV RNA, the phosphatases-treated RNA induced less interferon gene production. Data are representative graph from 3 independent experiments. Bars represent the mean and error bars are +/- SD."


To evaluate the efficacy and safety of Ex-PRESS implantation (Ex-PRESS) compared to trabeculectomy in the treatment of patients with open-angle glaucoma (OAG).A comprehensive literature search using the Cochrane Methodology Register to identify randomized controlled clinical trials (RCCTs) comparing Ex-PRESS to trabeculectomy in patients with OAG. Efficacy estimates were measured by weighted mean difference (WMD) for the percentage intraocular pressure reduction (IOPR%) from baseline to end-point, and odds ratios (OR) for the complete success rate and postoperative interventions. Safety estimates were measured by OR for postoperative complications. Statistical analysis was performed using the RevMan 5.1 software.A total of four RCCTs were selected for this meta-analysis, including 215 eyes of 200 patients . There was no significant difference between Ex-PRESS and trabeculectomy in the IOPR% , −6.17–12.47; P = 0.51). The pooled OR comparing Ex-PRESS to trabeculectomy for the complete success rate at one year after surgery were in favor of Ex-PRESS . The Ex-PRESS procedure was found to be associated with lower number of postoperative interventions  and with a significantly lower frequency of hyphema than trabeculectomy , whereas other complications did not differ statistically.In OAG, Ex-PRESS and trabeculectomy provided similar IOP control, but Ex-PRESS was more likely to achieve complete success, with fewer postoperative interventions. Complication rates were similar for the two types of surgery, except for a lower frequency of hyphema in the Ex-PRESS group. Glaucoma is a leading cause of blindness. It has been estimated that over 5.9 million people worldwide will be bilaterally blind with open-angle glaucoma (OAG) by 2020, which is more prevalent in Europe and China compared to other parts of the world Trabeculectomy has been the standard IOP-lowering procedure for OAG. Its success rate and complications are well established The efficacy and safety of the Ex-PRESS compared to trabeculectomy has been studied before, with the majority of reports suggesting similar efficacy and complication rates for the two types of surgery Ex-PRESS, glaucoma and trabeculectomy. A manual search was performed by checking the reference lists of original reports and review articles to identify studies not yet included in the computerized databases. The final search was carried out on August 2013, without restrictions regarding publication year or language.We conducted searches of PUBMED, EMBASE, ISI Web of Science, and the Cochrane Library, using the terms Articles were considered eligible for inclusion in the meta-analysis if the studies met the following inclusion criteria: (i) study design: RCCT; (ii) population: OAG patients ; (iii) intervention: Ex-PRESS versus trabeculectomy; (iv) outcome variables: at least one of the outcomes of interest discussed below was included. Abstracts from conferences and full texts without raw data available for retrieval, duplicate publications, letters, and reviews were excluded. For sequential reports on the same cohort of patients, only the most recent report was included, and data that could not be obtained from this last publication were obtained from the previous reports.IOPR) were calculated as follows: IOPR = IOPbaseline−IOPend-point, SDIOPR = (SDbaseline2+SDend-point2−SDbaseline×SDend-point)1/2, then the IOPR% and the SD of the IOPR% (SDIOPR%) were estimated by IOPR% = IOPR/IOPbaseline, SDIOPR% = SDIOPR/IOPbaselineFor efficacy, the primary outcome measure was the percentage of the IOP reduction (IOPR%). When authors reported the mean value and the standard deviation (SD) of the IOPR%, we used these values directly. For studies that only reported absolute values for the IOP at baseline and at end-point, the IOP reduction (IOPR) and the SD of the IOPR (SDThe data were extracted independently by two reviewers (G.H.C. and W.S.L.). Disagreement was resolved by discussion. The information extracted from each study included the authors of each study, the year of publication, information on study design, location of the trial, duration of the study, number of subjects, IOP measurements, and success rate. The proportion of patients with postoperative interventions and complications was also recorded.The qualities of RCCTs were assessed by two independent observers (F.Z.J. and S.H.M.) using a system reported by Downs and Blacks 2 statistic was calculated to assess heterogeneity between studies . If there was heterogeneity between studies, a random-effects model was applied to the data. Alternatively, a fixed effects model was used for pooling the data. Begg's rank correlation test and Egger's linear regression test were employed to quantitatively assess publication bias .The quantitative data were entered into Cochrane Review Manager . For continuous variables , the weighted mean difference (WMD) was measured, while the odds ratios (OR) were measured for dichotomous variables . Both outcomes were reported with a 95% confidence interval (CI). P<0.05 was considered statistically significant on the test for overall effect. The IA total of 74 articles were initially identified. Of these, 69 were rejected according to the exclusion criteria listed above. The five remaining articles with full texts that met the inclusion criteria were assessed 2 = 0%; P = 0.78)   , with no heterogeneity identified (I = 0.78) . Complet = 0.34) . Of the  = 0.42) . Begg's Postoperative complications comparing Ex-PRESS to trabeculectomy are shown in Glaucoma treatments are directed at reducing IOP. Traditionally, trabeculectomy is considered the standard IOP-lowering procedure for OAG. Ideally, any procedure seeking to replace trabeculectomy should offer at least the same success rate, without increasing the level of complications. In recent years, Ex-PRESS has been introduced as an alternative to trabeculectomy. Numerous studies have reported on the biocompatibility, safety, and efficacy of Ex-PRESS during its evolution over the last decade In this meta-analysis, we reviewed four RCCTs, including 215 eyes of 200 patients . In assessing the IOP, Ex-PRESS was associated with IOP-lowering efficacy comparable to that of trabeculectomy, demonstrating slightly bigger IOPR% reduction from baseline, but this difference that was not statistically significant. Our analysis of the IOPR% was based on data pooled from trials of different durations, ranging from six months to five years. The longest follow up was five years by de Jong et al This meta-analysis may have some limitations. First, we cannot fully exclude publication bias. Although the Begg's and Egger's tests demonstrated no evidence of publication bias, the results should be interpreted with caution. Second, the studies were carried out with small or very small sample sizes and it is impossible to mask the surgical technique as trabeculectomy is easily differentiated from Ex-PRESS during postoperative follow-ups. These factors can affect the interpretation of the results. Third, a potential source of heterogeneity is trials duration and lack of data reported in all phases of follow-up.Our findings indicate that Ex-PRESS and trabeculectomy provide similar IOP control, but Ex-PRESS is more likely to achieve complete success, with fewer postoperative interventions. Complication rates were similar for the two types of surgery, except for a lower frequency of hyphema noted in the Ex-PRESS group. RCCTs with longer duration and larger sample size are needed to provide more definitive information indicating whether Ex-PRESS is an effective and safe procedure for the treatment of OAG.Checklist S1PRISMA Checklist.(DOC)Click here for additional data file.


