However, the resultant gene ranking is often not reproducible among different data sets. Such irreproducibility may be caused by disease heterogeneity.Detection of disease-associated markers plays a crucial role in gene screening for biological studies. Two-sample test statistics, such as the t-statistics were often reversed. Focusing on such instability, we proposed a sign-sum statistic that counts the signs of the t-statistics for all possible subsets. The proposed method excludes genes affected by heterogeneity, thereby improving the reproducibility of gene ranking. We compared the sign-sum statistic with the t-statistic by a theoretical evaluation of the upper confidence limit. Through simulations and applications to real data sets, we show that the sign-sum statistic exhibits superior performance.When we divided data into two subsets, we found that the signs of the two t-statistic. Using simulated data sets we show that the sign-sum statistic excludes hetero-type genes well. Also for the real data sets, the sign-sum statistic performs well in a viewpoint of ranking reproducibility.We derive the sign-sum statistic for getting a robust gene ranking. The sign-sum statistic gives more reproducible ranking than the The online version of this article (doi:10.1186/s12920-016-0214-5) contains supplementary material, which is available to authorized users. In this field, statisticians seek to identify informative genes as candidates for further investigation. To this end, it is desirable to correctly rank genes according to their degree of differential expression. In such efforts, two-sample test statistics, such as the t-scores of data1 were positive without loss of generality, and then ranked genes using the t-statistic in data1 and data2 separately. Figure t-scores were mismatched. This may be due to statistical variations caused by a sample size or heterogeneous factors in breast cancer. Thus, the simple t-statistic or correlation results in an unstable estimation that strongly depends on the dataset used.However, the resultant gene rankings are often not reproducible among different data sets. Such irreproducibility may be caused by disease heterogeneity . In factSuch heterogeneity was also discussed in a context of cancer outlier , 5 whichlasso are able to determine both highly ranked genes and classifiers simultaneously. However, [Novel methods such as However,  supporteHowever, , which tt-statistics and correlations strongly fluctuate due to sample variation, combining a sampling method with a two-sample test statistic should improve reproducibility. The effects of sampling method have been demonstrated by multiple studies, both theoretical [t-test exhibits superior performance in these regards. Dabney [t-statistic is more accurate than the modified t-statistic and shrunken centroids. However, those authors’ conclusions are based on empirical studies in the context of particular data. We argue that the sampling method is effective from the standpoint of robustness with respect to heterogeneous factors; this is because heterogeneity represents a mixture of two or more classes, and we can easily imagine that sampling is the best way to capture heterogeneity to integrate information from many small subsample sets. To stabilize the performance of the simple t-statistic, we derived a sign-sum statistic that improves ranking reproducibility. This novel statistic repeatedly counts the sign of mean difference between subsets of the normal and disease groups. The sign-sum statistic is an extension of the Wilcoxon sum-rank statistic, which itself has superior robustness but an inferior power relative to the t-statistic [Because oretical  and apploretical . Meanwhioretical  presente. Dabney  also argtatistic . We showXij be gene expression levels for samples i=1,⋯,n, genes j=1,…,p. We assume that all samples fall into either of two groups, 0 or 1, which denote normal and disease groups consisting of n0 and n1 samples respectively. Then, a two-sample t-statistic is defined by Let y for gene j. There are two options for sj, a pooled Student’s type or a non-pooled Welch’s type. In this paper, we use Welch’s t-statistic; therefore, sj is written as syj is the sample standard deviation of gene j and y. Without loss of generality, we can assume that where t-statistics can fluctuate between two divided data sets, and even the signs of t-statistics can be mismatched. Therefore, we focus our attention to the signs of the t-statistics. If the t-statistics are evaluated by the full sample, the signs are positive over all genes by the assumption above. However, if the t-statistics are evaluated by subsets of the full sample, the signs may change, as shown in Fig. t-statistics for all possible subsets.However, as discussed in Background, the a and b from groups 1 and 0 respectively; thus, there are We pick samples of sizes x<0 or 1 otherwise, and j in the t-th subset of group y. A larger value of the sign-sum statistic means that the signs of the t-statistics evaluated by subsamples are more stably positive. We can show that the sign-sum statistic is an extension of Wilcoxon’s sum-rank statistic; in fact, if a and b are equal to 1, then they are equivalent.where t-statistic evaluated by subsamples. In a manner similar to the derivation of the sign-sum statistic, it is defined as For comparison, we derived a U because they are members of t-statistics, as shown in Additional file t-statistic evaluated by subsamples . Because the U-statistic has the property of asymptotic normality, the asymptotic confidence interval is described as U and Zα/2 is the 100α/2 upper percentile of a standard normal distribution. Because UT and US are members of U-statistics, these statistics are evaluated by the interval estimators as shown in Theorem 1.Let t-statistic evaluated by subsamples with level α is The asymptotic confidence interval of the π1+π0=1 and Zα/2 is 100α/2 upper percentile of a standard normal distribution.if α is The asymptotic confidence interval of the sign-sum statistic  with levZα/2 is 100α/2 upper percentile of a standard normal distribution, and if Gy(v)=Pr(Wy≤v) for y=0,1, and where X1s and X0s are independently distributed with F1 and F0, which denote the distribution functions of gene expression levels of the disease and normal groups, respectively.Here V1−W1 and V0−W0 represent the mean differences in the disease and normal groups, respectively. A property of U-statistics allows us to evaluate the asymptotic variance of the sign-sum statistic by the conditional distribution of Wy given Vy for each group y. The difference between these two statistics is mainly due to the fact that the sign-sum statistic is the sum of the non-linear functions of the t-statistic evaluated by subsamples. As a result, information about F1 and F0 is strongly reflected in the sign-sum statistic as a result of changing a and b. We can discriminate hetero genes from homo genes by this property, as shown in the next subsection.A proof of the Theorem 1 is given in Additional file a and b used in the sign-sum statistic, or equivalently controlling the subsample sizes from the disease and normal groups. If the t-value of a homo gene is larger than that of a hetero gene, then the t-statistic easily distinguishes the homo gene from the hetero gene. However, if the t-values of the two genes are equal, then the t-statistic will confuse these genes, because their confidence intervals are equal. Such confusing homo genes will be top-ranked by the sign-sum statistic if we find a and b such that Here we aim to remove heterogeneous factors by choosing a value, chosen such that the sample mean of the disease group fluctuates. This consideration is supported through numerical evaluations of specific situations, as described below.The difficulty and importance of considering such hetero genes is also discussed in  in the cN without loss of generality. Then, the homo gene expression levels of the disease group follow τ1 and τ2 are mixing proportions with τ1+τ2=1. Moreover, we constrain expectations of the t-statistics of these genes by equality in the limiting sense of probability convergence. That is, Let all gene expression levels in the normal group follow t-statistics fluctuates because the interval estimators of the hetero and homo genes are almost overlapping.where t-statistic cannot. Figure a , a larger value of b (sampling size from disease group) is better at discriminating these scenarios, and (ii) a smaller value of a tends to be superior. This is because disease heterogeneity affects the difference between a sensitive estimator of the mean in the disease group and a stable estimator of the mean in the normal group. Although it would be ideal to obtain an optimal setting for the sampling size in general situations, based on these observations we fixed a as 1 and allowed b to be 1,5, or 10 below. Below, the sign-sum statistic for each a and b is described as sa, b.The asymptotic confidence interval of the sign-sum statistic is not evaluated analytically because it has an integral form. In some situations, we can confirm that upper confidence limits of the sign-sum statistic differ between the homo and hetero genes. Figure N without loss of generality. Homo gene expression levels in the disease group were drawn from a normal distribution τ1N+τ2N, where τ1,τ2 are positive values with τ1+τ2=1. The mixture model suggests that a proportion τ1 of gene expression levels in the disease group cannot be discriminated from those in the normal group, as in real data.We carried out simple simulation studies to evaluate the performance of the sign-sum statistic. With the number of genes set at 1000, we generated expression levels for 100 homo genes and 100 hetero genes; the remaining 800 were non-informative genes whose expression level distributions were equal in the disease and normal groups. Gene expression levels in the normal group were assumed to be drawn from a standard normal distribution t-statistic confuses the homo and hetero genes by the constraint as . For p genes, the ORRS for the top k-ranking is defined as For these data, the measure of reproducibility is given below. First, we divided the original data randomly into two data sets while maintaining the sample ratio of the disease and normal groups at the same value as in the full data set. After gene rankings were performed by the St2 are the top k-ranked genes sets for two divided data on the t-th trial; in this case, k=100 and T=100. Np, k refers to the expected overlap in gene number for a random selection. A larger ORRS value means that the selection is more reproducible than the random selection.where t-statistic. Overall, s1,10, the sign-sum statistic with sampling size 1 from the disease group and 10 from the normal group, performed the best in Situations (I) and (II). s1,1,s1,5,s1,10 were competitive and performed better than the t-statistic in Situation (III). These results confirmed the stable behavior of the sign-sum statistic, as shown in Fig. t-statistic. The ranking yielded better results in a large sample size (n=1000) than in a small sample size (n=200). When the sample size is 1000, almost all homo genes were ranked higher than hetero genes. Table s1,10 performed the best, and this result corresponds to the simulation study, as shown in Table Table e size n=00. When t-scores in the context of a classification problem. We often found that the genes with large t-scores in the training data had small or sign-reversed t-scores in the test data. The sign-sum statistic was developed based on these two motivations. Using numerical simulation, we proved that the sign-sum statistic improves the robustness with respect to heterogeneity relative to the t-statistic. Furthermore, the sign-sum statistic allowed us to obtain a reproducible ranking in applications to real data. These conclusions were validated by an evaluation of the upper confidence