Clinically, metastatic rectal cancer has been considered a subset of left-sided colon cancer. However, heterogeneity has been proposed to exist between high and middle/low rectal cancers. We aimed to examine the efficacy of anti-epidermal growth factor receptor (EGFR) treatment for middle/low rectal and left-sided colon cancers.This study enrolled 609 patients with metastatic colorectal cancer who were treated with anti-EGFR therapy. They were divided into groups based on primary tumour locations: the right-sided colon, the left-sided colon or the middle/low rectum. The efficacy of first-line and non-first-line anti-EGFR treatment was analysed. Genomic differences in colorectal cancer data from The Cancer Genome Atlas (TCGA) were investigated and visualised with OncoPrint and a clustered heatmap.On first-line anti-EGFR treatment, patients with middle/low rectal tumours had significantly lower progression-free survival, overall survival, and overall response rates  than those with left-sided colon cancer . Similar outcomes were also identified on non-first-line anti-EGFR treatment. In TCGA analysis, rectal tumours displayed genetic heterogeneity and shared features with both left- and right-sided colon cancer.Anti-EGFR treatment has lower efficacy in metastatic middle/low rectal cancer than in left-sided colon cancer. RAS-wild-type (RAS-wt) left-sided mCRC [Colorectal cancer (CRC) originating from the right and left side of the colon differs in embryologic, epidemiologic, genetic and molecular aspects –4. In thded mCRC .In terms of mCRC, the rectum has been commonly categorised as part of the left-sided colon in previous trials and research and treated according to the same treatment principles described in the ESMO and NCCN guidelines , 16. HowThe definition of rectal cancer itself, which could differ surgically, anatomically, and/or biologically, remains inconclusive , 23. AccIn our study, we aimed to compare the efficacy of anti-EGFR therapy in treating metastatic middle/low rectal cancers and left-sided colon cancers. Right-sided colon cancers were also added for a complete analysis of the impact of primary tumour locations. CRC data from The Cancer Genome Atlas (TCGA) were analysed to validate our clinical results at a molecular level.RAS and BRAF mutations were excluded due to their unsuitability for anti-EGFR treatment. Patients were classified based on their primary tumour locations, including the right-sided colon , the left-sided colon , and the middle/low rectum . Patients with different primary tumour locations were treated with chemotherapy plus anti-EGFR (cetuximab or panitumumab) or anti-vascular endothelial growth factor  agents. They were divided into two groups based on their treatment sequences: one (the first-line group) receiving anti-EGFR first and the other (the non-first-line group) receiving anti-EGFR following anti-VEGF. All patients were administered at least two cycles of anti-EGFR regimens. The inclusion flowchart is presented in detail in Supplemental Fig. th Edition stage at first presentation (AJCC stage), metastasectomy , pathology, histological grade, mucinous component, signet cell component, lymphovascular invasion status, perineural invasion status, carcinoembryonic antigen  level and carbohydrate antigen 19-9  level, was collected. All materials and protocols in this study were compliant with the Declaration of Helsinki and Good Clinical Practice guidelines. The study protocols were approved by the ethics committee and institutional review board of the Taipei Veterans General Hospital.This is a retrospective large-scale cohort study. Data were collected between May 2005 and December 2019 at the Taipei Veterans General Hospital, Taiwan. Patients with pathologically confirmed mCRC were eligible for enrolment, while those with Median overall survival (OS) was defined as the time from diagnosis of metastasis to cancer death or loss to follow-up. Median progression-free survival (PFS) represented the time from the beginning of anti-EGFR treatment to disease progression, as confirmed by radiological images  or intolerable toxicity. The overall response rate  and disease control rate  of metastatic tumours were recorded based on the Response Evaluation Criteria in Solid Tumours (RECIST) version 1.1 . PatientIn order to clarify the genomic differences among left-sided colon, right-sided colon and rectal cancers, TCGA colorectal data were utilised as the training set to explore the differential expression of genomic features between left- and right-sided colon cancer. The significantly different expression of genomic features in the training set was compared with the potentially preventive genomic features in rectal cancer as a validation model.RAS or BRAF mutations, non-primary tumour samples or unspecified tumour locations were excluded from the