Osteosarcoma is an aggressive malignancy of the bone and an increase in serum alkaline phosphatase concentration has clinical prognostic value in both humans and canines. Increased serum alkaline phosphatase concentration at the time of diagnosis has been associated with poorer outcomes for osteosarcoma patients. The biology underlying this negative prognostic factor is poorly understood. Given that activation of the Wnt signaling pathway has been associated with alkaline phosphatase expression in osteoblasts, we hypothesized that the Wnt/β-catenin signaling pathway would be differentially activated in osteosarcoma tissue based on serum ALP status. Archived canine osteosarcoma samples and primary canine osteosarcoma cell lines were used to evaluate the status of Wnt/β-catenin signaling pathway activity through immunohistochemical staining, western immunoblot analyses, quantitative reverse-transcription polymerase chain reaction, and a Wnt-responsive promoter activity assay. We found no significant difference in β-catenin expression or activation between OSA populations differing in serum ALP concentration. Pathway activity was mildly increased in the primary OSA cell line generated from a patient with increased serum ALP compared to the normal serum ALP OSA cell line. Further investigation into the mechanisms underlying differences in serum ALP concentration is necessary to improve our understanding of the biological implications of this negative prognostic indicator. Osteosarcoma (OSA) is the most common primary bone malignancy of humans The expression of ALP is frequently used to identify cells of the osteoblastic lineage and is a hallmark of osteoblastic activity. Studies have suggested that ALP is a transcriptional target of the Wnt/β-catenin signaling pathway, as activation of this pathway in osteoblasts has been associated with increased ALP expression The Wnt/β-catenin signaling pathway is crucial for normal bone development and its transient activation is required for mesenchymal stem cell (MSC) commitment to the osteoblastic lineage Few studies have investigated the extent of active Wnt/β-catenin signaling in canine OSA, and no studies address specifically the potential role β-catenin may have in the increased serum ALP phenotype. As a prognostic indicator, serum ALP is aligned with poor outcome, and understanding the mechanism behind this phenomenon will allow us to better understand the biological relevance of altered serum ALP concentrations in OSA pathogenesis. This insight can be used for future clinical and therapeutic interventions for both humans and animals.Our overarching aim within this study was to determine if Wnt/β-catenin signaling is differentially activated in OSA tissue from dogs with increased serum ALP concentration. We achieved this by: (a) determining if β-catenin is differentially expressed, with respect to both quantity and cellular localization, between canine OSA patient populations with increased and normal serum ALP concentrations; and (b) determining if cell lines derived from normal and increased serum ALP OSA tissue have differential β-catenin activity as determined by: (i) the nuclear localization of β-catenin, and (ii) TCF binding site reporter plasmid activity. We hypothesized that the increased serum ALP levels associated with some canine OSA populations would be due to increased Wnt/β-catenin pathway activity.Our first aim was to determine if β-catenin mRNA was differentially expressed in OSA tumor tissue from dogs with increased serum ALP concentration (n = 10) compared to OSA tumor tissue from dogs with normal serum ALP concentration (n = 9). The assignment of a patient to the increased serum ALP concentration group was based on the patient's serum ALP concentration being above the upper limit of normal as defined by the reporting institute see . The med2 = 0.02, p = 0.61; Axin2: R2 = 0.02, p = 0.63; Sox9: R2 = 0.01, p = 0.70; data not shown).Next, we aimed to determine if differences existed in the expression of downstream targets of the Wnt/β-catenin signaling pathway between OSA tissue from dogs with increased serum ALP concentration and those with normal serum ALP concentration. Again, qPCR was used to assess relative mRNA expression of Axin2 and Sox9, two downstream targets of Wnt/β-catenin activity. Axin2 gene expression is positively regulated by active Wnt/β-catenin signaling therefore an increase in Axin2 expression would be expected with active Wnt/β-catenin signaling. Conversely, Sox9 gene expression is negatively regulated by Wnt/β-catenin activity and a reduction in Sox9 gene expression would be expected with active Wnt/β-catenin signaling. Consistent with the lack of difference in relative expression of β-catenin mRNA between the two populations, no difference was found in the relative expression of Axin2 or Sox9 in tumor tissue associated with increased serum ALP concentration compared to normal serum ALP concentration . AdditioAs β-catenin is highly regulated post-translationally, protein expression of pooled tissue lysates from patients with normal (n = 5) and increased (n = 5) serum ALP concentration was assessed to confirm the mRNA expression data. Samples used for tissue lysates were a randomly chosen sub-population of those used for qPCR analysis. β-catenin was identified in the lysates from both patient populations. There was no difference in the β-catenin expression of the normal serum ALP lysate compared to the increased serum ALP lysate .To assess the nuclear translocation of β-catenin between the two patient populations, western blot analysis was performed on the nuclear fraction of cell lysate from a single primary cell line derived from either increased serum ALP concentration patient tissue  and normal serum ALP concentration patient tissue . The nuclear fraction lysate from the iALPOS cell line displayed a slight decrease in β-catenin expression compared to the nALPOS cell line .Both the downstream gene target qPCR and the western blot of the nuclear fraction indicate that the Wnt/β-catenin pathway is not differentially activated in patients with increased serum ALP; to further confirm this data, a TCF-responsive luciferase reporter assay was performed. β-catenin acts as a transcriptional co-factor and binds to members of the TCF/LEF transcription factor family (reviewed in 25) resulting in expression of genes downstream of the TCF sites. In the TOPflash plasmid, luciferase is downstream of several TCF sites, thus increased Wnt/β-catenin activity would be indicated by higher luciferase expression, or a higher TOP/FOP ratio. The iALPOS cell line was found to have a higher TOP/FOP ratio  compared to the nALPOS cell line  .Given the apparent lack of differential expression or pathway activity in the small number of frozen samples and cell lines, a larger group of patients was used to confirm these findings. Immunohistochemistry was performed to detect β-catenin expression and localization in tumor tissue from patients with a known serum ALP status. As discussed above, serum ALP concentrations were determined at multiple institutions resulting in differing reference ranges. To account for differences in reference ranges, patients were