limit (Theorem 1).Gene ranking procedures are not reproducible among different studies . To obtaFDR  has been studied by novel methods such as SAM [q-values [In the context of gene screening, h as SAM  and rankq-values  for decit-statistic and sign-sum statistic have comparable predictive performance, although the DLDA predictor is constructed from each t-value for all genes. Thus the sign-sum statistic improves the ranking reproducibility without loss of predictive performance of the resultant genes.In this paper, we focused on robustness with respect to heterogeneity. However, we should still confirm that resulting genes are informative. In fact, the cancer outlier methods, which focus on the hetero genes, provide high reproducibility. However, we consider that the top-ranked differentially expressesd genes in any rankings should be effective for the latter prediction problem. Although further study is needed for such discussion, we performed a simple examination to ensure a certain degree of predictive power. Table Gene ranking is an essential in biological investigations. In this study, we were motivated by the desire to identify robust and predictive biomarkers. Hetero genes may be informative for some patients, but uninformative in others. In this sense, hetero genes should be extracted from gene rankings if these predictive performance is eqaul to or less than that of homo genes.t-statistic confuses homo and hetero genes as shown in the simulation study. The ranking irreproducibility would be caused by such heterogeneity also in the real data analysis. In fact, even the signs of t-statistics of many genes mismatch in the real data. We present the sign-sum statistic for getting robust ranking. Robustness for heterogeneity of the sign-sum statistic is shown by the evaluation of the upper confidence limit. We can get more reproducible ranking by the sign-sum statistic for simulated data which assumes that there are heterogeneous factors, for the breast cancer data which is known as the hetero disease and the data which includes different disease statuses.The http://bioinformatics.nki.nl/data/van-t-Veer_Nature_2002/ and GEO under the accession number of GSE31568.The data sets supporting the results of this article are provided at the following database AUC, area under curve; DLDA, diagonal linear discriminant analysis


The current study is an extension of our previous work, which demonstrated that water extract of brewers’ rice (WBR) induced apoptosis in human colorectal cancer (HT-29) cells. We also identified that brewers’ rice was effective in reducing the tumor incidence and multiplicity in azoxymethane (AOM)-injected colon cancer rats. Our present study was designed to identify whether WBR confers an inhibitory effect via the regulation of upstream components in the Wnt signaling pathway in HT-29 cells. To further determine whether the in vitro mechanisms of action observed in the HT-29 cells inhibit the downstream signaling target of the Wnt/β-catenin pathway, we evaluated the mechanistic action of brewers’ rice in regulating the expressions and key protein markers during colon carcinogenesis in male Sprague–Dawley rats.Brewers’ rice, is locally known as The mRNA levels of several upstream-related genes in the Wnt signaling pathway in HT-29 cells treated with WBR were determined by quantitative real-time PCR analyses. Caspase-3 and −8 were evaluated using a colorimetric assay. Male Sprague–Dawley rats were administered two intraperitoneal injections of AOM in saline (15 mg/kg body weight) over a two-week period and received with 10, 20, and 40 % (w/w) brewers’ rice. The expressions and protein levels of cyclin D1 and c-myc were evaluated by immunohistochemical staining and western blotting, respectively.CK1) and adenomatous polyposis coli (APC) mRNA levels. We discovered that the treatment of HT-29 cells with WBR resulted in the induction of apoptosis by the significant activation of caspase-3 and −8 activities compared with the control (P < 0.05). In vivo analyses indicated that brewers’ rice diminished the β-catenin, cyclin D1, and c-myc protein levels.The overall analyses revealed that the treatment of HT-29 cells with WBR inhibited Wnt signaling activity through upregulation of the casein kinase 1 (in vivo. We suggest that brewers’ rice may be a useful dietary agent for colorectal cancer.We provide evidence that brewers’ rice can induce apoptosis and inhibit the proliferation of HT-29 cells through regulation of caspase-dependent pathways and inhibit the Wnt/β-catenin downstream signaling pathway  Colorectal cancer has become a global health problem, represents the third most common cancer after lung and breast cancers and contributes for nearly 10 % of the total cases of cancer and approximately 8 % of the total cancer deaths . The intAPC gene and stimulating mutations in β-catenin, lead to the nuclear accumulation of β-catenin and subsequently result in the formation of a complex with T-cell factor (TCF)/lymphoid enhancing factor (LEF) to stimulate gene transcription [Emerging evidence reveals that Wnt/β-catenin signaling plays critical roles in human biology . Inapprocription . The TCFcription , result temukut, is typically used as an animal feed and brewing material [in vitro mechanisms of action observed in HT-29 cells inhibit the downstream signaling target of the Wnt/β-catenin pathway, we evaluated the mechanistic action of brewers’ rice in regulating the expressions and key protein markers during colon tumorigenesis in male Sprague–Dawley rats.Rice is one of the main staple foods in approximately half of the world’s population . The milmaterial . Severalmaterial  have demCK1 and APC mRNA levels. In addition, the treatment of HT-29 cells with WBR caused in the induction of apoptosis by the significant activation of caspase-3 and −8 activities compared with the control (P < 0.05). In vivo analyses also showed that brewers’ rice diminished the β-catenin, cyclin D1, and c-myc protein levels.We observed that the treatment of HT-29 cells with WBR inhibited Wnt signaling activity through upregulation of the Azoxymethane (AOM), 10 % (v/v) neutral buffered formalin, TRI Reagent®, and specific primers were bought from Sigma . Dulbecco’s Modified Eagle Medium (DMEM), RPMI-1640 medium, Mycoplex™ fetal bovine serum (FBS), penicillin and streptomycin (100×), and trypsin EDTA (1×) were obtained from PAA Laboratories GmBH . Colorimetric assay kit for caspase was bought from Genscript Corporation Inc . High capacity RNA-to-cDNA Kit and SYBR® Select Master Mix (CFX) were purchased from Applied Biosystems . Western blotting reagents were purchased from Bio-Rad . All other chemicals and reagents used were of analytical grade and bought from Sigma-Aldrich .Freshly milled brewers’ rice samples (MR 219) were obtained from BERNAS Milling Plant . Stabilized samples were carried out as previously described by Tan et al. . BrewersStabilized brewers’ rice was extracted with water following Yu et al. .2 atmosphere. The cells were treated with three different concentrations of WBR  for 72 h.The human colorectal cancer (HT-29) cells were bought from American Type Culture Collection , and the cells were cultured in DMEM supplemented with 10 % (v/v) FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. HT-29 cells were maintained and incubated at 37 °C in a humidified atmosphere with 5 % COACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S rRNA were used as housekeeping genes to normalize the target genes expression.The TRI Reagent® was used to isolate the total RNA, according to the manufacturer’s protocol. Two micrograms of RNA per 20 μL was reverse-transcribed using the High-Capacity RNA-to-cDNA Kit according to the manufacturer’s protocol. The reverse-transcription reaction was performed using an Authorized Thermal Cycler . The cDNA was then ready for use as a template for amplifications through real-time PCR reactions. Quantitative real-time PCR was carried out using SYBR® Select Master Mix (CFX). The nucleotide primer sequences originating from human cell lines are shown in Table pnitroanilide (pNA) after cleavage from the specific substrates DEVD-pNA (for caspase-3) and IETD-pNA (for caspase-8). Initially, HT-29 cells were treated with three different concentrations  of WBR for 72 h. The cells were centrifuged at 370 × g for 5 min to remove the medium and washed twice with phosphate-buffered saline (PBS) (pH 7.4). The cell pellets were then lysed with 50 μL of cold lysis buffer, followed by 1 h incubation period on ice. The resulting cell lysates were centrifuged at 9300 × g and 4 °C for 1 min, and the supernatants were collected. The protein concentration was quantified using a Bradford protein assay kit, according to the manufacturer’s protocols. Fifty μL of 2 × reaction buffers were mixed with 50 μL supernatant containing 200 μg of proteins in each tube, followed by 5 μL of caspase-3 or caspase-8 substrate. The suspension was collected and transferred to a 96-well microtiter plate and incubated at 37 °C for 4 h before measured at 405 nm using ELISA microplate reader.The caspase-3 and −8 activities were assessed spectrophotometrically using a commercial colorimetric assay kit, based on spectrophotometric detection of the chromophore n = 6 rats for each group): (G1), normal; (G2), AOM alone; (G3), AOM + 10 % (w/w) brewers’ rice; (G4), AOM + 20 % (w/w) brewers’ rice; and (G5), AOM + 40 % (w/w) brewers’ rice. During the acclimatization period, the animals were fed an American Institute of Nutrition (AIN)-93G diet and tap water ad libitum. Beginning at six weeks of age, the rats in G2-G5 were received intraperitoneal injections of azoxymethane (AOM) at a dose of 15 mg/kg body weight once weekly over a 2-week period. The rats in the normal group (G1) received an equal volume of normal saline (vehicle control). The animals were administered fresh food and tap water every day. The experimental diets were prepared weekly and kept at 4 °C. The components of an AIN-93G diet  of the Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (UPM) Serdang, Selangor (Reference number: UPM/FPSK/PADS/BR-UUH/00461). A total of 30 four-week-old male Sprague–Dawley rats were housed in plastic cages (two rats per cage) and maintained in an animal facility at a controlled temperature of approximately 25–27 °C and a relative humidity of 50 ± 10 % with 12-hour light/dark cycles. Hygienic condition was maintained by weekly changes of the woodchip beds. The animals were acclimatized for seven days prior to receiving the following defined diets for twenty weeks  neutral buffered formalin. The colon tissue was processed for further analyses.The colonic tissue sections were dehydrated, embedded in paraffin, and sectioned at 4-6-μm thickness. The detailed analyses for immunohistochemical evaluation were described previously . The priThe proteins from rat colon tissue (100 mg) were extracted with 300 μL of radio immune precipitation assay (RIPA) lysis buffer and 3 μL of protease inhibitor cocktail. The colon tissue was homogenized for 30 s prior to incubation at 4 °C for 1 h with agitation. The protein concentration was quantified using a Bradford protein assay kit according to the manufacturer’s protocol using bovine serum albumin as the standard. The protein suspensions were aliquoted into PCR tubes and stored at −80 °C prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Equal amounts (50 μg) of protein were separated by electrophoresis with 1× running buffer . The protein lysates and protein ladder were loaded onto gels, and the gels were run at 75 V for 15 min and then at 120 V for 45 min. After the protein samples were run on the SDS-PAGE gel, the gel was removed from the glass plate and rinsed in 1× Towbins transfer buffer  methanol; pH 8.3) at room temperature for 10 min. Protein transfer from the SDS-PAGE gel to a PVDF membrane was achieved by running the transfer apparatus at 