evaluation .Statistical comparisons were based on nonparametric tests. The correlations between clinicopathological variables and treatment responses were analysed using the chi-square test or Fisher’s exact test, where appropriate. PFS and OS curves were calculated through the Kaplan–Meier method. As for univariable and multivariable analyses, Cox proportional hazards regression was used to determine the effects of primary tumour locations and particular factors and to obtain hazard ratios (HRs) with corresponding two-tailed 95% confidence intervals (CIs). Cox regression models were conducted in the subgroup analysis to evaluate the impact of anti-EGFR treatment in primary tumour locations and sequences stratified by baseline variables. A two-sided n = 125), the left-sided colon (n = 313) and the middle/low rectal (n = 171) tumour groups are presented in Table A total of 609 patients were included in this study. The baseline characteristics of patients in the right-sided colon n = 25, the lThe PFS, OS, ORR and DCR results from different groups based on primary tumour location and lines of anti-EGFR treatment are presented in Figs. P = 0.007), OS , and ORR  were significantly higher in the left-sided CRC group than in the middle/low rectal tumour group. Significantly longer PFS  and DCR  were also observed in patients with left-sided tumours compared to middle/low rectal tumours under non-first-line anti-EGFR therapy. Conversely, a comparable OS outcome was observed between left-sided tumours and middle/low rectal tumours  under non-first-line anti-EGFR therapy. The group of patients with right-sided CRC showed the worst PFS, OS, ORR and DCR in both first-line and non-first-line anti-EGFR treatment. The characteristics of treatment responders and non-responders among middle/low rectal cancer patients are summarised in Supplemental Table In terms of the first-line anti-EGFR therapy group, PFS  and DCR  between the left-sided colon and rectum groups under first-line anti-EGFR treatment  revealed no significant differences in PFS  had more SNP mutations than left-sided colon (n = 1944) or rectal (n = 379) tumours. There were more SNP overlaps between rectal and right-sided colon tumours (n = 590) than between rectal and left-sided colon tumours (n = 251). The genomic distribution and types of SNPs based on tumour locations are detailed in Fig. Genetic bioinformation in the mutation annotation format from 161 tumour samples with 1–100% SNP mutations was grouped by primary tumour location and visualised. The Venn diagram Fig.  and UpSeFor the epigenomic DNA methylation analysis, a volcano plot Fig.  and a liPrimary tumour location has been considered a useful biomarker to predict the prognosis and the treatment effects of bioagents in mCRC. Although the rectum is routinely grouped in the left-sided colon in current treatment guidelines, this large-scale retrospective study, nevertheless, demonstrates that anti-EGFR therapy has lower efficacy in metastatic middle/low rectal cancer than in left-sided colon cancer, potentially leading to better clinical practice principles for mCRC management.k-means clustering, k = 3) are presented in Fig. KRAS mutation rates in rectal cancer compared with right- and left-sided colon cancer were reported [In our TCGA investigation, distinctions in a genomic level between the rectal and the left-sided colon tumours are identified. The molecular differences serve some possible explanations to our clinical findings. In Fig. reported , 35. Leereported .RAS-wt mCRC patients and mentioned that the benefit of anti-EGFR agents against rectal cancers may be overestimated [P = 0.028) and ORR  of rectal tumours in the AVF2107g trial [Clinically, the differential efficacy of anti-EGFR therapy between the rectal and the left-sided colon tumours was proposed in the previous literature. The analysis of a Phase III trial of panitumumab (PICCOLO) reported similar PFS outcomes between rectal and right-sided colon cancers in stimated . Loupaki7g trial . Another7g trial . Several7g trial , 26, 28.P = 0.010) and ORR  than left-sided colon tumours, while no significant differences in PFS  and DCR  were seen in the first-line anti-EGFR treatment group [Analyses of left-sided colon and rectal tumours were also performed using the traditional cut-off point  with the aim of testing our hypothesis . Thus, the guiding principles for mCRC treatment selection according to primary tumour location, especially tumours in the middle/low rectum, need further evaluation and confirmation.In several previous studies, anti-EGFR as a first-line treatment has shown higher efficacy and a greater survival benefit than anti-VEGF in treating left-sided, patients . HoweverSubgroup analyses for all patients  remain to be investigated. Specific genomic features affecting the efficacy and resistance rate of anti-EGFR therapy should also be explored to demonstrate their relationship with primary tumour locations.In conclusion, the differential efficacy of anti-EGFR treatment between the middle/low rectal cancers and the left-sided colon cancers has been established. Moreover, TCGA genomic analysis partially supported our findings that rectal cancer is not a homogeneous group and that it shares features with both left-sided colon and right-sided colon cancer. The usefulness of primary tumour location for the basis of anti-EGFR treatment decisions warrants further exploration.Supplemental informationDataset 1