assigned to the increased serum ALP concentration group based on being above the upper limit of normal for the reporting institute's reference range see . The medOsteosarcoma is a notably aggressive disease, and as such, prognostic indicators are extremely useful in the clinical setting. Serum alkaline phosphatase (ALP) concentration has been correlated with overall survival times and disease-free intervals, and an increased serum ALP concentration is a negative prognostic indicator for both human and canine OSA patients Cellular ALP is frequently used as a marker of osteoblasts and its expression is increased during latter stages of osteoblastic differentiation The major objective of this study was to determine if Wnt/β-catenin signaling is enhanced in canine OSA tissue from patients with increased serum ALP concentration compared to patients with normal serum ALP concentration. To address this, β-catenin expression was first examined at the mRNA level in frozen canine OSA samples from patients with known serum ALP concentration. No difference was found in the two patient populations at the mRNA level. This indicates that transcription of the β-catenin gene is not different between the two populations. However, β-catenin expression is highly regulated at the post-translational level and this may allow for differences to exist between the two populations at the protein level Next, western immunoblots were performed on pooled tissue lysates to assess if β-catenin is differentially regulated at the post-translational level between the two populations, which would result in altered total protein expression. Total β-catenin protein expression was similar between pooled canine OSA tissue from patients with normal or increased serum ALP. This indicates that there are no differences in post-translational regulation of β-catenin between the two tumor populations that would allow for the stabilization of the protein and result in increased β-catenin expression. Constitutive expression of β-catenin is a hallmark of cancers noted for activated Wnt/β-catenin signaling, most notably colorectal carcinomas and hepatocellular carcinomas  western immunoblot of the nuclear fraction of cell lysate from canine cell lines derived from either an increased serum ALP patient (iALPOS) or a normal serum ALP patient (nALPOS) and (ii) IHC of biopsies taken from canine patients of known serum ALP concentration. There was a slight increase in β-catenin in the nuclear fraction of the nALPOS cell line, though no discernable differences were observed in nuclear β-catenin expression as determined by IHC for these patient populations.The lack of difference in protein localization between the populations strongly suggests that Wnt/β-catenin pathway is not differentially activated in canine patients with increased serum ALP compared to normal serum ALP patients. However, neither western blots nor IHC are particularly quantitative, and we wanted to specifically determine the difference, if any, in pathway activity between these two patient populations. This was done using the TOPflash reporter assay, which is dependent upon the binding of transcription factors to TCF-binding sites (TOPflash) to drive luciferase expression. In this study there was a roughly 1.5-fold increase in luciferase activity in the increased serum ALP canine OSA cell line (iALPOS) compared to the normal serum ALP canine OSA cell line (nALPOS). While this value was statistically significant, the clinical significance of this difference is questionable, as there was no difference in downstream target mRNA production (neither Axin2 nor Sox9). Cell lines with mutations in the Wnt signaling pathway resulting in β-catenin stabilization demonstrate significantly increased corrected luciferase TOP/FOPflash ratios: SW480, a human colon adenocarcinoma cell line with a Wnt pathway activating mutation, has a TOP/FOPflash ratio of 10 Our study indicates that increases in serum ALP associated with some canine OSA patients is not due to differential Wnt/β-catenin signaling activity between the two populations. However, these finding are not meant to abandon the association of Wnt/β-catenin signaling with ALP expression. Rather it is possible that other intermediary signaling pathways, acting in concert with the Wnt/β-catenin pathway, are differentially activated which may amplify any Wnt/β-catenin activity. One such pathway that could be involved is the BMP/Smad signaling pathway. In this model, β-catenin prompts the expression of BMP4 and 6, which then stimulate the activation of Smad1 and Smad5 in an autocrine/paracrine fashion; this may result in differential ALP expression and/or activity It is possible the differences in ALP concentration may not be due to differences at the transcriptional level. Rather, the differing ALP concentrations may be due to differences in the post-transcriptional or post-translational regulation of ALP, as it is well established that ALP expression is regulated at many levels Two other possibilities as to why no differences in β-catenin expression or activity were identified between the two canine OSA populations is perhaps the increased serum ALP concentration was not due to the bone isoform of ALP. A limitation of the current study is the inability to verify the increased serum ALP concentration was due the bone isoform of ALP. A number of analyses were performed on archived tissue, both formalin-fixed and frozen, and as such we were unable do perform iso-enzyme analysis on serum collected concurrently. However, in the selection of samples attempts were made to reduce the likelihood that increased serum ALP was due to non-bone sources by selecting only cases known to be free of corticosteroid treatment pre-operatively and concurrent liver disease. Finally, some caution must be exercised in regards to the ex vivo cell line results as only two primary canine OSA cell lines were used. However, these cell lines were used due to knowledge of the serum ALP status of the patients from which they were generated. This information, to our knowledge, is unavailable for most canine and human OSA cell lines.To the authors' knowledge this is the first study investigating possible cellular mechanisms responsible for the differential serum ALP protein expression in canine OSA patient populations, though no significant difference in β-catenin expression or activity was found to exist between canine OSA samples associated with normal and increased serum ALP concentration. Further investigation into the mechanisms underlying differences in serum ALP concentration are important to improving our understanding of the biological implications of this negative prognostic indicator. The identification of altered signaling pathways unique to this negative prognostic factor may assist in developing therapeutic interventions based on this knowledge and potentially improve outcomes for this population of patients.