100 V and 400 mA for 2 h. The PVDF membrane was blocked with blocking buffer (5 % skim milk solution) at room temperature for 1 h. The membranes were washed three times with phosphate-buffered saline in 0.1 % Tween-20 (PBST) for 10 min each before incubation with specific primary antibodies in 5 % skim milk PBST. Rabbit monoclonal antibody (E247) to β-catenin, rabbit monoclonal antibody (EPR2241) to cyclin D1, and rabbit monoclonal antibody (Y69) to c-myc were used at a 1:10,000 dilution  overnight at 4 °C on a roller mixer. After overnight incubation with the primary antibodies, the membranes were washed three times with PBST for 10 min each before incubation with goat polyclonal secondary antibody to rabbit IgG conjugated to horseradish peroxidase (HRP)  at a 1:10,000 dilution at room temperature for 1 h. The protein band was developed and detected using a chemiluminescence blotting substrate kit (Clarity western ECL substrate), and a chemiluminescence imager  was used to view the protein bands on the membranes. Beta-actin  was used as a loading control to confirm that the protein concentrations in all of the loaded samples were equal. The densitometric analyses of the band intensities obtained were performed using the Image J Software .The statistical data were expressed as the mean ± standard deviation (SD) and analyzed using a one-way analysis of variance (ANOVA). Statistical analyses were conducted following the Statistical Package for Social Science (SPSS) version 19.0 . A P-value of < 0.05 was considered significant.50 less than 100 μg/mL [50 values of 38.33 ± 6.51 μg/mL and 54.00 ± 5.29 μg/mL, respectively [50 values of 21.88 ± 12.43 μg/mL and 34.50 ± 5.92 μg/mL for WBR and MBR, respectively [According to published guidelines, any extract that exerts potentially cytotoxic effects should have an IC00 μg/mL . We prevectively , as evalectively . The resAXIN1, CK1, GSK3β, and APC in response to WBR were examined using quantitative real-time PCR. Furthermore, to understand how WBR regulates the Wnt signaling, Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6), which is essential in signal transduction and is more likely to stimulate Wnt signaling when overexpressed, was analyzed. Given the broad cytotoxicity range of WBR against HT-29 cell line determined by the MTT and LDH assays, as reported in our earlier study [To obtain further insights into the growth inhibitory effects of WBR in HT-29 cells are caused by the mechanistic action of WBR, the mRNA levels of several upstream-related genes in the Wnt signaling pathway were subsequently determined by quantitative real-time PCR analyses. In general, the Wnt/β-catenin signaling activity depends on the amount of β-catenin in the cytoplasm, and β-catenin is targeted for degradation by a destruction complex consisting of AXIN1, APC, CK1, and GSK3β . Thus, ter study , 19, onlAPC [AXIN1 mRNA level needs to be modulated to ensure proper Wnt signaling pathway. In the present study, we found that no significant difference in AXIN1 gene between the control cells and the cells treated with 32 μg/mL WBR and between the control cells and the cells treated with 64 μg/mL WBR (P > 0.05)  Fig. . TherefoAXIN1, β-catenin is targeted for phosphorylation, in part via the regulation by CK1γ. Beta-catenin is targeted for degradation via ubiquitin-mediated proteolysis through the action of CK1α [CK1 mRNA level in HT-29 cells treated with 32 and 64 μg/mL WBR for 72 h was significantly increased compared with the control (P < 0.05). Thus, this finding suggested that treatment with WBR at concentrations of 32 and 64 μg/mL elevated the CK1 mRNA level, and this increase may be associated with the induction of apoptosis, which may result to the phosphorylation of β-catenin in a Wnt pathway. Because WBR promotes the CK1 mRNA level, the susceptibility of HT-29 cells to WBR may also be due to GSK3β. A previous study found that GSK3β may promote cancer development and tumorigenesis [GSK3β in response to WBR was analyzed in HT-29 cells.In addition to the effects observed on  of CK1α . As showigenesis . Thus, tBecause GSK3β modulates many substrates and signaling pathways, the mechanisms underlying its tumor suppressor or promoter activities are complex . One of GSK3β mRNA level was mainly present in untreated HT-29 cells , which are cancer chemopreventive agents, have also been reported to increase the phosphorylation of GSK3β and thereby induce the downregulation of β-catenin/TCF signaling [GSK3β between the control group and those from the groups treated with 16 μg/mL WBR or 32 μg/mL WBR (P > 0.05)  Fig. . Taken tAPC mRNA level compared with HT-29 cells treated with WBR. A previous study reported that most of the colorectal cancer cases present inactivating APC mutations [APC gene are observed in 70 % of colorectal cancer cases and are regarded as an initiating event due to an inability of the destruction complex to stimulate the degradation of β-catenin [APC mRNA level in a dose-dependent manner. This finding indicated that the APC gene in HT-29 cells was significantly elevated after 72 h of incubation with WBR compared with the untreated HT-29 cells (control) (P < 0.05)  Fig. .LRP6 in response of HT-29 cells was investigated to determine whether WBR can modulate LRP6 at the mRNA level.The interaction of a Wnt ligand with transmembrane co-receptors, LRP6, suppresses the phosphorylation of the transcriptional coactivator β-catenin, which allows the translocation of β-catenin to the nucleus and activates the Wnt target genes . Thus, tThe LRP6 is a crucial co-receptor in Wnt signaling . Wnt ligAXIN1, CK1, GSK3β, and APC, our data revealed that treatment with various concentrations  of WBR significantly reduced the mRNA level of LRP6 in HT-29 cells in a dose-dependent manner compared to the untreated cells (P < 0.05) . In addition to the upregulation of caspase-3, the caspase-8 activity in HT-29 cells was also significantly increased after treatment with WBR for 72 h (P < 0.05). Therefore, the findings may suggest that an increase in caspase-8 activity result in the stimulation of the downstream apoptotic executioner caspase-3, which subsequently activates the molecular cascade of apoptosis in HT-29 cells. Taken together, our data indicate that the caspase-3 and −8 activities are induced by WBR in a dose-dependent manner. WBR markedly increase in apoptosis, which was accompanied by the activation of caspase-3 and −8 activities. Collectively, these results demonstrated that the activation of caspase activities in HT-29 cells after treatment with WBR increments initiator caspase-8 and executioner caspase-3. Our data presented in this study demonstrated that WBR inhibits the proliferation of colon cancer in vitro, as reported in our earlier study [in vitro mechanisms of action observed in HT-29 cells could suppress the downstream signaling target of the Wnt/β-catenin pathway, we investigated whether brewers’ rice can reduce the expressions and key protein markers during colon carcinogenesis in male Sprague–Dawley rats using immunohistochemical and western blot evaluations. Thus, the levels of the downstream signaling targets cyclin D1 and c-myc in response to brewers’ rice were further evaluated in the colons of rats injected with AOM.As shown in Fig. er study , leadingThe current study is an extension of our earlier work, which determined brewers’ rice to be an effective dietary agent for the reduction of colon tumor incidence and multiplicity formation in AOM-treated rats . We alsoBecause cyclin D1 and c-myc are downstream signaling targets in Wnt signaling and the overexpression of cyclin D1 and c-myc are found in most patients with colorectal tumors, in whom lowering the expression of cyclin D1 has therapeutic significance , we inveP < 0.05). However, no significant difference in both cyclin D1 and c-myc was found between the AOM alone and 10 % (w/w) brewers’ rice treatments (P > 0.05)  Table . These fAPC gene [To investigate the inhibitory effects of brewers’ rice in regulating the Wnt signaling pathway, we employed AOM-induced colonic tumors, which are known to exert the wild-type β-catenin and truncated APC gene . We hypoAPC gene .The protein level of β-catenin was diminished accordingly when the concentration of brewers’ rice was increased, indicating that brewers’ rice increased the degradation of β-catenin in a dose-dependent manner Fig. . ExpecteP < 0.05)  compared with G2 (05) Fig. . This reTo evaluate whether the dietary administration of brewers’ rice on AOM-injected colon carcinogenesis mediates the inhibition of the Wnt/β-catenin downstream signaling pathway, we investigated the Wnt target genes at the protein level. Cyclin D1 is a well-recognized cell cycle protein targeted by β-catenin  that is P < 0.05) . These results suggested that the effects of the dietary administration of brewers’ rice on cyclin D1 and c-myc corroborated the potential of brewers’ rice for regulating the β-catenin level in colon tumors. Taken together, this finding indicated that the dietary administration of brewers’ rice inhibited the Wnt/β-catenin downstream signaling pathway.Expectedly, we observed no cyclin D1 and c-myc protein expression in the normal colon mucosa Fig.  and c. A05) Fig.  and c. HIn the present study, the colon tumor samples in each group were randomly selected for analysis. Only three determinations conducted for each group in both immunohistochemistry and Western blotting analyses were based on several previous studies , 51–53.Most studies have reported the additive and/or synergistic protective effects of several components . TherefoThis study provides clear and substantial evidence that brewers’ rice offers great potential against colorectal cancer through the inhibition of colon carcinogenesis by the modulation of upstream and downstream components in the Wnt signaling pathway. Our findings demonstrated that brewers’ rice downregulates β-catenin and the Wnt target proteins cyclin D1 and c-myc, which provides a good rationale for a further comprehensive study of dietary brewers’ rice against colon cancer. Collectively, these findings provide evidence that WBR can induce apoptosis and inhibit the proliferation of HT-29 cells through regulation of caspase-dependent pathways and suppression of the Wnt/β-catenin downstream signaling pathway in AOM-induced rats. We suggest that brewers’ rice may be a useful dietary agent for colorectal cancer.