Here, an MST4‐pβ‐cateninThr40 signaling axis essential for intestinal stem cell (ISC) homeostasis and CRC development is uncovered. In response to Wnt3a stimulation, the kinase MST4 directly phosphorylates β‐catenin at Thr40 to block its Ser33 phosphorylation by GSK3β. Thus, MST4 mediates an active process that prevents β‐catenin from binding to and being degraded by β‐TrCP, leading to accumulation and full activation of β‐catenin. Depletion of MST4 causes loss of ISCs and inhibits CRC growth. Mice bearing either MST4T178E mutation with constitutive kinase activity or β‐cateninT40D mutation mimicking MST4‐mediated phosphorylation show overly increased ISCs/CSCs and exacerbates CRC. Furthermore, the MST4‐pβ‐cateninThr40 axis is upregulated and correlated with poor prognosis of human CRC. Collectively, this work establishes a previously undefined machinery for β‐catenin activation, and further reveals its function in stem cell and tumor biology, opening new opportunities for targeted therapy of CRC.Elevated Wnt/ β‐cateninThr40 signaling axis essential for maintaining intestinal stem cell (ISC) homeostasis is identified. In response to Wnt signal, MST4 directly phosphorylates β‐catenin at Thr40 to actively block the GSK3β/β‐TrCP‐mediated phosphorylation/degradation of β‐catenin. Genetic activation of the MST4‐pβ‐cateninThr40 axis exacerbates ISC‐driven colorectal cancer (CRC); and hyperactivation of this axis is clinically correlated with poor prognosis.A novel MST4‐p Subsequently, transit‐amplifying cells differentiate into various intestinal epithelial cells (IECs), including enterocytes, tuft cells, goblet cells, and enteroendocrine cells.To maintain tissue integrity and homeostasis, the intestinal epithelium is routinely renewed or rapidly repaired in the case of damage, a process driven by intestinal stem cells (ISCs) in mucosal glands known as crypts. Growing evidence revealed that only a group of cells within tumors, namely, cancer stem cells (CSCs), have full potential for tumorigenesis; these are the genuine drivers of tumor development. For example, Lgr5+ cells not only serve as stem cells for tissue renewal and regeneration in intestinal homeostasis, they can also fuel the pool of CSCs in intestinal tumorigenesis. It is now understood that multiple signaling pathways control both ISCs and CSCs during this process, among which the Wnt/β‐catenin signaling has been extensively investigated over the past several decades.The intestinal epithelium is frequently vulnerable to malignant transformation and neoplasm. Colorectal cancer (CRC) is the third most common cancer worldwide, and is the fourth most common cause of cancer‐related death globally.β‐catenin in the nucleus. For example, deficiency of the tumor suppressor adenomatous polyposis coli (APC) protein, an upstream component of the Wnt pathway, acutely activates β‐catenin in ISCs, thusly promoting the development of CRC. Depletion of β‐catenin has been shown to induce apoptosis and ablation of crypts in intestinal organoids. Yet currently the regulatory mechanisms of β‐catenin in ISC homeostasis and tumorigenesis are not fully understood.The evolutionarily conserved Wnt pathway has been well documented for its essential role in individual development, tissue homeostasis, and tumorigenesis. In particular, over 90% of CRC patients show aberrant activation of this pathway with sustained accumulation of the downstream transcription coactivator β‐catenin at T45 by CK1α primes the subsequent phosphorylation of β‐catenin at S33/37/T41 by GSK3β, leading to β‐TrCP‐mediated degradation of β‐catenin. In contrast, when the Wnt signal is present, it interacts with Frizzled family receptors and LRP5/6 co‐receptors, and these interactions free β‐catenin from the APC‐containing destruction complex, generating a nonphosphorylated form of β‐catenin that can shuttle between the cytoplasm and nucleus. After entering the nucleus, β‐catenin binds to and transactivates the transcription factor TCF, which induces the expression of downstream target genes. Beyond this classic scenario, a number of studies have hinted at additional mechanisms for the regulation of β‐catenin activity. To date, however, such alternative molecular mechanism