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the UW-Madison's School of Veterinary Medicine Animal Care and Use Committee (Protocol: V01391-0-09-08). Owner consent was obtained prior to the collection of tumor tissue and generation of cell lines.For experiments involving immunohistochemistry, archived tissue blocks of canine OSA associated with normal (n = 32) and increased (n = 24) serum ALP from the Pathology Service at the University of Wisconsin-Madison Veterinary Medical Teaching Hospital (UWVMTH) were identified by medical records search. Patient requirements for inclusion into this portion of the study included no treatment with corticosteroids for a period of two weeks preceding the identification of increased serum ALP concentration and liver enzyme values within normal limits with the exception of ALP. Canine OSA tissue was collected at the time of diagnostic biopsy, surgical amputation or necropsy. One investigator (AM) from the histopathology service reviewed all slides to confirm the diagnosis of OSA.For experiments involving RNA analysis, tumor tissue was either collected from dogs at the UWVMTH or obtained from a tumor bank at Colorado State University's College of Veterinary Medicine. All tumor tissue collected at the time of a diagnostic biopsy or amputation for treatment of OSA was flash-frozen in liquid nitrogen and stored at −80°C. All tumor tissue is from dogs with known serum alkaline phosphatase concentration, drug history, and histological confirmation of OSA. Frozen normal canine bone was provided by the Comparative Orthopedics Research Laboratory at the University of Wisconsin-Madison School of Veterinary Medicine.Cell lines were generated from the tumor tissue of dogs undergoing amputation for treatment of OSA at the UWVMTH. Patient requirements for inclusion into this study included no treatment with corticosteroids for a period of two weeks preceding the identification of increased serum alkaline phosphatase concentration and tissue collection, histological confirmation of OSA, and liver enzyme values within normal limits with the exception of alkaline phosphatase.The determination of serum alkaline phosphatase concentration for patient samples utilized in this study occurred at multiple reference laboratories and at different times. Therefore, the normal reference ranges for serum alkaline phosphatase concentrations differed based on the reporting institute. For this reason each patient was classified as having normal or increased serum alkaline phosphatase according to the normal reference value range for that institute at the time of sampling. The reference ranges provided by institutions were as follows: 13–289, 20–157, and 20–142 U/L.2. All cell lines used in this experiment were beyond the 15th passage.Primary cell lines were generated from clinical tissue samples. Tumor tissue was surgically excised, placed on ice, and stored in phosphate-buffered saline (PBS) with PenStrepFungizone  to prevent microbial contamination. Mechanical and enzymatic digestion using collagenase I (200 units/ml) and DNase I (100 units/ml) was used in association with scalpel and scissors mincing, followed by filtration through a 40 mesh sieve until a single cell suspension was created. The resulting cell suspension was centrifuged for 7 min at 1400rpm. The pellet is then washed with sterile saline and re-centrifuged with the same conditions. To the pellet, 3 mL of complete modified eagle media (CMEM) is added, and the mixture is incubated in a flask with an additional 9 mL of CMEM. All cells were maintained in CMEM: MEM medium supplemented with 10% heat-inactivated cosmic calf serum, sodium pyruvate, l-glutamine, MEM vitamins, non-essential amino acids, and 1% Pen/Strep at 37°C in a humidified incubator with 5% COTotal RNA was isolated from clinical samples using Trizol , and purified by PureLink RNA Mini Kit  according to the manufacturer's instructions. cDNA was synthesized from 250 ng of total RNA using the High Capacity cDNA Reverse Transcription Kit  according to the manufacturers protocol. qPCR was performed using TaqMan Gene Expression Master Mix with TaqMan Gene Expression Assays  according to the manufacters protocol on a Bio-Rad iCycler machine with a Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. Assays include: canine β-catenin (Cf02667771_m1), canine Axin2 (Cf02631333_m1), and human SOX9 (Cf02631333_m1) . Ct values were normalized to 18S expression (4352930E). Relative difference in mRNA expression of clinical OSA samples was compared to normal canine bone obtained from the Comparative Orthopaedic Research Laboratory, School of Veterinary Medicine, University of Wisconsin – Madison, using the ΔΔCt method Total cell lysates of ten clinical canine OSA samples  were obtained by combining approximately 250–500 mg of tissue and 1 mL of Mammalian Protein Extraction Reagent (M-PER) in a 15 mL tissue grinder. Included samples were randomly chosen from the same patient population used for QPCR analysis. Tissue was ground until lysate appeared homogenous (5–10 min), then centrifuged for 7.5 min at 13,000rpm at 4°C. Lysates were stored at −20°C. Pooled increased serum ALP or normal serum ALP lysates were formed by combining 200 µg of protein lysate from each single sample.Nuclear fractions of two primary cell line lysates  were isolated using the Subcellular Protein Fractionation Kit . Manufacturers protocol was used to isolate membranous and cytoplasmic fractions; the remaining pellet (nuclear fraction) was lysed with M-PER. All fractions were stored at −20°C.Forty micrograms of protein lysate  were separated by electrophoresis on a 7.5% sodium dodecyl sulphate-polyacrylamide gel at 150 V for 45 min, transferred to nitrocellulose membranes at 100 V for 1 h, then blocked with tris-buffered saline/0.05% Tween20 (TBST) containing 5% non-fat dry milk and 1% bovine serum albumin for 1 h. The membranes were probed for overnight at 4°C with either (a) mouse anti-β-catenin antibody  diluted 1∶1000 in blocking solution, (b) goat anti-GAPDH  diluted 1∶200 in blocking solution, or (c) rabbit anti-laminin  diluted 1∶200 in blocking solution. Excess primary antibody was removed by washing three times for 5 min with TBST. Membranes were incubated with 50 ng/mL horseradish peroxidase-conjugated secondary antibody diluted in blocking solution for 1 h at room temperature, then washed three times for 5 min at TBST, and treated with SuperSignal West Pico Chemiluminescent Substrate . Blots were exposed to film, developed, and then imaged using a Gel Logic 100 Imaging System .Cell lines were plated in a six-well plate at a density to achieve 40–50% confluency within 24 h. Cells were then transiently transfected using Lipofectamine LTX and PLUS reagents , according to the manufacturers protocol, to introduce either 2.5 µg TOPflash, or 2.5 µg FOPflash reporter plasmid, along with 0.5 µg of TK-Renilla luminescent reporter plasmid . The TOPflash luciferase reporter plasmid contains TCF4 binding sites upstream of the luciferase gene, resulting in luciferase activity in the presence of active Wnt/β-catenin signaling, whereas the FOPflash reporter plasmid contains mutated TCF4 binding sites. The TK-Renilla plasmid serves as a control for transfection efficiency. Twenty-four hours after transfection, cells were harvested and luciferase and renilla luminescence were measured using the Dual-Luciferase Reporter Assay System  on a luminometer . The relative luciferase units for each transfection were adjusted by renilla activity in the same sample, and each corrected TOPflash luciferase value was normalized to the corresponding corrected FOPflash value. Three independent transfections were performed, with each sample assayed in triplicate.Immunohistochemical staining (IHC) was performed on slides made from archived osteosarcoma tissue to detect β-catenin protein. The process of β-catenin IHC staining and quantification of staining was performed as previously published 2 values and p values are reported.All statistical comparisons were performed using a Mann Whitney Test with commercially-available software ; p<0.05 was considered significant. All data are given as the median and range of values unless otherwise indicated. To determine the correlation between QPCR target gene expression and serum ALP concentration a linear regression analysis was performed using the same commercially-available software. R