Vasculature is present in all tissues and therefore is indispensable for development, biology, and pathology of multicellular organisms. Endothelial cells guarantee proper function of the vessels and are the original component in angiogenesis. Morphogenesis of the vascular system utilizes processes like cell adhesion, motility, proliferation, and survival that are closely related to the dynamics of actin filaments and actin-tethered adhesion complexes. Here we review involvement of actin cytoskeleton-associated junctional molecules of endothelial cells in angiogenesis and lymphangiogenesis. Particularly, we focus on F-actin binding protein afadin, an adaptor protein involved in broad range of signaling mechanisms. Afadin mediates the pathways of vascular endothelial growth factor- (VEGF-) and sphingosine 1-phosphate-triggered angiogenesis and is essential for embryonic development of lymph vessels in mice. We propose that targeting actin-tethered junctional molecules, including afadin, may present a new approach to angiogenic therapy that in combination with today used medications like VEGF inhibitors will benefit against development of pathological angiogenesis. Endothelial cells (ECs) in mature vascular system are quiescent, nonproliferating  heterogenic population. The endothelium generated by a single layer of ECs separates the blood and lymph from other components of the vessel wall and serves wide variety of functions, specific not only for the vascular bed but also for the tissue they populate , 2. ECs Intercellular junctions between the adjacent ECs and between ECs and surrounding non-EC wall components  maintain the organization of EC layer and vessel integrity. Their function is beyond just mechanical support involving at least inhibition of ECs proliferation and neovasculogenesis in mature vessels as well as regulation of ECs gene expression and survival . AdhesivIn this review we focus on the involvement of actin-associated molecules at the junctional apparatus in AG and LAG and, in particular, afadin, an adaptor protein with multiple roles in cellular physiology . Small GAfadin is an adaptor protein discovered in 1997 by Mandai et al. and holds two RA (Ras association), a FHA (forkhead-associated), a DIL (dilute), a PDZ , three PR (proline-rich), and F-actin structural domains  20]. Tw. Tw20]. Physiological AG, the formation of blood vessels from existing ones, occurs not only in the embryo but also in postnatal life . Pathological AG accompanies some chronic inflammatory diseases , cancer, and atherosclerosis , 33, 34.Undeniably, the prime molecular machinery that stimulates VG and sprouting AG is comprised of VEGF and VEGF receptor in ECs , 34, 36.VEGF signaling is also critical for tumor AG. At present the most extensively applied medication in human cancer treatment is VEGF inhibitors . One of  in vivo data validated the vital role of afadin in postnatal AG. Moreover, afadin is involved in maintaining epithelial and endothelial barrier function [We have also found that, in endothelial afadin cKO mice, postnatal development of retinal vessels is initially delayed, that small vessels network decreased, and that VE-cadherin staining in ECs became discontinuous. Even in heterozygous KO mice, recovery of blood flow and neoformation of capillary networks after hind limb ischemia were diminished compared to control mice. Thosefunction –48, migrfunction , 32, elofunction , and modfunction . All tho in vivo neovascularization (hind limb ischemia model in mice) [β1 integrin affinity, thereby stimulating ECs adhesion and migration .VEGF receptor-Rap1 activation signal in ECs is transmitted not only to afadin/Akt but also to p42/44 ERK1/2 and p38 MAPK . Those tin mice) . The rolThe mice with EC-targeted deletion of afadin  were borOur reports described above complement the knowledge about signaling in embryonic LAG and postnatal AG. Lack of afadin in lymphatic ECs of the mouse embryo does not impede vessel morphogenesis but compromises intercellular junctions of ECs. This effect is secondary to F-actin stress fiber synthesis and VE-cadherin dislocation. Contrary to adhesion structures in blood ECs, lymphatic ECs have “button-like” intercellular contacts that facilitate the transport of intercellular fluids . This fuParticipation of afadin in the signaling downstream of VEGF and S1P receptors as well as its involvement in tubulogenesis and apoptosis might focus attention on this molecule in the context of tumor AG and cancer therapy. Afadin is also important antiapoptotic guard in embryogenesis, and platelet-derived growth factor (PDGF) receptor binds PI3K regulatory subunit p85/afadin in a similar way as shown for VEGF and S1P receptors to activate Akt .Many transmembrane adhesion molecules in ECs and their intracellular partners that link the molecular assembly to actin filaments contribute to the complex sequence of events in the process of VEGF-induced AG. Studies with cKO mice that eliminate the function of selected molecule in ECs demonstrated the individual role of excluded junctional component in AG or LAG and revealed its unique phenotype.β-catenin, plakoglobin, and p120-catenin. β-Catenin and plakoglobin interact with α-catenin that anchors  VE-cadhestioned , 66. p12estioned . VE-cadhestioned . VG was achinery .αvβ3, syndecan-1, and insulin-like growth factor-1 receptor is activated. These molecular events facilitate early adhesion and migration of ECs in VEGF-induced AG [VE-cadherin is crucial for the functional integrity of endothelial layer  and contduced AG . During duced AG . It has duced AG . VE-cadhduced AG . VE-cadhduced AG .β-catenin in ECs die at E11.5–E13.5 [β-catenin cKO mice, immunofluorescence of plakoglobin was increased, α-catenin decreased, and desmoplakin was found at EC contacts but not at those of control mice. The shift of molecular composition of junctional proteins in the β-catenin cKO mice has led to the authors' interesting hypothesis: lack of β-catenin forces creation of extra VE-cadherin/plakoglobin complexes and plakoglobin/desmoplakin/vimentin interaction analogous to that in complexus adhaerentes in lymphatic vessels [β-catenin [β-catenin transcriptional activity through interaction with T-cell factor (Tcf)/Lef transcription factors [β-catenin cKO mice may partially be a result of Wnt signal failure. Interestingly, the constitutively active β-catenin mutant in ECs also impairs embryonic AG because of overactivity of Notch-related pathways [Mice embryos with inactivated .5–E13.5 . VG and  vessels . In addi-catenin . ECs exp-catenin . Canonic factors . Wnt can factors  and the pathways .p120-Catenin stabilizes membrane localization of VE-cadherin by preventing endocytosis . Deletioα and β subunits that upon activation cluster to form focal adhesions and assemble intracellular signaling molecules that are linked to actin cytoskeleton and specific cellular functions. In addition, integrins regulate VE-cadherin/catenin complexes during cell movement [αvβ3 is coreceptor of VEGF receptor in AG [αv, α3, α5, β1, and β3 demonstrate their obligatory role in AG [β subunit of integrins and mediates coupling to actin, in ECs of mice produced severe defect in AG with diffuse hemorrhages and disrupted vascular trees, leading to embryonic death [β subunit partner important for integrin activation [β3-dependent adhesion, migration, and tube formation. Another key component of integrin activation, focal adhesion kinase (FAK), induced tumor AG [Integrins are large family of adhesive proteins that interact with extracellular matrix components and some adhesion molecules in the cell membranes and are critical for cell motility and survival . They armovement . A vast movement , 84, 85.or in AG  and nullle in AG –89. Defiic death . Kindlintivation , is alsotumor AG .α9β1 appears to have specific role in LAG. Integrin α9-null mice suffered insufficiency of the valves in collecting lymph vessels, developed chylothorax, and inexorably died by postnatal day 12 because of respiratory failure [α9β1/fibronectin interaction that is necessary for the leaflets matrix organization [α9β1 has unique property of binding directly VEGF to promote ECs adhesion and migration [Integrin  failure . The valnization . Integriigration .ECs in lymphatic microvasculature have specialized junctions that apparently reflect specific functionality of lymph capillaries . Those “αvβ3, essential molecule for tumor AG. At present, several groups of pharmacological inhibitors of integrin αvβ3 are in the different stages of testing and the results show favorable effects in preclinical and clinical settings [Pathological AG and LAG are considered dysregulated processes utilizing similar molecular repertoire as AG and LAG during embryogenesis . Howeversettings .AG and LAG are attractive targets for influencing pathological conditions that present with excessive/abnormal vascular proliferation  or ischemia (atherosclerosis). The growth of vessels in AG demands fundamental processes of cell biology and morphogenesis: cellular adhesion, migration, proliferation, and survival. All of these functions utilize as essential effector actin filaments and associated junctional molecules that assure intercellular or cell-matrix communication. As a part of the same machinery, actin-tethered molecular complexes appear to be highly integrated, and they cooperate to regulate organization or disorganization of particular adhesion structure. Furthermore, the main switch of AG, VEGF signaling, is intimately dependent on these molecular complexes. Because of these properties, molecular assemblies including VE-cadherin, integrin, and nectin as well as actin may be considered as (co)targets of VEGF-related pro- or antiangiogenic therapy.Afadin functions downstream of angiogenic signals translated by VEGF and S1P in ECs and also may represent target for modulating AG. Afadin has multiple roles in cellular processes, which may broaden the effects of its inhibition. Not only physiological AG but also pathological AG  might be affected by modulating the afadin-related signals. However, there are many unanswered questions at present to justify this strategy: compromising intercellular adhesions may promote metastasis in cancer, whereas dysfunctional actin-related machinery might have opposite effects by preventing cell adhesion and promoting apoptosis; fundamental roles of cell junctions most probably will require targeted approach, delivery of active substances only to the diseased location; mechanisms involving afadin in pathological AG could have altered effects. Nevertheless, the proposed hypotheses might be worth testing in translational studies with hope that it will help in reducing the resistance to VEGF receptor inhibitors in VEGF-induced (or other) pathological AG.


Knockdown of VE-cadherin expression with siRNA did not affect UTP-induced activation of extracellular signal-regulated kinases 1/2 (ERK1/2) but led to a loss of UTP-induced Rac1 activation and tyrosine phosphorylation of p120 catenin, a cytoplasmic protein known to interact with VE-cadherin. Activation of the P2Y2R by UTP also caused a prolonged interaction between p120 catenin and vav2 (a guanine nucleotide exchange factor for Rac) that correlated with the kinetics of UTP-induced tyrosine phosphorylation of p120 catenin and VE-cadherin. Inhibitors of VEGFR-2 (SU1498) or Src (PP2) significantly diminished UTP-induced Rac1 activation, tyrosine phosphorylation of p120 catenin and VE-cadherin, and association of the P2Y2R with VE-cadherin and p120 catenin with vav2. These findings suggest that the P2Y2R uses Src and VEGFR-2 to mediate association of the P2Y2R with VE-cadherin complexes in endothelial adherens junctions to activate Rac1.Vascular endothelial cadherin (VE-cadherin) mediates homophylic adhesion between endothelial cells and is an important regulator of angiogenesis, blood vessel permeability and leukocyte trafficking. Rac1, a member of the Rho family of GTPases, controls VE-cadherin adhesion by acting downstream of several growth factors, including angiopoietin-1 and vascular endothelial growth factor (VEGF). Here we show that UTP-induced activation of the G The vascular endothelium separates circulating blood from the underlying tissue and provides a semipermeable barrier for normal fluid, nutrient and waste exchange. During inflammatory and allergic reactions, however, the endothelial barrier is disrupted causing tissue swelling (edema) that is often accompanied by leukocyte infiltration. It is well known that the integrity of the endothelial barrier is controlled by the formation and disruption of intercellular adhesion structures comprised of adherens junctions and tight junctions and also by contractility provided by the actomyosin cytoskeleton . The Rho2 nucleotide receptor (P2Y2R), a Gq protein-coupled receptor activated equally well by ATP and UTP, has emerged as an important regulator of blood vessel permeability and leukocyte recruitment. In vivo studies focusing on the P2Y2R have shown that activation of this receptor transiently increases microvascular leakage to macromolecules . In. In2 nucr et al. , who shoand RhoA  14] and and2 nucand RhoA  16]. Si. Si2 nuctivities –[11] . VE. VE21]. -catenin . Modulat-catenin –31]. In. In2+-de-catenin  33], an, an2+-de-catenin . These i-catenin .2R in human coronary artery endothelial cells (HCAECs) affects receptor distribution and association with