essential for full activation of β‐catenin remains partially understood and yet to be established in vivo. Moreover, these ideas about an expanded scope of β‐catenin biology have not been examined in ISCs or CSCs, leaving any possible role in tissue homeostasis and tumorigenesis unexplored.In the absence of the Wnt signal, phosphorylation of  Despite their structural similarities, the MST4 kinase is evolutionarily more distant to other members. MST4 has been implicated in biological processes including cell polarity, migration, autophagy, oxidative stress, and inflammation, as well as tumorigenesis. For example, MST4 can phosphorylate Ezrin and thereby inducing cell polarity. Also, MST4 can directly phosphorylate TRAF6 in macrophage to prevent excessive inflammation, therefore avoiding tissue damage. Recently, we discovered that MST4 acts as a noncanonical branch of the Hippo pathway to limit stress‐induced activation of the transcriptional coactivator YAP during gastric tumorigenesis.The serine/threonine kinase MST4 belongs to the mammalian Ste20 family, members of which also include MST1/2 known as central kinases of the Hippo signaling pathway.β‐catenin, which phosphorylates β‐catenin at Thr40 to inhibit GSK3β‐mediated phosphorylation at Ser33, therefore preventing its interaction with β‐TrCP. In response to Wnt signaling, this MST4‐pβ‐cateninThr40 axis is required for the process that actively blocks β‐TrCP‐mediated degradation, causing subsequent accumulation and full activation of β‐catenin. Disruption of the MST4‐pβ‐cateninThr40 axis in mice not only sharply decreased the number of ISCs/CSCs, but also suppressed CRC development; while constitutive activation of this axis by either forced activation of MST4 kinase or mutation of β‐catenin mimicking Thr40 phosphorylation led to increased number of ISCs/CSCs and exacerbated tumorigenesis. Consistently, the MST4‐pβ‐cateninThr40 signaling was markedly enhanced in CRC and associated with poor prognosis. Overall, this work uncovered a Wnt signaling‐induced MST4‐pβ‐cateninThr40 axis essential for ISC‐mediated tissue homeostasis and tumorigenesis, opening a window for novel targeted therapies.In this study, we characterized MST4 as a direct bona‐fide kinase of 22.1β‐catenin is known to play a dominant role. We first analyzed transcription of MST kinases in CRC specimens from GEO Database (GSE 20842) and TCGA Database, and found in both cases that the expression levels of the MST4 kinase are significantly elevated in CRC samples compared with healthy tissues  of MST4 on β‐catenin. Consistent with MST4's positive regulation of Wnt signaling observed above, knockdown of MST4 in 3T3‐L1 cells substantially impaired Wnt3a‐induced nuclear translocation of β‐catenin  , and a phosphomimic T178E mutant (MST4TE). We first depleted endogenous MST4 based on sgRNA targeting of the MST4 UTR region in HEK293FT cells. As expected, MST4 deficiency abrogated Wnt3a‐induced transcription of AXIN2  assay in HEK293FT cells showed an interaction of endogenous MST4 with endogenous Wnt signaling key components LRP5/6, GSK3α/β, and β‐catenin but not E3 ligase β‐TrCP; note that Wnt3a stimulation especially enhanced this interaction of MST4 with β‐catenin ‐tagged MST4 and His‐tagged β‐catenin and analyzed their interaction using a pull‐down approach, and our results indicate that MST4 directly interacts with β‐catenin  of β‐catenin phosphorylated by MST4, we performed mass spectrometry‐based phosphoproteomics analysis, which identified several residues including Thr40, Thr556, and Thr653 as candidate sites  and purified the corresponding β‐catenin proteins. Our in vitro kinase assay showed that mutation of Thr40 nearly blocked β‐catenin phosphorylation by MST4, while mutation of T556 and 653 reduced such phosphorylation  in HEK293FT cells that were transfected with wildtype β‐catenin or β‐cateninT40A mutant. A total of 468 unique proteins were identified in our data analysis when using a protein false discovery rate equal to or lower than 1%. We then applied a manual thresholding approach and a probabilistic PPI prediction algorithm (SAINTexpress) to compute the most likely associations between each of these 468 proteins and β‐catenin (T40A). These methods yielded 40 high‐confidence candidates  database indicated that 25% of these proteins are cytoskeletal proteins and that 15% of them have annotated functions relating to protein degradation  and the other mimicking MST4 phosphorylation based on β‐catenin3A . We then obtained β‐catenin KO cells using CRISPR‐Cas9 method and performed a rescue assay in HEK293FT cells. Confirming the results of the in vitro kinase assay, we found wildtype β‐catenin rescued AXIN2 transcription, and β‐cateninT40D further increased AXIN2 expression  of MST4 on the activation of n Figure , as wellβ‐cateninThr40 axis actively prevents the