IRS1 mRNA and protein resulted higher, relative to paired mucosa, in adenomas of familial adenomatous polyposis patients and in CRCs that overexpressed c-MYC, ß-catenin, InsRß, and IGF1R. Analysis of IRS1 immunostaining in 24 cases of primary CRC with paired colonic epithelium and hepatic metastasis showed that staining intensity was significantly higher in metastases relative to both primary CRC (P<0.01) and colonic epithelium (P<0.01). Primary and metastatic CRCs, compared to colonic epithelium, contained significantly higher numbers of IRS1-positive cells . Pathologic correlations in 163 primary CRCs revealed that diffuse IRS1 staining was associated with tumors combining differentiated phenotype and aggressive markers . In Caco 2 IRS1 and InsR were maximally expressed after polarization, while IGF1R was highest in pre-polarized cells. No nuclear IRS1 was detected, while, with polarization, phosphorylated IRS1 (pIRS1) shifted from the lateral to the apical plasma membrane and was expressed in surface cells only. In HT29, that carry mutations constitutively activating survival signaling, IRS1 and IGF1R decreased with polarization, while pIRS1 localized in nuclear spots throughout the course. Overall, these data provide evidence that IRS1 is modulated according to CRC differentiation, and support a role of IRS1 in CRC progression and liver metastatization.Colorectal cancer (CRC) is associated with lifestyle factors that affect insulin/IGF signaling, of which the insulin receptor substrate 1 (IRS1) is a key transducer. We investigated expression, localization and pathologic correlations of IRS1 in cancer-uninvolved colonic epithelium, primary CRCs with paired liver metastases and  Colorectal cancer (CRC) has been linked to lifestyle risk factors, most notably diets based on energy-dense foods and low physical activity APC) gene, encoding a central component of the cytosolic multi-protein complex that controls ß-catenin degradation APC-mutated cells show high cytoplasmic and nuclear ß-catenin; the latter, after binding to TCF/LEF transcription factors, forms complexes that, by switching on several cancer-related genes, impose a proliferative crypt progenitor phenotype (reviewed at http://www.stanford.edu/~rnusse/wntwindow.html) Intestinal epithelial differentiation is regulated by several pathways, particularly ß-catenin-dependent WNT signaling IRS1, encoding one of the two major insulin receptor substrates (IRS1 and IRS2), that integrate signaling from the InsR, IGF1R and other cytokine and growth factor receptors IRS1 by TCF/LEF-ß-catenin complexes. Furthermore, IRS1 is necessary for transformation in cells that ectopically express oncogenic ß-catenin and for maintainance of the neoplastic phenotype in APC-mutated cells IRS1 mutant acts as a tumor suppressor Apc(Min/+) mouse model, intestinal tumorigenesis is attenuated by irs1 knock-out Interestingly, recent evidences link the ß-catenin and the insulin/IGFs signaling pathways. In fact, ex vivo, in cancer-uninvolved human colonic epithelium, primary CRCs and paired liver metastases, and in vitro, in two CRC cell models capable of spontaneous in vitro polarization, Caco2 and HT29 In the present study we investigated the expression, localization and clinicopathologic correlations of IRS1 p53 and KRAS mutations A formalin-fixed, paraffin-embedded (FFPE) series of 24 primary CRCs with paired cancer-uninvolved colonic mucosa and synchronous liver metastasis was retrospectively identified at the Department of Surgical and Oncological Sciences, University of Palermo, Palermo, Italy. For this series standard whole sections were used for immunohistochemistry (IHC). An additional FFPE series, consisting only of primary CRCs, provided by the Digestive Disease Research Center (DDRC), Tehran University of Medical Sciences, Tehran, Iran, included 163 of the 205 CRC cases described in Bishehsari et al. and in Mahdavinia et al. APC mutation (respectively Glu1309fsX1312 and Ser843fsX860). Collection and analysis of samples and clinico-pathological data were approved by the G. D'Annunzio University Ethical Committee and by the Institutional Review Board of the DDRC, Shariati Hospital, University of Tehran (protocol dated 17/03/2004). All cases were anonymized.Samples of tumor and paired colonic mucosa, snap-frozen or rapidly fixed in RNAlater , were collected at the Department of Clinical Physiopathology, University of Florence, Florence, Italy, from 8 unselected CRC cases and 2 familial adenomatous polyposis (FAP) patients with molecularly-identified germline TMA and standard whole tissue sections were cut at 4 µm and stained with anti-IRS1 rabbit polyclonal  at 1∶300 dilution for 30 min, after antigen retrieval by microwave treatment at 750 W for 10 min in 10 mM sodium citrate buffer pH 6.0 . The anti-rabbit EnVision kit  was used for signal amplification. Serial TMA sections were also incubated with the following mouse monoclonal antibodies: anti-ß-catenin , anti-p53  and anti-Ki67 , for which antigen retrieval was performed by thermostatic bath at 96°C for 40 min in sodium citrate buffer (Dako), and anti-EGFR pharmDx , according to manufacturer's instructions. All immunoreactions were revealed by a streptavidin-biotin-enhanced peroxidase system . Positive and negative controls were included for each antibody and in each batch of staining.P values are two-sided; P<0.05 was considered as statistically significant.For each marker the percentages of positive cells were estimated in four fields at 400× magnification (≈1000 cells). IRS1, Ki67, p53, EGFR and ß-catenin were considered positive when >1% of the tumor cells were stained, ß-catenin was scored separately for immunostaining in the cytoplasm, nucleus and along the cell membrane. The independent samples t-test was used to evaluate differences in IRS1 expression (% of positive cells) according to pathological and mutational features. Expression of IRS1 was correlated to that of each of the other markers by Spearman's rho test. The SPSS (version 15.0) program  was used for all statistical analyses. All cited http://rsbweb.nih.gov/ij/). Images were acquired at standardized bright-field settings (400× magnification). Captured color jpeg images were converted to greyscale 8 bit images, then the