VE-cadherin. Our previous work demonstrated that activation of the P2Y2R promotes monocyte adhesion and extravasation into rabbit carotid arteries and increases the development of atherosclerotic plaques . VE. VE47]. f Tyr685 . Althougf Tyr731 . In addicadherin . We, howcadherin , suggestprotease , phosphoprotease  and focaprotease , and uprprotease  and inteprotease . As mentprotease . Phosphoprotease  and FAK protease . Moreoveprotease  59]..in vivo 2R in endothelial cells is known to promote cell proliferation via protein kinase C (PKC), phosphoinositide 3-kinase (PI3K) and ERK1/2 signaling  [61 [6160] . The P2Yignaling . Here, w2R and VE-cadherin induced by UTP was prevented by pretreatment of endothelial cells with PP2 or SU1498, compounds that inhibit Src and VEGFR-2 kinase activity, respectively. This suggests that Src and VEGFR-2 kinases regulate assembly of a VE-cadherin-associated complex with the P2Y2R that is important for downstream signaling to Rac1. In support of this idea, both Src and VEGFR-2 have been found to associate with the activated P2Y2R [Interaction between P2Yed P2Y2R  16] and and2R an2R interaction, UTP also caused a rapid and transient association between VEGFR-2 and VE-cadherin, suggesting that P2Y2R activation recruits VEGFR-2 to the VE-cadherin/ P2Y2R complex. VEGFR-2 is known to associate with VE-cadherin in response to not only VEGF stimulation but also shear stress in vascular endothelial cells , it, it2R/VE2R with UTP in confluent HCAEC cultures activates both RhoA and Rac1 and in vivo studies showed that the Rho-associated protein kinase inhibitor Y27632 blocks the increase in microvascular permeability induced by UTP [2R in quiescent endothelial cells is unclear, it is known that Rac1 regulates the integrity of adherens junctions and participates in cytoskeletal rearrangements important for endothelial permeability induced by histamine, thrombin and VEGF [2R. Interestingly, the expression of both dominant-negative and constitutively active forms of Rac1 increases the permeability of unstimulated endothelial cells [Intracellular signaling transduced by VE-cadherin is complex and varies depending on whether the vasculature is growing (angiogenic) or established . In confd by UTP . Althougand VEGF  and tranand VEGF . Thus, Ral cells , suggest2R induces the Src- and VEGFR-2-dependent activation of VE-cadherin in adherens junctions, which leads to the p120 catenin- and vav2-dependent activation of Rac1 (2R can associate with VE-cadherin as well as VEGFR-2 [Vβ3/β5 integrins [2R participates in a multi-receptor complex to regulate the function of adherens junctions. This study elucidates a novel mechanism whereby nucleotides act as signaling cues to modulate adherens junctions and activate Rac1 signaling in endothelium, an important process for regulating vascular permeability and responses to inflammation.Results presented here demonstrate that in endothelial cells, activation of the G protein-coupled P2Y of Rac1 . Since t VEGFR-2  and αVβ3ntegrins , these r01


Dickkopf-1 (DKK1) is an antagonist of Wnt/β-catenin signaling implicated in tumorigenesis. However, the biological role of DKK1 and β-catenin involved in chondrosarcoma has not been sufficiently investigated. This study was designed to investigate the expression profiles of DKK1 and β-catenin, and to clarify their clinical values in chondrosarcoma.The mRNA and protein levels of DKK1 and β-catenin in fresh chondrosarcoma and the corresponding non-tumor tissues were analyzed by Real-time PCR and Western blot, respectively. The protein expression patterns of DKK1 and β-catenin were investigated by immunohistochemistry. The associations among DKK1 level, β-catenin accumulation, clinicopathological factors and the overall survival were separately evaluated.Both DKK1 and β-catenin levels were remarkably elevated in chondrosarcoma compared with the corresponding non-tumor tissues. High DKK1 level and positive β-catenin accumulation in chondrosarcoma specimens were 58.7% and 53.9%, respectively. Elevated DKK1 level significantly correlated with positive β-catenin accumulation, and they were remarkably associated with histological grade and Musculoskeletal Tumor Society stage. Furthermore, DKK1 level and β-catenin accumulation had significant impacts on the prognosis of chondrosarcoma patients. Multivariate analysis revealed that DKK1 level was an independent prognostic factor for overall survival.Elevated DKK1 levels associated with β-catenin accumulation play a crucial role in chondrosarcoma. DKK1 can serve as a novel predictor of poor prognosis in patients with chondrosarcoma. Chondrosarcoma is a malignant cartilage-forming tumor, accounting for approximately 20% of bone malignancies and presenting with a wide spectrum of clinical behaviors The WNT signal transduction cascade is implicated in several important physiological and pathophysiological conditions such as tumorigenesis Abnormalities in Wnt/β-catenin signaling are implicated in a variety of human cancers The potential association of deregulation of Wnt/β-catenin signaling with chondrosarcoma development provides the rationale for deeply exploring the underlying role of DKK1 and β-catenin in chondrosarcoma in vivo, and determines whether DKK1 and beta-catenin can be predictive for overall survival of patients with chondrosarcoma. Our findings suggest that elevated levels of DKK1 and β-catenin play a role in the pathogenesis of chondrosarcoma, and DKK1 can be recognized as an independent factor to predict the prognosis of chondrosarcoma patients. Thus it could be a potential biological target for the management of chondrosarcoma.Signed informed consent from all patients was obtained for sample collection and analysis. All samples were anonymously coded in accordance with local ethical guidelines. This study was approved by the institutional review board of the Peking University Third Hospital and Tianjin Hospital and was in full compliance with national legislation and the ethical standards of the Chinese Medical Association (IRB00001052-08044).Sixty-three cases of conventional chondrosarcoma, seventeen cases of benign cartilage tumors including osteochondromas and enchondromas based on accepted clinicopathological and radiological criteria were enrolled in this study 5′-GATCATAGCACCTTGGATGGG-3′ and 5′-GGCACAGTCTGATGACCGG-3′) 5′- AGCCGAGATGGCCCAGAAT-3′ and 5′-AAGGGCAAGGTTTCGAATCAA-3′) 5′-ATCATCCCTGCCTCTACTGG-3′ and 5′-CCCTCCGACGCCTGCTTCAC-3′). PCRs were optimized for the number of cycles to ensure product intensity to be within the linear phase of amplification. The cycles were as follows: 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 55°C for 35 s and 72°C for 30 s. Data were analyzed according to the relative standard curve method with normalizing the values of GAPDH expression in each sample.RNA extraction, cDNA synthesis, and quantitative polymerase chain reactions (PCR) were performed as described previously Protein extraction and Western blot were performed as previously described Detailed experimental procedures for immunohistochemistry have been described previously All the stained slides were simultaneously scored blinded by two, independent observers without prior knowledge of clinicopathologic data, and a consensus score was reached for each slide. For the assessment of the intensity of DKK1 staining, DKK1 staining was evaluated using the following criteria as described previously P<0.05 was considered statistically significant. All reported P values were two-sided.Data are presented as the Mean ± SD. Differences between groups were analyzed using the Student’s t-test for continuous variables, or the Pearson’s Chi-square test/Fisher’s Exact Test for categorical variables. Curves for overall survival were drawn according to the Kaplan-Meier method, and differences between the curves were analyzed by applying the log-rank test. Univariate and multivariate analyses of clinicopathological factors and overall survival were done using Cox proportional hazards regression model. Statistical analyses were performed using SPSS  and P = 0.006, P = 0.001, P = 0.014, P = 0.012, To identify whether the expression levels of DKK1 and β-catenin are involved in the pathogenesis of chondrosarcoma, we measured the mRNA and protein levels of DKK1 and β-catenin in chondrosarcomas and the corresponding non-tumor tissues. Our results shown that the mRNA levels of DKK1 and β-catenin in chondrosarcomas were significantly increased compared with the corresponding non-tumor tissues (P = 0.035) and MSTS stage (P = 0.020). Similarly, β-catenin accumulation was markedly correlated with advanced clinicopatholgoical parameters such as higher histological grade (P = 0.037) and higher Musculoskeletal Tumor Society stage (P = 0.011). Furthermore, we found that both DKK1 level and β-catenin accumulation had no significant correlation with other clinicopathological factors such as gender, age and anatomical location.The associations among DKK1 level, β-catenin accumulation and clinicopathological factors are presented in P = 0.000, Previous studies have documented that DKK1 was significantly associated with β-catenin expression in other tumors P = 0.015, P = 0.018, P = 0.010, Previous studies have documented that DKK1 and β-catenin can be predictive for the prognosis of patients with several solid tumors P = 0.000, P = 0.003, P = 0.004, P = 0.005, respectively). In the multivariate Cox regression analysis, histological grade , MSTS stage  and DKK1 level  had a significantly independent impact on patient prognosis, whereas β-catenin accumulation failed to demonstrate independency (P = 0.248).To identify the potential independent and significant factors for the overall survival, we performed Cox regression analysis . UnivariOur observations have implicated DKK1 and β-catenin in the pathogenesis and prognosis of chondrosarcoma. Elevated levels of DKK1 significantly correlated with nuclear β-catenin accumulation could be involved in the development of chondrosarcoma, and DKK1 can be identified as an independent prognostic factor for the overall survival in chondrosarcoma. These findings suggest that disruption of DKK1 suppressing nuclear β-catenin accumulation could be a novel attractive target for chondrosarcoma treatment.Previous reports have demonstrated that DKK1 can suppress the Wnt-induced signaling that is frequently activated in tumor biology P = 0.035, Aberrant activation of the Wnt/β-catenin signaling pathway has been implicated in tumorigenesis including sarcoma P = 0.000, No reports have been documented regarding as the relationship between DKK1 levels and β-catenin accumulation in chondrosarcoma. In this study, elevated level of DKK1 positively correlated with β-catenin accumulation in chondrosarcoma tissues  for 2 hr, followed by SDS-PAGE and immunoblot analysis for total β-catenin (BD Transduction Laboratories), active β-catenin  dephosphorylated on Ser37 or Thr41 (Millipore) and β-actin (Sigma). B. SW1353 chondrosarcoma cells were transfected with either empty vector or DKK1-HA. Expression of tagged DKK1 was assessed by immunoblot analysis of lysates with an anti-HA antibody. Total β-catenin, active β-catenin, and β-actin were detected by Western blot as described above. C. DKK1 suppressed the TCF-reporter transcriptional activity in SW1353 cells. Cells were cotransfected with either TOP-FLASH or Fop-FLASH plasmid (Upstate Biotechnology), and the pRL-CMV plasmid (Upstate Biotechnology) encoding Renilla luciferase as an internal control for transfection efficiency. Luciferase activity was measured 48 h after transfection with the Dual-luciferase reporter assay system (Promega). The values represent the mean (±SD) of three independent experiments, and the ratio of the activity obtained with the wild-type TOP-FLASH plasmid to the activity observed with the mutant FOP-FLASH plasmid was shown.(TIF)Click here for additional data file.