phosphorylation of β‐catenin by GSK3β and therefore blocks β‐TrCP‐mediated degradation, leading to the accumulation and full activation of β‐catenin.Taken together, these results demonstrate that in response to Wnt signaling, an MST4‐p2.5β‐cateninThr40 singling axis, we carried on to investigate this axis in vivo. Given the realized importance of the classic Wnt signaling in intestinal tissue homeostasis and tumorigenesis, we first detected the expression of MST4 and β‐catenin in intestine and found both MST4 and β‐catenin were highly expressed at the bottom of small and large intestinal crypts . Initial observations successfully experimentally confirmed the expected recombinant alleles of fl/flVillin‐Cre;Mst4 mice and absence of the MST4 protein after its Villin‐Cre‐mediated excision in IECs  in the small intestinal crypt cells of fl/flVillin‐Cre;Mst4 mice as compared to control littermates, so were large intestinal crypts . First, we selectively killed proliferating cells in intestinal epithelium using 5‐fluoruracil (5‐FU) as reported previously. Staining for Ki67 confirmed that proliferating cells completely disappeared after 2 days post intraperitoneal injection of 5‐FU . Consistent with the observations in fl/flVillin‐Cre;Mst4 mice, MST4 deficiency in Sox9+ cells markedly weakened the number of stem cells (Olfm4+ cells) and proliferating cells (Ki67+ cells)  Figure . Besidese Figure . Moreovee Figure , findinge Figure .β‐cateninThr40 axis in CRC, we first examined CRC tissue samples from mice treated with azoxymethane/dextran sodium sulfate (AOM/DSS). As shown in Figure fl/flSox9‐CreERT2;Mst4) significantly decreased colorectal tumorigenesis  of the MST4‐ps Figure . H&E stas Figure . MeanwhiC Figure .β‐cateninThr40 axis in ISCs/CSCs homeostasis and CRC development.Taken together, these results clearly demonstrate an essential role for the MST4‐p2.7β‐cateninThr40 axis, we created T178EMst4 conditional knockin mice and crossed them with Sox9‐CreERT2 mice  to endorse constitutive kinase activity of the MST4 in ISCs  using the CRC mice model. Results showed that constitutive activation of MST4 in ISCs/CSCs significantly increased AOM/DSS‐induced colorectal tumorigenesis  mimicking constitutive phosphorylation of β‐catenin by MST4 would cause functional and/or pathological consequences similar to those observed for constitutive activation of MST4. To test this point, we created T40DCtnnb1 conditional knockin mice and crossed them with Sox9‐CreERT2 mice  to specifically mimic phosphorylation of β‐catenin by MST4 in ISCs. Indeed, we observed more accumulation of β‐catenin and stronger signal for Ki67 staining in crypt cells of the T40D/+Sox9‐CreERT2;Ctnnb1 mice than in wildtype control mice  and crossed them with tdTomato mice  to genetically trace cells expressing MST4 during AOM/DSS‐induced colorectal tumorigenesis. The results of tdTomato lineage tracing showed that MST4+ cells were mainly found at the bottom of the crypts for normal part of mouse colon  staining of tissue microarrays showed that MST4 protein levels were sharply increased in tumor tissues of CRC patients when compared with that of healthy control , tumor stage (p = 0.0205), and lymph node metastasis (p = 0.0365). Also, the levels of β‐catenin Thr40 phosphorylation were positively correlated with tumor size (p = 0.046), tumor stage (p = 0.029), and lymph node metastasis (p = 0.035) (Table β‐catenin Thr40 phosphorylation (p = 0.011) (Table p = 0.0093) and β‐catenin Thr40 phosphorylation levels (p = 0.0204) were both negatively correlated with the 7.5‐year survival rate for CRC patients . Mechanistically, MST4 phosphorylates β‐catenin at Thr40 to competitively inhibit GSK3β binding/phosphorylating β‐catenin, preventing β‐TrCP from binding and degrading β‐catenin.The classic Wnt signaling triggers the release of β‐cateninThr40 axis confirms the importance of restricting β‐TrCP activity toward β‐catenin, and further highlights multiple‐layered regulations at this step. That said, we also observed, using IP‐MS, that the β‐cateninT40A variant underwent increased associations with cytoskeleton‐related proteins compared to wildtype β‐catenin. At this stage, we cannot rule out the possibility of MST4 regulating β‐catenin via some adhesion‐based mechanism . Further study will be needed to fully address the MST4‐mediated stabilizing mechanism of β‐catenin, especially in a context of multiple substrates  intertwined with each other. Nonetheless, our findings that the MST4‐pβ‐cateninThr40 axis is required for β‐catenin accumulation support the usage of Thr40 phosphorylation as a new marker for Wnt/β‐catenin signaling.Our finding of the MST4‐pβ‐cateninThr40 axis, we found that MST4 was hyperactivated in both mouse and human CRC tissues, and that MST4 expression levels were positively correlated with CRC progression but negatively