Region Of Interest manager function was used to outline cytoplasmic areas, each comprising 5–15 cells. Unwanted nuclear or stromal elements were edited out. The areas analyzed in 4 matched sets of crypt epithelium, primary CRC and liver metastasis were 11 for bottom crypt epithelium, 10 for top crypt epithelium, 78 for total colonic epithelium, 84 for primary CRC, and 84 for metastatic CRC. Using default ImageJ settings, the units measured for each area were density value, total area in square pixels, average size and area fraction. Density values for crypt epithelium, primary CRC and metastatic CRC, normalized per area sizes, were analysed using unpaired Student's t-test. A value of P<0.05 was considered statistically significant The density of IRS1 immunostaining in paired cancer-uninvolved colonic epithelium, primary CRC and liver metastasis was determined by semiquantitative digital analysis using ImageJ software , treated with DNAase-1 (Ambion), checked by spectrophotometry and agarose gel electrophoresis, and retro-transcribed with the High Capacity DNA Archive Kit (Applied Biosystems), following manufacturer's instructions. RTqPCR assays were performed in duplicate using 96-well optical reaction plates and an ABI 7500HT machine (Applied Biosystems). Baseline amplification plot values were set automatically and thresholds were kept constant to obtain normalized cycle times and linear regression data. The reaction mix per well contained 10 µl Power Syber Green (Applied Biosystems), 2.4 µl of primers , 4.6 µl RNAase-free water, 3 µl cDNA (60 ng). For all experiments the PCR protocol was: denaturation at 95°C for 10 min, then 40 cycles at 95°C for 15 sec and at 60°C for 60 sec. Quantification was performed relative to in vitro differentiation APC point mutation, with second hit by loss of heterozygosity (LOH), a missense mutation in ß-catenin exon 5 (which does not appear to affect degradation), and is wild-type for KRAS, BRAF, PIK3CA and PTENAPC mutations , as well as mutations in SMAD4, BRAF, TP53, and PI3KCA, encoding the p110α catalytic subunit of the class I phosphatidylinositol 3-kinases (PI3K) http://www.sanger.ac.uk/cosmic).Modulation of IRS1 and of the insulin/IGF1 axis was investigated in the Caco-2 and HT29 CRC cell lines, which, under specific culture conditions, undergo spontaneous ATCC . Caco-2 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), L-glutamine (2 mM), penicillin (100 units/ml), and streptomycin (100 µg/ml) under a humidified atmosphere with 5% CO2 at 37°C. Cells were plated on 10 cm Petri dishes and allowed to grow at confluency. Upon confluency (designated as zero time point) Caco-2 culture was carried out in DMEM with 20% FBS. The entire time course was performed twice and whole cell lysates were obtained at 3, 7 and 14 days from confluency. HT29 cells were maintained in DMEM with 10% FBS and allowed to grow for 3 (preconfluent), 7 (confluent) and 14 (post-confluent) days, at which times whole cell lysates were obtained.Caco-2 and HT29 cells were obtained from http://rsbweb.nih.gov/ij/). The data were normalized for ß-actin and expressed as percentage of the maximum value.Whole cell or whole tissue lysates were prepared using ice-cold lysis buffer  supplemented with protease inhibitors . Lysates were cleared by centrifugation (10000× g for 20 min) and protein content was determined by the Bradford method. Fifty micrograms (50 µg) of total proteins were resolved under reducing conditions on 7.5% SDS-PAGE and transferred to reinforced nitrocellulose. The membrane was blocked with 3% not-fat dry milk in PBS with 0.01% Tween 20 for 1 hr at room temperature and then incubated overnight with the following primary antibodies: anti-IRS1 rabbit polyclonal , diluted 1∶500; anti-tyrosine 632-phosphorylated IRS1 (pIRS1 Tyr632) polyclonal antibody (Santa Cruz), diluted 1∶200; anti-ß-catenin monoclonal , diluted 1∶50 or anti-ß-catenin polyclonal , diluted 1∶1000; polyclonal against the ß subunit of the InsR (InsRß)  diluted 1∶200; polyclonal against the ß subunit of the IGF1R  diluted 1∶800; anti-actin monoclonal  diluted 1∶10000. The membrane was then washed in PBS and incubated for 1 h at room temperature with the corresponding horseradish peroxidase-conjugated secondary antibody, diluted 1∶2000 . Bound antibodies were detected using the enhanced chemiluminescent (ECL) method . Quantification of western blot signals (mean ± SE from at least two independent experiments) was obtained analyzing digitized signals with ImageJ software  and incubated with anti-ß-catenin monoclonal , anti-IRS1 rabbit polyclonal , diluted 1∶50; and anti-pIRS1 (Tyr632) polyclonal (Santa Cruz), diluted 1∶50. Nuclei were stained with 4,6-diamido-2-phenylindole  and cell membranes with wheat germ agglutinin . Primary antibodies were visualized using goat anti-mouse IgG fluorescein isothiocyanate-conjugated  or goat anti-rabbit IgG-Texas-Red-conjugated  for 30 min at room temperature. Cells were analyzed using an Apotome Axio Observer Z1 inverted microscope  equipped with an AxioCam MRM Rev.3 at 40× magnification. Colocalization of the fluorescence signals was analyzed with AxioVision 4.6.3 software. Image analysis was performed using Adobe Photoshop.Cells were fixed in a mixture of 2% paraformaldehyde-2% glutaraldehyde in PBS (pH 7.4), post-fixed in 1% osmium tetroxide in veronal acetate buffer (pH 7.4) for 1 h at 25°C, stained with 0.1% tannic acid in the same buffer for 30 min at 25°C and with uranyl acetate (5 mg/ml) for 1 h at 25°C, dehydrated in acetone and embedded in Epon 812. Thin sections were finally examined under a Philips CM10 transmission electron microscope, after post-staining with uranyl acetate and lead citrate.IRS1 and of c-MYC, key WNT target and effector IRS1 relative to paired mucosa. Overall, the mRNA levels of IRS1 were in good agreement with those of c-MYC.We determined by RTqPCR the constitutive expression of IRS1 and c-MYC mRNA. These CRCs also overexpressed InsRß, IGF1R, and ß-catenin, while in the other cases the mucosal levels of InsRß, IGF1R, and ß-catenin were similar or above those of the paired CRC , and in paired colonic mucosa and adenoma samples from two unrelated FAP patients ired CRC . In the ired CRC .P = 0.013 and P = 0.014, respectively, P<0.01) and colonic epithelium (P<0.01) (P = 0.001), while CRCs with mucinous/signet-ring phenotype were associated with focal or no IRS1 (P<0.001). Poorly differentiated CRCs often manifested nuclear staining in addition to cytoplasmic reactivity. We further assessed IRS1 protein expression by IHC in individually-matched paraffin-embedded sections of colonic mucosa, primary and metastatic CRC. Twenty-four cases with paired primary CRC and liver metastasis, also including cancer-uninvolved colonic mucosa, were available for analysis. Immunoreactive IRS1 was clearly detectable in crypt epithelium, as well as in primary and metastatic CRC . Primary(P<0.01) . Differe(P<0.01) . OverallP = 0.008), p53 (P = 0.032), membrane (P = 0.001) and cytoplasmic (P<0.001) ß-catenin. Other associations involved cell membrane ß-catenin, positively correlated with cytoplasmic ß-catenin (P<0.001) and EGFR (P = 0.005), and cytoplasmic ß-catenin, positively