Glucagon-like peptide-1 (GLP-1) plays a role in the regulation of adipogenesis; however, the precise underlying molecular mechanism has not been fully defined. Wnt was recently identified as an important regulator of adipogenesis. This study aimed to investigate the involvement of the Wnt signaling pathway in the effects of GLP-1 on adipocyte differentiation. 3T3-L1 cells were induced to differentiate. The changes in the expression levels of adipogenic transcription factors and Wnts and the phosphorylation level and subcellular localization of β-catenin were quantified after GLP-1 treatment. GLP-1 stimulated adipocyte differentiation and lipid accumulation, which were accompanied by the expression of adipocyte marker genes. The expression of Wnt4 was upregulated in the process of adipocyte differentiation, which was further enhanced by treatment with GLP-1. β-catenin, an important mediator of the Wnt pathway, was immediately dephosphorylated and translocated from cytoplasm to nucleus when differentiation was induced. In the presence of GLP-1, however, β-catenin was redirected to the cell plasma membrane leading to its decreased accumulation in the nucleus. Knockdown of Wnt4 blocked the effect of GLP-1 on the cellular localization of β-catenin and expression level of adipogenic transcription factors. Our findings showed that GLP-1 promoted adipogenesis through the modulation of the Wnt4/β-catenin signaling pathway, suggesting that the GLP-1-Wntβ-catenin system might be a new target for the treatment of metabolic disease. Chronic nutrient overload causes an increase in adipose depots. This effect has often been associated with the development of type 2 diabetes, atherosclerosis, and hyperlipidemia ;2. The gThe stimulation of adipogenesis consists of the sequential activation of a transcription factor cascade . The traThe Wnts are a family of secreted proteins that act through paracrine and autocrine mechanisms to regulate many aspects of cell fate and development . The canThe incretin system and Wnt-signaling pathway interact at different stages. Activation of the incretin hormone glucagon-like peptide-1 (GLP-1) signaling pathway by its receptor agonists increases Wnt member 4 (Wnt4) expression and activates the canonical Wnt signaling pathway to promote β-cell proliferation –19. On tTM RT reagents kits and TaKaRa SYBR premix Ex Taq were acquired from TaKaRa Bio . Antibodies for β-catenin, (Ser37/Thr41) non-phospho-β-catenin and FITC-conjugated anti-rabbit and anti-mouse IgG antibodies were purchased from Cell Signaling Transduction . Antibodies for Wnt4 and GAPDH were acquired from Santa Cruz Biotechnology . Lifectamine2000 and siRNA control were purchased from Life Technologies . All other chemicals of analytical grade were acquired from Dingguo Bio .Cell culture reagents, 5-bromo-2'-deoxyuridine (BrdU), anti-BrdU antibody, DNase I and TRIzol, were purchased from Life Technologies . GLP-1, Exendin9-39 (Ex9), isobutylmethylxanthine (IBMX), dexamethasone (Dex), 4',6-diamidino-2-phenylindole (DAPI), 3--2,5-diphenyltetrazolium bromide (MTT) and Oil Red O were acquired from Sigma Chemical . Complete Protease Inhibitor Cocktail was purchased from Roche Applied Science . TaKaRa PrimeScript3T3-L1 pre-adipocytes were cultured as previously described . BrieflyLipid accumulation of differentiated 3T3-L1 adipocytes was determined by quantitative Oil Red O staining. Briefly, 14 days after the induction of differentiation, the cells were fixed for 20 min with 4% formaldehyde, followed by incubation with Oil Red O for 30 min. The dye was eluted by the addition of 100 μl (96-well plate) of isopropanol per well and then was measured by reading its absorbance at 540nm.TM RT reagents kits, according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qPCR) was performed with ABI Prime 7500, using approximately 2–4 μl reverse-transcribed reaction diluted 10 times each. Samples were prepared using TaKaRa SYBR premix Ex Taq, according to the manufacturers’ instructions. After the reaction, each PCR product was verified for its single amplification by melting curve analysis. The gene-specific primers for amplification are listed in Total RNA was extracted from 3T3-L1 adipocytes using TRIzol reagent. After the RNA was treated with DNase I, 1.0 μg of RNA was reverse-transcribed using TaKaRa PrimeScriptTwo target regions (1:591–611 and 2:651-671ref) for Wnt4 were selected using the QIANGEN siRNA online design tool. The scrambled fragment 5’-AAGAGGAGCATATTGGGAAGA-3’, which does not have similarity with any mRNA listed in Genebank, was used as a negative control. Transfection of siRNA into 3T3-L1 cells was performed with Lifectamine 2000 according to the manufacturer’s instruction.2, 1 mM dithiothreitol and complete protease inhibitor cocktail). The lysate was passed through a 25 G needle 10 times using a 1 ml syringe and was left on ice for 20 min. The nuclear pellet was centrifuged out at 720 g at 4°C for 5 min, and the supernatant was collected for membrane fraction by ultracentrifugation. The nuclear pellet was further washed with fractionation buffer and was passed through a 21 G needle 21 times. Then, the nuclear fraction was collected after centrifugation at 3000 rpm for 5 min at 4°C. For the membrane fraction, the supernatant was centrifuged at 150,000 G for 1 hr. The supernatant was the cytosolic fraction, and the pellet was washed and centrifuged again at 100,000 G for 45 min to obtain the membrane protein pellet. The fractionated proteins were lysed with RIPA buffer , 25 mM β-glycerophosphate, 5 mM sodium orthovanadate, and 5 mM NaF). The cell lysates were resolved by SDS-PAGE as previously described [Cytoplasmic and nuclear protein fractions were prepared as described previously . Brieflyescribed . The proCells were induced for differentiation for the indicated amount of time as described in the legend, with or without GLP-1. After fixation, the cells were permeabilized and then stained with anti-β-catenin antibody. Finally, FITC-conjugated anti-rabbit IgG antibody was added. Images were obtained using a Nikon fluorescence microscope.At 60% confluence, 3T3-L1 pre-adipocytes were kept in serum-free DMEM for 6 h and then induced in DMEM with 0.5% BSA medium for proliferation, with or without GLP-1. For MTT assay, the cells were incubated with MTT for 4 h, and DMSO was then added to dissolve the MTT formazan crystals. Absorbance was read at 590 nm.For Brdu incorporation assay, cells were incubated with 10 μM BrdU for 2 h and fixed with Carnoy's fixative  for another 20 min. Subsequently, the cells were denatured by 2 M HCl and were stained with anti-BrdU monoclonal antibody. FITC-conjugated anti-mouse IgG antibody was added, followed by DAPI counterstaining.t-test and, in cases of multiple time points and treatments, by one-way ANOVA. P values <0.01 were considered to be highly significant and <0.05 were considered to be significant.The values are presented as means ±S.E.M.s. Comparisons between groups were performed with Student’s unpaired We investigated whether GLP-1 regulated Wnt synthesis in vitro. 3T3-L1 pre-adipocytes were cultured with or without GLP-1 (10nM and 50 nM) for 17 h. As demonstrated in We next examined the effects of GLP-1 on the proliferation of pre-adipocytes. GLP-1 increased the gene expression levels of cyclin D1 at doses of 10 nM and 50 nM . MTT assWe found that the lipid accumulation in differentiated adipocytes treated with GLP-1 was increased in a dose-related fashion . The expWnts is reported to be an important regulator of adipogenic differentiation; we investigate whether GLP-1 exerted its action on adipogenesis via regulating the expression of Wnts in pre-adipocytes undergoing differentiation. After 3 h of incubation with MDI, we noticed an increase in Wnt4 expression, with maximum mRNA levels occurring between the 1st day and 4th day after cell induction . In contWnt4 is known to be an important accelerator of adipogenesis. Silencing Wnt4 via siRNA  signific37 and Thr41 [37 and Thr41 (np-β-catenin). When differentiation was initiated, the level of np-β-catenin protein rapidly increased (at 6h time point)   and the e point)  due to te point) . GLP-1 te point) . These rβ-catenin is reported to be a transcription repressor during adipogenic differentiation. Wnt4 is able to antagonize canonical Wnt signaling by specifically redirecting β-catenin to the plasma membrane . We testTranslocation to membrane by Wnt4 avoids β-catenin to be degradated in the cytoplasm , which mGLP-1 exerts a variety of biological functions, including enhancing glucose-dependent insulin secretion, suppressing glucagon secretion, slowing gastric emptying, reducing food intake and stimulating satiety . Using aSuppression of adipogenesis has been linked to increased insulin resistance , while eWnt signaling possibly serves to regulate adipose expansiontightly, to meet energy storage demands . The finThe phosphorylation level of β-catenin is tightly regulated by Wnt signaling. When adipogenesis was initiated, the expression levels of Wnt6 and Wnt10b rapidly and sharply decreased. In addition, expression levels of the Wnt receptors Fz1, Fz2 and Fz5 also decreased upon the induction of differentiation . Thus, βWnt4 is essential for cell proliferation and development in a broad range of tissues ;39. In pIn conclusion, our data demonstrated that GLP-1 promoted adipogenesis, in part by enhancing Wnt4 expression. On the molecular level, we revealed a novel pathway of β-catenin based on its translocation and accumulation in the nucleus during the early stages of differentiation. GLP-1 redirected β-catenin to the plasma membrane via the Wnt4 pathway. Therefore, GLP-1 could exert part of its effects on adipocytes by upregulation of Wnt4, modulating β-catenin signaling in pre-adipocytes undergoing differentiation.S1 FigThe transcription level of Wnt6 and Wnt10 were quantified by qPCR at the indicated time points after MDI induction. n = 3–6.(TIF)Click here for additional data file.


The results showed that GSK-3β activation and osteogenic marker expression levels were downregulated by ephrinB2-FC treatment, but these effects were inhibited by blocking integrin αvβ3 in MSCs. Our findings demonstrate that EphB4 can promote osteogenic differentiation of MSCs via upregulation of POSTN expression. It not only helps to reveal the interaction mechanism between EphB4 and Wnt pathway but also brings a better understanding of EphB4/ephrinB2 signaling in bone homeostasis.Erythropoietin-producing hepatocyte B4 (EphB4) has been reported to be a key molecular switch in the regulation of bone homeostasis, but the underlying mechanism remains poorly understood. In this study, we investigated the role of EphB4 in regulating the expression of periostin (POSTN) within bone marrow-derived mesenchymal stem cells (MSCs) and assessed its effect and molecular mechanism of osteogenic induction in vitro. Treatment with ephrinB2-FC significantly increased the expression of POSTN in MSCs, and the inhibition of EphB4 could abrogate this effect. In addition, osteogenic markers were upregulated especially in MSCs overexpressing EphB4. To elucidate the underlying mechanism of cross talk between EphB4 and the Wnt pathway, we detected the change in protein expression of phosphorylated-glycogen synthase kinase 3 Osteogenic differentiation of bone marrow-derived mesenchymal stem cells (MSCs) is essential for normal bone homeostasis and the repair of bone defects. Thus, the molecules that have osteoinduction activity play a critical role in these processes. Among them, erythropoietin-producing hepatocyte B4 (EphB4) and its ligand ephrinB2 have attracted much interest for use in regulating bone homeostasis , 2. Boneαvβ3 and regulating Wnt/β-catenin signaling [β), and bone morphogenetic protein-2 (BMP-2) as well as mechanical stimulation [β-catenin signaling during osteogenesis in vitro.In addition to osteoinductive molecules, the local microenvironment also plays critical roles in the