correlated with CRC patient survival. Importantly, the levels of phosphorylation of Thr40 of β‐catenin were dramatically increased, as detected by using a fully verified homemade Thr40‐phosphor‐specific antibody in CRC patients, indicative of hyperactivation of the MST4‐pβ‐cateninThr40 signaling in these patients. In this regard, whether the levels of Thr40 phosphorylation of β‐catenin could be considered as a diagnostic marker for CRC warrants further investigation using a larger sample size, and perhaps different subtypes of CRC.Supporting a protumorigenic role of the MST4‐pβ‐catenin activity in stem cell biology, our work established an essential role of the MST4‐pβ‐cateninThr40 axis in maintaining ISCs/CSCs homeostasis in contexts of both physiology and tumorigenesis. Yet whether and how this axis governs other types of stem cells warrants further investigation. As aberrant activation of the β‐catenin in ISCs promotes adenomatous growth, it is likely that the MST4‐pβ‐cateninThr40 axis in ISCs became hyperactivated during tumorigenesis, and then directly promote the expansion of CSCs during CRC development. Supporting this notion, human and mouse CSCs indeed have severe upregulation of the MST4‐pβ‐cateninThr40 axis. More importantly, multiple means of genetic manipulation of this axis specifically in ISCs markedly altered the progression of CRC in mice models. For example, constitutive activation of MST4  or heterozygous mutation of β‐catenin T40D mimicking its phosphorylation by MST4 in ISCs substantially increased the number of ISCs/CSCs and exacerbated CRC. Thus, hyperactivation of the MST4‐pβ‐cateninThr40 axis may represent a critical event for the transition from ISC‐mediated homeostasis to CSC‐mediated tumorigenesis. At this stage, we have not yet observed aberrant crypt foci or adenoma in T40DCtnnb1 mutant mice, possibly due to the heterozygosity of the mutation. Whether homozygous mutation of β‐catenin T40D would cause autonomous colorectal adenoma warrants further investigation in the future.Consistent with the importance of Stk26‐Cre;R26‐tdTomato mice suggest a subpopulation of ISCs with high levels of MST4 might develop into colorectal cancer stem cells. It would also be intriguing to assess, potentially using an organoid system, the stemness of isolated CRC cells expressing high levels of MST4. In addition to characterizing the potential function of the MST4‐pβ‐cateninThr40 axis in CSCs, another unresolved but important question concerns the molecular mechanisms for dysregulation of this axis during CRC development.Currently, the cell origin of CRC and CRC stem cell are not well understood. Our current work suggests cells expressing high levels of MST4 could represent an origin of CRC. Genetic tracing of MST4‐expressing cell lineage via  Besides, our previous study observed that MST4 phosphorylates YAP and regulates Hippo/YAP signaling to suppress gastric tumorigenesis. Here, in the intestinal crypts of mice, we did not observe an apparent effect of MST4 on YAP under physiological condition; but did observe a positive role of MST4 on YAP protein levels during AOM/DSS‐induced tumorigenesis (data not shown). Thus, the regulatory function of MST4 appears to be highly context‐dependent. Different mice models could be useful for gaining overall insights into the pathological functions of MST4. In addition to the AOM/DSS and Apc mutation/knockout‐induced CRC mice models, we created here in this work a T40DCtnnb1 conditional knockin mice, which could mimic hyperactivation of β‐catenin and therefore possibly be used for modeling CRC.Finally, it is worth mentioning that MST4 may phosphorylate multiple substrates, and could therefore exert in a context‐dependent manner complex influences and orchestrate different pathways during tumorigenesis. For example, we previously reported that MST4 targets the E3 ligase TRAF6 to limit innate inflammatory responses.β‐cateninThr40 axis required for accumulation of β‐catenin, and demonstrated an essential role for this axis in controlling ISCs/CSCs homeostasis and colorectal tumorigenesis.In summary, this work uncovered an MST4‐p4floxedMst4 and T178EMst4 mice were generated using Cre/LoxP homologous recombination system with the principle, targeting strategies, and screening mechanisms briefly described in the previous study and Figure T40D/+Ctnnb1 mice were obtained by homologous recombination system and the exon 2–15 of Ctnnb1 was replaced by mutated exon 2–15 (T40D mutation at exon 3) by Shanghai Model Organisms Center . The experimental procedure and approaches were previously outlined. Chimeric mice were crossed with Flpe‐deleter mice to obtain floxedMst4 and T178EMst4 mice. Villin‐Cre mice were obtained from Shanghai Model Organisms Center . Stk26‐Cre mice were developed using Cre/LoxP recombination in Shanghai Model Organisms