correlated with nuclear ß-catenin (P<0.001), Ki67 (P = 0.002) and p53 (P = 0.009), while nuclear ß-catenin positively correlated with p53 (P = 0.049). Predictably, a positive correlation was found between Ki67 and p53 (P = 0.029).As shown in These results suggest that high IRS1 is associated with CRCs combining well/moderately differentiated histological phenotype with immunohistochemical markers of poor prognosis .KRAS, BRAF, PIK3CA and PTENhttp://www.sanger.ac.uk/cosmic). Caco-2 cells are capable of spontaneous differentiation, documented by the expression of microvilli, enzymes and transporters characteristic of polarized enterocytes and by the development of tight junctions, which, in the in vivo setting, are necessary for upward migration of crypt epithelial cells toward the mucosal surface Caco-2 carries an inactivating APC mutation with second hit by LOH, but is known to be negative for mutations in Transmission electron microscopy of the Caco-2 cultures documented at day 3 the formation of localized electron-dense areas of close opposition between adjacent lateral plasma membranes, characteristic of forming tight junctions, and at day 14 the presence of complete intercellular junctional complexes, as well as polarization of the absorptive apical brush border . OverallImmunofluorescence analysis demonstrated differences in the subcellular distribution of total IRS1 and of pIRS1 Tyr632 during the Caco-2 culture time course . At day In conclusion, the immunofluorescence data indicate that in the Caco-2 model the intensity of IRS1 and pIRS1 staining increases with polarization, and that, during such process, pIRS1 Tyr632 becomes expressed in surface cells only, switching in subcellular distribution from the cytoplasm and basolateral membranes, where it colocalizes with ß-catenin, to the apical plasma membranes. This parallels the tightening of the intercellular junctions, as evidenced by electron microscopy, which makes the basolateral membranes inaccessible to freely-diffusing extracellular molecules.APC mutations, as well as mutations in SMAD4, BRAF, TP53, and, particularly, PI3KCA, which constitutively activate and deregulate intracellular signaling http://www.sanger.ac.uk/cosmic). Therefore HT29 cells provide a distinct in vitro model for the study of intestinal epithelial differentiation and polarity HT-29 cells carry double-hit IRS1 protein expression was analyzed by western blotting in HT29 cultures during spontaneous polarization, together with the InsRß and IGF1Rß subunit proteins. Western blot analysis showed that IRS1 levels were highest at day 3 (pre-confluent) and markedly decreased at day 7 (confluent), with only a slight increase (relative to day 7) at day 14 (post-confluent) . IGF1Rß Transmission electron microscopy analysis of pre-confluent (day 3), confluent (day 7) and post-confluent (day 14) HT29 cells demonstrated gradual polarization during the time course . FormatiImmunofluorescence analysis clearly revealed that in pre-confluent, and, with lesser intensity, post-confluent HT29 cells IRS1 was detectable in the cytoplasm, particularly in the perinuclear region, as well as in discrete spots within the nuclei . NuclearThese data indicate that in HT29 cells, known to carry mutations that constitutively activate intracellular signaling, IRS1 is highest in pre-confluent cells and pIRS1 localizes in discrete nuclear spots, with little variation during the polarization course.Of the 3 human IRS genes, IRS1, IRS2 and IRS4, the ubiquitously expressed IRS1 and IRS2 mediate the major metabolic, proliferative and anti-apoptotic functions of InsR and IGF1R signaling IRS2 RNA increases with spontaneous differentiation in both HT29 and Caco-2 cells and is downregulated in ß-catenin-dependent intestinal tumorigenesis We previously showed that IRS2 is directly controlled by the caudal-related homeobox protein 2 (CDX2) and significantly expressed in top crypt intestinal epithelium In contrast, several lines of evidence, summarized in the introduction, implicate IRS1 in intestinal carcinogenesis, under the direct control of TCF/LEF-ß-catenin complexes IRS1 mRNA and protein levels resulted higher, relative to paired mucosa, in CRCs that overexpressed c-MYC, ß-catenin, InsRß, and IGF1R. Overexpression of IRS1, together with ß-catenin, InsRß, and IGF1R, in FAP-associated adenomas was in agreement with data reported for the Apc(Min/+) mouse model In the present study, The immunostaining of the primary CRCs was in the range of that of the cancer-uninvolved top crypt in terms of intensity, but more diffuse in terms of percentages of positive cells, which may account for the above-discussed higher mRNA and protein levels in CRC relative to mucosa. In the hepatic metastases, IRS1 positivity was similar to that of the primary tumors in percentages of stained cells, but the staining intensity resulted significantly higher. The increase of IRS1 staining intensity in hepatic metastases is consistent with the fact that the liver is the major site of IGFs synthesis IGF/INS/IRS axis has a key anti-apoptotic role Human CRC is a molecularly heterogeneous disease Unlike Bommer et al. in vitro CRC models, we investigated the expression of IRS1, InsRß and IGF1R in Caco-2 and HT29 cells. In Caco-2, that mimics crypt to villus axis differentiation To correlate IRS1 with differentiation in PI3KCA mutation cooperates with other mutations in increasing proliferative and survival capacities IRS1 inhibition attenuates tumorigenicity in HT29 cells, that express low cytoplasmic IRS1 In HT29 cells, where a gain of function ex vivo in primary CRCs versus mucosa. Furthermore, diffuse IRS1 appears to be associated with CRCs that, despite their moderately to well differentiated histologic features, express markers of biological aggressiveness. The increase in IRS1 immunostaining in hepatic secondaries is consistent with a possible role in liver metastatization In conclusion, our results provide further evidence that IRS1 is differentially modulated, together with InsRß, IGF1R, and ß-catenin, during differentiation in the Caco-2 and HT29 cell models and Figure S1IRS1, insulin receptor, IGF1 receptor and ß-catenin in colonic mucosa and adenomas from familial adenomatous polyposis coli (FAP) patients. Panel A compares the western blot expression levels of IRS1, beta subunit of the insulin receptor (InsRß), beta subunit of the insulin-like growth factor 1 receptor (IGF1Rß), ß-catenin and, as loading control, ß-actin, in paired mucosa (M) and adenoma (T) samples from two unrelated FAP patients. In both cases, the levels of IRS1, InsRß, IGF1Rß, and ß-catenin are distinctly higher in the adenoma versus the paired mucosa sample. Panel B, detailing the edge of an adenoma, highlights the difference in IRS1 immunostaining associated with the transition between normal-appearing (downward pointing arrow) and dysplastic (upward pointing arrow) colonic crypts. The hyperplastic and mucin-depleted epithelium of the dysplastic crypts shows diffuse cytoplasmic IRS1, while mostly perinuclear/nuclear IRS1 is evident in non-dysplastic crypts. Panel C shows diffuse cytoplasmic IRS1 in a severely dysplastic adenoma.(TIF)Click here for additional data file.