regulation of the bone homeostasis and repair. Extracellular matrix (ECM) is an important part of the microenvironment and is vital to the regulation of cell adhesion, migration, and differentiation via cell-ECM interactions. In bone, the ECM is composed primarily of several molecular complexes of proteins, such as collagen (CO), laminin (LM), osteopontin (OP), fibronectin (FN), osteonectin (ON), and other glycoproteins , 5. A reignaling . It alsomulation –10. Base6 cells per 37.5 cm2 flask in human MSC basal medium containing 10% fetal bovine serum  at 37°C in 5% CO2. When cells reached 90% confluence, they were detached using 0.25% trypsin/EDTA  and replated at a density of 1.8 × 105 cells per 37.5 cm2 flask. UltraCULTURE Serum-Free Medium  was used when necessary.MSCs were isolated and cultured using previously described methods . Briefly5 cells were incubated with a phycoerythrin- (PE-) labeled monoclonal antibody to CD34, CD45, CD73, CD90, or CD166 or fluorescein isothiocyanate- (FITC-) labeled monoclonal antibody to CD105  for 30 min in room temperature. Cells were washed and resuspended in PBS, and data were acquired on a FACS Calibur (BD Biosciences). MSCs were confirmed based on the expression of CD73, CD90, CD105, and CD166 and the lack of CD34 and CD45 expression.Cells were harvested at the third passage for identification of surface antigens by flow cytometry. About 5 × 10SABC IHC kits  were used for immunohistochemical staining with a primary antibody to POSTN , and nuclei were counterstained with hematoxylin. Images were captured using a Leica Microsystem microscope .MSCs were infected with recombinant adenoviruses expressing the EphB4 gene or siRNA sequences at a multiplicity of infection(MOI) of 40 for 72 h as previously described . The effμg/mL, R&D Systems, Minneapolis, MN, USA) and POSTN  were added individually to promote osteogenic differentiation. NVP-BHG-712  was used as a EphB4 kinase inhibitor. Integrin αvβ3 on MSCs was blocked by incubation with a mouse monoclonal antibody  at 37°C for 2 h before exposure to ephrinB2-FC or POSTN as a control treatment.EphrinB2-FC , which was then refreshed every 3 days. Alkaline phosphatase (ALP) staining was performed with a FAST BCIP/NBT tablet  after 9 days in culture, and calcium nodules were stained by 0.4% Alizarin red S staining (Sigma-Aldrich) after 21 days in culture.μg total RNA using the PrimeScript 1st Strand cDNA Synthesis Kit , and real-time quantitative PCR was carried out using the 7500 qPCR System  with SYBR Premix EX Taq II (TaKaRa). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for reference.Total RNA was extracted using the QIAGEN Rneasy Mini kit  and quantified using an ultraviolet spectrophotometer . Then, cDNA was synthesized from 4  POSTN: forward primer, GCCATCACATCGGACATA, and reverse primer, CTCCCATAATAGACTCAGAACA. Runx2: forward primer, AGATGATGACACTGCCACCTCTG, and reverse primer, GGGATGAAATGCTTGGGAACTGC. Osterix: forward primer, ACTGGCTAGGTGGTGGTCAG, and reverse primer, GGTAGGGAGCTGGGTTAAGG. α-SMA: forward primer, CATGGCATCATCACCAACTG, and reverse primer, GCTGGGACATTGAAAGTCTC. ALP: forward primer, ACCATTCCCACGTCTTCACATTTG, and reverse primer, AGACATTCTCTCGTTCACCGCC. COL1: forward primer, CCTGGAAAGAATGGAGATGATG, and reverse primer, ATCCAAACCACTGAAACCTCTG. GAPDH: forward primer, ACCCATCACCATCTTCCAGGAG, and reverse primer, GAAGGGGCGGAGATGATGAC.The primer sequences used were as follows:αvβ3 were added as previously described. After 5 days, total protein was prepared in radioimmunoprecipitation (RIPA) lysis buffer  and quantified by bicinchoninic acid assay . Then, 20 μg protein was separated by 8% sodium dodecyl sulfate- (SDS-) polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes . Membranes were blocked in 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) and incubated overnight at 4°C with primary antibodies against POSTN, COL1 , GSK-3α/β, phosphorylated-GSK-3α/β (Ser21/9), β-catenin, Runx2 , and GAPDH . Membranes were washed with PBS containing 0.1% Triton X-100 and incubated with horseradish peroxidase- (HRP-) anti-rabbit IgG or anti-mouse IgG  at a dilution of 1 : 5000 as a secondary antibody for 1 h at room temperature. The signals on the membranes were visualized by ChemiDoc XRS  with an enhanced chemiluminescence kit (Amersham Biosciences) and quantitatively analyzed using Image J2x software .Cells were starved for 12 h, and then the medium was replaced with fresh serum-free medium. Antibodies to ephrinB2-Fc, POSTN, NVP-BHG-712, and integrin POSTN concentrations in serum-free medium collected from MSC which were treated by ephrinB2-Fc or BHG712 as previously described were measured using the Human Periostin ELISA Kit  according to the manufacturer's instructions.The concentrations of phosphorylated EphB4 were measured using the Human Phospho-EphB4 DuoSet IC ELISA kit (R&D Systems) according to the manufacturer's instructions and as previously described .P < 0.05 was considered statistically significant.Data are expressed as mean ± standard deviation (SD) values. Statistical analysis was conducted with two-way analysis of variance (ANOVA) using the GraphPad Prism 5.0 statistical software package .  in vitro  was performed. The data showed that, with stimulation by ephrinB2-FC, the sum IOD of ALP staining was increased significantly in wild-type MSCs and MSCs overexpressing EphB4. However, this phenomenon was not observed in the BHG712-treated, integrin  Figures . The sum Figures . These dαvβ3-blocked and POSTN-treated MSCs in serum-free medium for 3 days, and the level of p-GSK-3β-Ser9 was significantly greater in the POSTN-treated group than in the other groups. In addition, the expression of β-catenin and other osteogenesis markers, including Runx2 and COL1, also was greater in POSTN-treated MSCs (β (p-GSK-3β-Ser9/GSK-3β) and β-catenin showed that POSTN could decrease the amount of active GSK-3β and increase β-catenin expression significantly, and this effect was not observed in MSCs after blockage of integrin αvβ3  [β-catenin by suppressing the activation of GSK-3β. Combined, these findings indicate the possibility of cross talk between EphB4 and the Wnt pathway through upregulation of POSTN expression to promote osteogenic differentiation of MSCs. We know that the Wnt signaling pathway is vital for promoting osteogenic differentiation of MSCs, and that it can suppress chondrogenic and adipogenic differentiation among these cells [β-catenin, which is a key factor in the canonical Wnt signaling pathway, is also known to be crucial for the osteogenic differentiation of MSCs. As a downstream signaling molecule of GSK-3β, β-catenin is ubiquitinated and degraded upon phosphorylation of active GSK-3β [β reduces the activation of GSK-3β [β by increasing the level of p-GSK-3β-Ser9 in wild-type MSCs as well as MSCs overexpressing EphB4, and the expression of β-catenin and osteogenic markers were affected accordingly. However, this effect was not observed in MSCs treated with EphB4 siRNA or BHG712, which functions as an EphB4 kinase inhibitor [β-Ser9 level in MSCs after blocking integrin αvβ3 also was deceased, as was the expression of β-catenin and osteogenic markers. These findings suggest that the cross talk between EphB4/ephrinB2 signaling and the Wnt pathway is mediated by POSTN.Eph (erythropoietin-producing hepatocyte) receptors, the most numerous subfamily of receptor tyrosine kinases (RTKs), and their ephrin ligands are known to be involved in cell-cell communication responsible for the regulation of cell proliferation and differentiation as well as cancer progression –28. Ephsne (PTH) . Our resse cells –38. β-cae GSK-3β , and thef GSK-3β , 41. Ournhibitor . These r in vitro, and the exact mechanism by which activation of EphB4 increases POSTN expression was not determined. In addition, further studies are needed to determine the specific mechanism by which POSTN affects the Wnt pathway in promoting osteogenic differentiation and the osteogenic inductive effect of EphB4-POSTN should be investigated in vivo for greater biological relevance. However, our study provides a platform for exploring the molecular osteogenic mechanism of EphB4/ephrinB2 signaling.It should be noted that this study only concentrated on the effect of upregulation of POSTN expression induced by EphB4 signaling with cell-based systemsIn conclusion, we showed that the activation of EphB4 in MSCs treated with ephrinB2-FC can increase the expression of POSTN, and POSTN can promote osteogenic differentiation through cross talk with the Wnt pathway. This is the first report to unravel the relationship between EphB4 signaling, POSTN expression, and the Wnt pathway, and our results provide a novel perspective for understanding the osteogenic mechanism of EphB4/ephrinB2 signaling in human MSCs.


KCNQ1OT1, an imprinted antisense lncRNA in the human KCNQ1 locus on chromosome 11p15.5, is involved in cis-limited silencing within an imprinted KCNQ1 cluster. Furthermore, aberration of KCNQ1OT1 transcription was observed with a high frequency in colorectal cancers. However, the molecular mechanism of the transcriptional regulation and the functional role of KCNQ1OT1 in colorectal cancer remain unclear. Here, we show that the KCNQ1OT1 transcriptional level was significantly increased in human colorectal cancer cells in which β-catenin was excessively accumulated in the nucleus. Additionally, overexpression of β-catenin resulted in an increase in KCNQ1OT1 lncRNA-coated territory. On the other hand, knockdown of β-catenin resulted in significant decrease of KCNQ1OT1 lncRNA-coated territory and an increase in the mRNA expression of the SLC22A18 and PHLDA2 genes that are regulated by KCNQ1OT1. We showed that β-catenin can promote KCNQ1OT1 transcription through direct binding to the KCNQ1OT1 promoter. Our evidence indicates that β-catenin signaling may contribute to development of colorectal cancer by functioning as a novel lncRNA regulatory factor via direct targeting of KCNQ1OT1.Long noncoding RNAs (lncRNAs) have been implicated in many biological processes through epigenetic mechanisms. We previously reported that  INK4A locus (ANRIL), the HOX transcript antisense RNA (HOTAIR) and the KCNQ1 opposite strand/antisense transcript 1  can epigenetically regulate the expression of neighboring genes in cis or of distant genes in trans4545A number of long non-coding RNAs (lncRNAs) have recently been identified through rapid advances in high-throughput analyses of transcriptomes1KCNQ1OT1 as an imprinted gene at the KCNQ1 cluster on human chromosome 11p15.5 by using a novel in vitro system that was developed for the screening of imprinted genes using human monochromosomal hybridsKCNQ1OT1 is stably accumulated at its own gene region and that some imprinted genes on the KCNQ1 cluster, which lie outside of the KCNQ1OT1 lncRNA transcriptional domain throughout the cell cycle, suggesting that KCNQ1OT1 may play a significant role as a regulatory factor at a specific domain such as at an imprinted cluster68KCNQ1OT1 lncRNA may control gene expression by accumulation at a sub-chromosomal region in a way that resembles X chromosome inactivation (XCI) by XIST RNAKCNQ1OT1 transcription and epigenetic statuses including histone modifications and DNA methylation at the KCNQ1 clusterKCNQ1OT1 transcription may be closely related to initiation and/or progression of colorectal cancer. However, as yet little is known regarding the mechanism by which KCNQ1OT1 lncRNA is regulated and its functional role in cancer development.We previously identified the antisense lncRNA APC), axis inhibitor (AXIN), casein kinase 1α (CK1α) and glycogen synthase kinase 3β (GSK3-β)1213E2F4 transcription was directly activated by the β-catenin that accumulated in the nucleus in colorectal cancers, resulting in cancer progressionKCNQ1OT1 lncRNA in colorectal cancers could be caused by an effect of β-catenin activity.The majority of colorectal cancers are driven by aberration of the Wnt/β-catenin signaling pathwayKCNQ1OT1 