Center . min/+Apc mice were generated as described previously. Lgr5‐EGFP‐ires‐CreERT2 mice,Sox9‐CreERT2 mice, and flox/+Apc mice were kindly provided by Dr. Yi Arial Zeng , Dr. Bin Zhou , and Dr. Zhengquan Yu , respectively. All mice of both sexes were of a C57BL/6 background, and except where noted were 2 months old when subjected to the beginning of the experiments. The mice were cultured in automated watered and ventilated cages in a specific pathogen‐free environment on a 12 h light/dark cycle. All of the mice were randomly allotted to experimental groups in all experiments, which were conducted based on the guidelines of the Institutional Animal Care and Use Committee of the Institute of Biochemistry and Cell Biology.The approval ID for animals breeding was SIBCB‐NAF‐14‐004‐S329‐023 issued by the Animal Core Facility of Center for Excellence in Molecular Cell Science (CAS), Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.The intestinal tracts were flushed with cold PBS, followed by 4% paraformaldehyde at 4 °C, and embedded in paraffin wax for sectioning. The sections (3 µm) were stained with indicated primary antibodies. The intestinal fractions were from at least four mice per group. Images were captured using laser confocal scanning microscopes equipped with either Olympus FV3000  60× NA 1.36 oil immersion objective, or Zeiss 880  20× objective and 63× NA 1.4 oil immersion objective. Four lasers were used with wavelengths of 405, 488, 568, and 647 nm. Images were processed by using Imaris software and ImageJ software. and analyzed with the aid of an Olympus BX51 dissecting microscope .For detailed histologic analysis, sections were stained with hematoxylin & eosin (H&E) or periodic acid–Schiff,Axin2 in intestinal tissues was detected in situ hybridization followed by the manufacturer's instructions . Briefly, the tissue sections were deparaffinized, retrieved, and then hybridized with Axin2 specific probes . Then, the signal of hybridized probes was amplified using a cascade of signal amplification molecules and further visible by RED dye‐labeled probes. Subsequently, the sections were redyed with Gill II hematoxylin and mounted with VectaMount Permanent Mounting Medium , and photographed using an Olympus BX53 photomicroscope .The transcription of  Mice at ages of about eight weeks were injected intraperitoneally with 10 mg kg−1 AOM . Then, the mice were treated with 1 g/100 mL DSS in drinking water  for 7 days after 7 days of AOM injection. After that, mice were given regular drinking water for 7 days, followed by three cycles  for T178E/T178ESox9‐CreERT2;Mst4 mice and T40D/+Sox9‐CreERT2;Ctnnb1 mice; four cycles for Sox9‐CreERT2;fl/flMst4 mice. At day 75  or at day 98 , the mice were sacrificed at the end of the model. There were at least four mice per group in the experiments. Section of colons were fixed in 4% paraformaldehyde and embedded in paraffin for histopathological analysis.An AOM‐DSS induced colorectal cancer in mice was generated as described previously with modifications.−1 penicillin, and 100 µg mL−1 streptomycin at 37 °C with no CO2 in a humidified incubator ; the other cells were grown in DMEM medium  supplemented with 10% heat‐inactivated FBS, 100 µg mL−1 penicillin, and 100 µg mL−1 streptomycin at 37 °C with 5% CO2 in a humidified incubator . All of the cells were obtained from the indicated sources and were passaged for less than 3 months. The identities of cell lines SW480 and MC38 were confirmed by carrying out fingerprinting . The cells were regularly detected for mycoplasma using MycoAlert Mycoplasma Detection Kits .HEK293FT, SW480, MC38, and 3T3‐L1 cells  were obtained from the Shanghai Life Academy of Sciences cell library . SW480 cells were cultured in L‐15 medium  supplemented with 10% heat‐inactivated fetal bovine serum (FBS), 100 µg mLtN/N0, where tN is the value of ATP contents at time t h and N0 is the value of ATP contents at time 0 h.For cell proliferation assay, cells (2000 per well) were seeded in 96‐well plates after siMST4 RNAs transfection for 48 h. Then, cell numbers were measured and analyzed using an ATP‐based CellTiter‐Lumi Plus kit (Beyotime) based on the manufacturer's guidelines. The intracellular ATP contents were detected using a BioTek Synergy NEO multidetector microplate reader (Thermo). Cell growth was analyzed using the equation fold change = −1 penicillin, 100 µg mL−1 streptomycin, 1× B‐27 (Invitrogen), 20 ng mL−1 basic FGF (Peprotech), and 20 ng mL−1 EGF (Peprotech) and seeded into 6‐well ultralow attachment plates  for three repeats after siMST4 RNAs transfection for 48 h. At day 10, the number of sphere (area of sphere ≥70 µm2) was calculated and analyzed.For sphere formation assay, cells were suspended with DMEM/F12 medium (Gibco) with 100 µg mL6 cells per mice, n = 5 per group) and MC38 cells  were respectively