The Wnt/β-catenin signaling pathway plays a key role during hepatocellular carcinoma (HCC) genesis and development. The present study aimed to investigate the effects of the Wnt/β-catenin signaling pathway on the expression of angiogenic growth factors involved in HCC. The HCC HepG2 cell line was transfected with small interfering RNA (siRNA) against β-catenin. After 72 and 96 h, protein was extracted and the expression levels of β-catenin, matrix metalloproteinase (MMP)-2, MMP-9, vascular endothelial growth factor (VEGF)-A, VEGF-C and basic fibroblast growth factor (bFGF) were detected by western blot analysis. β-catenin protein expression was inhibited at both time points. Notably, MMP-2, MMP-9, VEGF-A, VEGF-C and bFGF protein expression levels decreased at 72 h and then increased at 96 h after transfection. Our results demonstrated that in HCC cells, the Wnt/β-catenin signaling pathway may regulate the protein expression of the angiogenic factors, MMP-2, MMP-9, VEGF-A, VEGF-C and bFGF. These proteins were downstream of β-catenin signaling and were also regulated by other factors. In conclusion, the Wnt/β-catenin signaling pathway may contribute to the regulation of HCC angiogenesis, infiltration and metastasis through regulating the expression of these angiogenic factors. This transcription complex activates the expression of downstream target genes, resulting in abnormal cell proliferation and cell carcinogenesis  –3, gastrogenesis . To dateogenesis ,8, cycliogenesis ,9, surviogenesis  and peroResearch on tumor angiogenic factors was initiated in 1968 . To datePrevious studies have identified that the Wnt/β-catenin signaling pathway is involved in the regulation of angiogenic factors. It was reported that the β-catenin pathway regulated the expression of VEGF, MMP-7 and other factors in human colorectal cancer . AdditioHCC has a strong tendency to infiltrate and metastasize, and angiogenic factors have key roles in tumor angiogenesis, infiltration and metastasis. Therefore, in the present study, β-catenin expression was silenced using RNA interference (RNAi) technology in order to examine the association between the Wnt/β-catenin signaling pathway and angiogenic factors in HCC. We aimed to further elucidate the role of the Wnt/β-catenin signaling pathway in the pathogenesis of HCC.The HCC HepG2 cell line was purchased from the cell bank of the Chinese Academy of Sciences . Lipofectamine™ 2000 was purchased from Invitrogen Life Technologies . Small interfering RNA (siRNA) directed against β-catenin was designed and synthesized by the Shanghai GenePharma Co., Ltd. . The siRNA sequences were as follows: Sense, 5′-GGGUUCAGAUGAUAUAAAUTT-3′; and antisense, 5′-AUUUAUAUCAUCUGAACCCAG-3′. Dulbecco’s modified Eagle’s medium (DMEM) with high glucose was purchased from the Shanghai Hecoly Automatic Control Equipment Co., Ltd.  and fresh fetal calf serum (FCS) was purchased from TBD Biotechnology Corp. . The primary antibodies used were mouse anti-β-catenin, mouse anti-β-actin, rabbit anti-MMP-2, rabbit anti-MMP-9, mouse anti-VEGF-A, goat anti-VEGF-C and mouse anti-bFGF. The secondary antibodies of horseradish peroxidase (HRP)-conjugated goat anti-rat, goat anti-rabbit and rabbit anti-goat were used. All antibodies were purchased from Santa Cruz Biotechnology, Inc. .2. Cells were replated when they reached confluency and digested with 0.25% trypsin for 1 min in order to maintain the cell line.HepG2 cells were plated in culture flasks and were cultured in DMEM supplemented with 10% (vol/vol) fresh FCS at 37°C in a humidified atmosphere with 5% CO5 cells were plated in six-well plates in triplicate and grown to ~30–50% confluency. For transfection, 500 μl of siRNA in Lipofectamine 2000 was added to each well containing cells and 2 ml of DMEM without FCS, and plates were gently rocked back and forth. After 6 h of transfection, the media were replaced with DMEM containing 10% (vol/vol) FCS. The cells were harvested after 72 and 96 h, and mRNA and proteins were isolated for further analyses. Control groups without the addition of transfection reagents were also included in this study. All experiments were performed in triplicate and representative results were reported.A total of 3×10Cells were lysed in radioimmunoprecipitation assay buffer 72 and 96 h after transfection for protein isolation . Protein concentrations were determined using the bicinchoninic acid assay (Beyotime). Protein samples were electrophoresed on 10% SDS-PAGE gels  or 15% SDS-PAGE gels (VEGF-A and bFGF) and transferred onto polyvinylidene difluoride membranes. The membranes were blocked by incubation in phosphate-buffered saline (PBS) containing 5% skimmed milk at 37°C for 1 h and then incubated with specific primary antibodies (1:200 dilution) at 4°C overnight. The membranes were rinsed three times with PBS, followed by incubation with HRP-conjugated secondary antibodies  at 37°C for 1 h. Membranes were then rinsed three times with PBS and developed in 3,3′diaminobenzidine. When protein bands were visible on the membranes, color development was discontinued and membranes were rinsed in distilled water. Images were captured by the Gel Imaging system  and images were analyzed by Quantity One software . The quantitative results of gray-scale analysis were used for the statistical analysis.Data were compared by Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.Protein expression was assessed by western blotting at 72 and 96 h after the transfection of siRNA against β-catenin into HCC HepG2 cells. Our results demonstrated that the expression levels of β-catenin were decreased after 72 and 96 h, although the levels were slightly higher after 96 h than those at 72 h . MMP-2 and -9 protein expression was inhibited at 72 h, but that of MMP-2 had returned to normal levels after 96 h . MMP-9 expression levels were also increased at 96 h, but were less than the levels observed in the control group  . The expIn conclusion, following the transfection of siRNA against β-catenin into HCC HepG2 cells, β-catenin protein expression was inhibited after 72 and 96 h. MMP-2, -9, VEGF-A, -C and bFGF expression levels were also decreased at 72 h, but began to recover to normal levels at 96 h. Based on these observations, we hypothesized that the expression of these important factors was regulated through the Wnt/β-catenin signaling pathway.et al in 1998 (RNAi, first reported by Fire  in 1998 , is a ge in 1998 . To date1/TCF binding in T cells (Following knockdown of β-catenin in HCC HepG2 cells for 72 h, the protein expression levels of MMP-2, -9, VEGF-A, -C and bFGF also decreased. These findings indicated that the Wnt/β-catenin signaling pathway can regulate the expression of these proteins and that they are downstream target proteins of β-catenin signaling. However, the underlying mechanisms involved in this regulation have not yet been fully determined. Previous studies have reported that the Wnt/β-catenin signaling pathway acted directly on the MMP promoter through LEF T cells , but thiMMPs promote angiogenesis and contribute to tumor infiltration and metastasis not only through degradation of the extracellular matrix and vascular basilemma, but also through the active regulation of transforming growth factor-β, bFGF, VEGF and other important signaling molecules. The VEGF family regulated the formation of blood vessels and lymphatic vessels, vascular permeability and endothelial cell survival . MoreoveIn addition, MMP-2, -9, VEGF-A, -C and bFGF protein expression levels increased after blocking β-catenin expression for 96 h in HepG2 cells. These results demonstrated that the Wnt/β-catenin signaling pathway is not the only factor regulating the expression of these proteins and that a number of other factors are also involved in their regulation. These findings were consistent with other previous studies. Several studies have reported that the STAT3 signaling pathway influenced tumor angiogenesis, infiltration and metastasis by regulating the expression of VEGF, MMPs or bFGF in pancreatic cancer , colorecIn conclusion, this study demonstrated that the Wnt/β-catenin signaling pathway contributed to HCC angiogenesis, infiltration and metastasis through regulating the expression of angiogenic factors. The results of the present study require further validation in other cell lines and in animal studies. In addition, the target genes of the Wnt/β-catenin signaling pathway and the regulation of angiogenic factors have not yet been fully elucidated. Our results aid us to better understand the complex network underlying HCC pathogenesis and provide the basis and methods for multiple aspects of HCC research, including prevention, diagnosis and treatment. With the continual progression of HCC research, a number of concepts will be updated and bring us closer to curing HCC.