transcription in colorectal cancer cell lines is driven by direct binding of β-catenin to its promoter region. Moreover, both KCNQ1OT1 transcription and the extent of KCNQ1OT1 lncRNA-coated territory changed remarkably depending on β-catenin activity. These data provided additional novel evidences that the regulation of KCNQ1OT1 lncRNA by β-catenin signaling may be involved in the multiple processes of colorectal cancer development.In the present study, we showed that KCNQ1OT1 transcription is frequently observed in colorectal cancer tissues and cell linesKCNQ1OT1 expression status in four colorectal cancer cell lines using quantitative reverse transcription PCR (qRT-PCR). The KCNQ1OT1 transcription level was increased 1.8-fold in HCT15 and SW480 cells compared to that in HCT116 and DLD-1 cells (p < 0.05).We have previously reported that aberration of KCNQ1OT1 lncRNA transcription status depends on the amount of β-catenin protein in the nucleus, we performed immunofluorescence staining analysis using a β-catenin antibody and measured the relative intensities of the fluorescence signals of β-catenin in the nuclei of HCT116, DLD-1, HCT15 and SW480 cells (p < 0.001). These results indicated that KCNQ1OT1 lncRNA transcription levels could be increased by nuclear accumulation of β-catenin in colorectal cancer, suggesting that KCNQ1OT1 transcription could be regulated by β-catenin activity. Next, we performed single polymorphism nucleotide (SNP) analysis of KCNQ1OT1 transcript (bdSNP: rs231359) in order to validate the status of the allelic expression of KCNQ1OT1 in HCT116 and HCT15 cells which exhibit differential distribution of β-catenin to cytoplasm and nuclear compartments of cells. Both the G and T alleles were detected in the genomic DNA. In contrast, only G allele was detected in cDNA samples (KCNQ1OT1 locus are frequently observed in HCT116 and HCT15 cells by DNA fluorescence in situ hybridization (DNA-FISH) analysis using a KCNQ1OT1 specific DNA probe . Analysis by qRT-PCR and western blotting indicated that the transcription and protein level of β-catenin, respectively, was markedly increased in the β-catenin overexpressing clone compared with that in the control clone (p < 0.001). To investigate whether the KCNQ1OT1 lncRNA signal in the nucleus, i.e., lncRNA-coated territory, was increased by β-catenin activity, we performed RNA-fluorescence in situ hybridization (RNA-FISH). We used a specific probe to detect KCNQ1OT1 lncRNA, in β-catenin overexpressing HCT116 cells and measured the area of the lncRNA-coated territory. KCNQ1OT1 lncRNA-coated territory expanded in β-catenin overexpressing HCT116 cells compared with that in the cells transfected with control vector . These results suggested that an increase in KCNQ1OT1 transcription through β-catenin activity eventually leads to an increase in its lncRNA-coated territory.l vector . Figure 0.10 μm2 . To inveKCNQ1OT1 lncRNA-coated territory, we performed knockdown of β-catenin transcription using short interfering RNA (siRNA) in the HCT15 cells that showed the highest amount of nuclear β-catenin protein of the colorectal cancer cell lines tested . In contrast, no remarkable change in the CDKN1C mRNA level was observed  for transcription factor binding sites that supports Genomatix software. Second, we constructed human KCNQ1OT1 promoter-luciferase reporter plasmids; plasmid Kp2022 contained a 2020-bp fragment from within the KCNQ1OT1 promoter region that included this TCF binding site, and plasmid Kp1080 contained a truncated fragment of the 5′ region of the KCNQ1OT1 promoter that did not include this TCF binding site . This result suggested that β-catenin regulated KCNQ1OT1 lncRNA transcription through an effect on the KCNQ1OT1 promoter.To investigate whether β-catenin regulates ing site . We thenKCNQ1OT1 promoter, we performed chromatin immunoprecipitation (ChIP) analysis. In this assay, crosslinking of β-catenin to the TCF binding site in the KCNQ1OT1 promoter in cells of the colorectal cancer cell lines HCT15 and SW480 was assayed  associated with imprinted clusters function as important regions for maintenance of the parent-origin-specific gene expression pattern, which is known as the imprinting control region (ICR). The ICR of the ed genes ,3 and 4.ed genes 13. It isKCNQ1OT1 lncRNA resembles that of Xist RNA in terms of chromatin association and cis-limited epigenetic silencingXist RNA is essential for X chromosome inactivation (XCI). Xist RNA accumulates and spreads along the X chromosome, which expresses its RNA, and then recruits gene silencing complexes that include histone methyltransferases and the polycomb group proteins Eed/Ezh2/Suz12, which ultimately establishes XCI by induction of heterochromatin formationJarid1c or Utx that escape from XCI are always located at the periphery or outside of the Xist RNA-coated territory in C2C12 normal mouse myoblast cellsKcnq1ot1 lncRNA-coated territory area in placental derived cells is largely expanded compared to that of embryonic cells17Kcnq1ot1 lncRNA-silencing target locus differs in the placenta and the embryo17cis-limited lncRNA-coated territory is coordinated with the silencing behavior of its coated-genes. In the present study, we demonstrated that the mRNA expression of SLC22A18 and PHLDA2, which are regulated by KCNQ1OT1 lncRNA spreading, was increased by the knockdown of β-catenin in colorectal cancer cells , HOTAIR, E2F4 antisense transcript and metastasis associated lung adenocarcinoma transcript 1 (MALAT1) has been observed in colorectal cancer that harbors alteration of Wnt/β-catenin signaling pathways915212223E2F4 antisense transcript and of MALAT1 transcription, which cause accumulation of β-catenin in the nucleus, strongly contributes to the development of colorectal cancer15KCNQ1OT1 transcription through binding to its promoter region . HCT116, DLD-1, HCT15 and HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum . SW480 cells were cultured in Leibovitz’s L-15 medium  supplemented with 10% FBS.6 HCT116 cells were seeded in each well of six wells plates and were transfected 24 h after seeding with 4 μg of β-catenin expression vector or XL-5 vector. Both the β-catenin expression vector and XL-5 were purchased from OriGene . For knockdown of β-catenin, 5 × 105 HCT15 cells were seeded in each well of 6 plates and were transfected 72 h after seeding with 100 pmol of β-catenin siRNA  or control .Cells were transfected with plasmid or siRNA using Lipofectamine 2000 . For overexpression of β-catenin, 1 × 10KCNQ1OT1 promoter region containing the predicted binding site for TCF-1 based on the Transcription Element Search System (http://www.cbil.upenn.edu/tess), Genomatix GEMS Lancher (http://www.genomatix.de) and Genetyx Ver.10 software  was PCR-amplified from cDNA and inserted into a BglII/Acc65I-digested luciferase reporter vector pGL3-basic . The PCR primer sets were designed to amplify the upstream KCNQ1OT1 promoter region from -2022 to -294. The PCR primer sets used were: forward primer: 5′-GGGGTACCCCAGGTGACAAGGTGCAGGCGC and reverse primer: 5′-ACAGAGTTCCTCGTTGGGAGCTTGAAGATCTTC. For the truncated luciferase reporter construct without the TCF-1 site (Kp1080), deletion of the upstream KCNQ1OT1 promoter region −2022 from −1080 was performed using PCR-based site-directed mutagenesis . Kp1080 was PCR-amplified from the Kp2022 luciferase reporter construct according to the manufacturer’s protocol. The PCR primer sets used were: forward primer: 5′-CGGAGGTGGGAATCCCCGTTG, and reverse primer: 5′-GGGGTACCTATCGATAGAGAAATG.The Luciferase assay was performed as described previouslyMethylation-sensitive southern hybridization was performed using DIG High Prime DNA Labeling and Detection Kit  according to the manufacturer’s instructions. In brief, 20 μg of genome DNA from normal and colorectal cancer cell lines were digested with BamHI and NotI overnight, resolved by gel electrophoresis, and transferred to High bond N + . The PCR products were labeled by Digoxigenin-11-dUTP and used as probe. The PCR primer sets used were: forward primer: 5′- TCTCTCTGGGAGGGTTTGAA and reverse primer: 5′- TTACTTCGCCCCCTAATTCCT. Immunoreactive bands were analyzed using a Luminescent Image Analyzer LAS-4000 .Western blotting was performed as described previouslyKCNQ1OT1 locus was used dbSNP website (http://www.ncbi.nlm.nih.gov/SNP/) and PCR products spanning SNP from colorectal cancer cell lines were sequenced . The PCR primer sets were: forward primer: 5′-GGGTAGGCTGGTCACGTTTA and reverse primer: 5′-AGTCCCCTGTAGATTCTGGG. The sequence data was analyzed using Finch TV (http://www.geospiza.com).Search of allelic specific SNP on ChIP assay was performed as described previously4) were seeded on 24 × 60 mm micro cover glass and were incubated at 37 °C for 24 h. The cells were then fixed with 4% paraformaldehyde at room temperature for 10 min. After two cold PBS washes, the cells were treated 0.5% saponin (Sigma-Aldrich) and 0.5% Triton X-100 (Sigma-Aldrich) at room temperature for 20 min and were then kept in 20% glycerol  /PBS for 2 h. Subsequently, the cells were passed six times through liquid nitrogen. After two PBS washes, the cells were blocked with 5% bovine serum albumin (Sigma-Aldrich) in PBS (5% BSA/PBS). Rabbit monoclonal antibody against the human β-catenin antigen diluted to 1:500 in 3% BSA/PBS was applied to the cells. The cells were washed twice in PBS with 0.05% Tween-20 , followed by addition of the secondary antibody Alexa Fluor 488 goat-anti-rabbit IgG  diluted to 1:600 in 3% BSA/PBS. After a PBST wash, the cells were stained with DAPI . Immunofluorescence staining was observed by using the confocal microscope LSM780 . Intensity was measured using the ZEN 2010 software (Carl Zeiss). Note that all Immunofluorescence staining experiments were performed in parallel and, for detection, exposure and interval time was kept the same in several experiments.Cells . Images were minimally enhanced for brightness and contrast to resemble that which was seen by eye through the microscope. In setting the confocal microscope, the objective lens used was the Plan-Apochromat 63x/1.40 Oil DIC M27, and the lasers used were 405 nm (DAPI) and 488 nm (Alexa 488). The image sizes were: x, 512; y, 512; z, 13, and 12-bit. Area was measured using the ZEN 2010 software (Carl Zeiss). Note that all RNA-FISH experiments were performed in parallel and, for detection, exposure and interval time was kept the same in several experiments.RNA-FISH analysis was performed as previously describedKCNQ1OT1 locus were counted.DNA-FISH analysis was performed as previously describedKCNQ1OT1 transcription, and β-catenin and SLC22A18 mRNA expression were detected using qRT-PCR. KCNQ1OT1 transcription, and β-catenin and SLC22A18 mRNA expression, were analyzed using the following specific primers. KCNQ1OT1: forward, 5′-CTTTGCAGCAACCTCCTTGT; reverse, 5′-TGGGGTGAGGGATCTGAA. β-catenin: forward, 5′-TCTGATAAAGGCTACTGTTGGATTGA; reverse, 5′-TCACGCAAAGGTGCATGATT; SLC22A18: forward, 5′-CATCTTGCTTACCTACGTGCTG; reverse, 5′-CCCAGTTTCCGAGACAGGTA. PHLDA2: forward, 5′- TCCAGCTATGGAAGAAGAAGCRNA isolation and reverse transcriptase (RT)-PCR were performed as described previouslyCDKN1C: forward, 5′- CTCCGCAGCATCCACGAT; reverse, 5′- GGTGCGCACTAGTACTGGGA. cDNA was amplified using an Applied Biosystems StepOne thermal cycler system and a SYBR green PCR kit . The mRNA level was normalized to human GAPDH mRNA .; reverse, 5′- GTGGTGACGATGGTGAAGTACA. P-values less than 0.05 using an unpaired Two-tailed Student’s t test. In RNA-FISH analyses, we used the Chi-squared test to determine whether there was any significant difference in the distribution of area ratio between control cells and β-catenin expressing plasmid or siRNA transfected-cells.Data from more than two separate experiments are presented as means ± S.D. Significance was established at How to cite this article: Sunamura, N. et al. Regulation of functional KCNQ1OT1 lncRNA by β-catenin. Sci. Rep.6, 20690; doi: 10.1038/srep20690 (2016).