injected subcutaneously into BALB/c nude mice for at least four mice per group. After 4 days, the siRNAs were intratumorally injected every three days. The mice were sacrificed with CO2 after subsequent two weeks, and tumor volumes were then calculated as V = LL × Ls2/2 .Healthy male BALB/c nude mice were maintained in specific pathogen‐free conditions in available watered and ventilated cages on a 12 h light/dark cycle. During tumor formation, HCT116 cells (2 × 10α‐tubulin (T6199), and β‐actin (A2228) were acquired from Sigma . Those antibodies for Olfm4 (39141), β‐TrCP (4394), and HA (3724) were acquired from Cell Signaling Technology ; those for Ki67 (ab15580), Lgr5 (ab75732), and lysosome (ab2408) were from Abcam ; those for β‐catenin (610153) were from BD Biosciences ; those for SOX9 (AB5535) were from EMD Millipore ; those for GSK3α/β (sc‐7291) and Laminin A/C (sc‐7292) were from Santa Cruz ; and those for MST4 and phospho‐β‐catenin (Thr40) were produced by Shanghai Immune Biotech . Secondary antibodies for Alexa Fluor 568 goat antirabbit IgG (H+L) (A11011), Alexa Fluor 647 donkey antigoat IgG (H+L) (A21447), and Alexa Fluor 488 goat antirabbit IgG (H+L) (A11001) were acquired from Invitrogen .Specific antibodies for Flag (M3165), β‐catenin and its variants (Flag‐tagged) were subcloned into a pCDH1 vector. The siRNAs targeting of MST4, β‐catenin, and the negative control was designed and synthesized by Genepharma . The sequences of these siRNAs were: for human siMST4‐1, the forward was 5′‐GCCUGAUCCAAAGAAAGUATT‐3′, and the reverse was 5′‐UACUUUCUUUGGAUCAGGCTT‐3′; for human siMST4‐2 targeting the UTR region of MST4, the forward was 5′‐AUUGUUACAAUAUAGGCACCATT‐3′, and the reverse was 5′‐UGGUGCCUAUAUUGUAACAAUTT‐3′; for mouse siMst4‐1, the forward was 5′‐CCAGCAGCAGGGAAAGUAATT‐3′, and the reverse was 5′‐UUACUUUCCCUGCUGCUGGTT‐3′; for mouse siMst4‐2, the forward was 5′‐GGGAAUUACUGCUAUUGAATT‐3′, and the reverse was 5′‐UUCAAUAGCAGUAAUUCCCTT‐3′; for human siβ‐catenin‐1, the forward was 5′‐CUGGUUGUUAUGUGAUCAUTT‐3′, and the reverse was 5′‐AUGAUCACAUAACAACCAGTT‐3′; for human siβ‐catenin‐2 targeting the UTR region of β‐catenin, the forward was 5′‐CCCUAGCCUUGCUUGUUAATT‐3′, and the reverse was 5′‐UUAACAAGCAAGGCUAGGGTT‐3′; for human siβ‐catenin‐3 targeting the UTR region of β‐catenin, the forward was 5′‐GGGUAAAUCAGUAAGAGGUTT‐3′, and the reverse was 5′‐ACCUCUUACUGAUUUACCCTT‐3′; and for the negative control, the forward was 5′‐UUCUCCGAACGUGUCACGUTT‐3′, and the reverse was 5′‐ACGUGACACGUUCGGAGAATT‐3′.Flag‐MST4, its variants, and shMST4 were described previously. Briefly, HEK293FT cells were transfected with lentiCRISPRv2‐sgRNAs plasmids for 48 h and selected using 2 µg mL−1 puromycin for 1 week. Then, knockout efficiency of the indicated proteins was detected using western blotting assay. MST4 sgRNAs were previously described. And CTNNB1 sgRNAs were designed based on human GeCKO library (https://www.genscript.com/CRISPR‐gRNA‐library.html). The sequences of CTNNB1 sgRNAs were: for human sgCTNNB1‐1, the forward was 5′‐GAAACAGCTCGTTGTACCGC‐3′, and the reverse was 5′‐GCGGTACAACGAGCTGTTTC‐3′; for human sgCTNNB1‐2, the forward was 5′‐GACACGCTATCATGCGTTCT‐3′, and the reverse was 5′‐AGAACGCATGATAGCGTGTC‐3′; for human sgCTNNB1‐3, the forward was 5′‐AATGCAGTTCGCCTTCACTA‐3′, and the reverse was 5′‐TAGTGAAGGCGAACTGCATT‐3′.Knockout cells were obtained according to the guidelines as previously described.siRNAs were transfected using Lipofectamine RNAiMAX Reagent  with manufacturer's guidelines. And the plasmids were transfected by Lipofectamine 2000  based on the manufacturer's reference manual.For luciferase reporter assay, HEK293FT cells or SW480 cells were transfected with TOP, FOP, Renilla, and indicated plasmids for 48 h for at least four repeats. Then, cells were lysed and the luciferase activities were measured using the Dual‐Luciferase Assay System (Promega).ACTB or Actb was used as an internal control. The primers used were:hMST4: 5′‐ATCTTGTGCAAACCCTGAGTTG‐3′ (F),5′‐TTCAATCGCCTGATTCCTGCT‐3′ (R);hAXIN2: 5′‐AGAAGCAGCAGATTGATTCC‐3′ (F),5′‐CCCCCATTACTCATGTAAGC‐3′ (R);hCCND1: 5′‐ CAATGACCCCGCACGATTTC ‐3′ (F),5′‐ CATGGAGGGCGGATTGGAA ‐3′ (R);hACTB: 5′‐ ATCATGAAGTGTGACGTGGA‐3′(F),5′‐ CTCAGGAGGAGCAATGATCT‐3′(R);mMst4: 5′‐TTTGGGAATTACTGCTATTGAACTT‐3′ (F),5′‐TGGATGCATGTCAGAATTCG‐3′ (R);mAxin2: 5′‐ATGAGTAGCGCCGTGTTAGTG‐3′ (F),5′‐GGGCATAGGTTTGGTGGACT‐3′ (R);mCcnd1: 5′‐GCGTACCCTGACACCAATCTC‐3′ (F),5′‐CTCCTCTTCGCACTTCTGCTC‐3′ (R);mLgr5: 5′‐CCTACTCGAAGACTTACCCAGT‐3′ (F),5′‐GCATTGGGGTGAATGATAGCA‐3′ (R);mSox2: 5′‐GCGGAGTGGAAACTTTTGTCC‐3′ (F),5′‐CGGGAAGCGTGTACTTATCCTT‐3′ (R);mSox9: 5′‐CAAGCGGAGGCCGAAGA‐3′ (F),5′‐CAGCTTGCACGTCGGTTT‐3′ (R);mActb: 5′‐ GGCTGTATTCCCCTCCATCG‐3′(F),5′‐ CCAGTTGGTAACAATGCCATGT ‐3′(R).Note: F, forward; R, reverse.Total RNAs were extracted from cells using Total RNA Extraction Reagent , and then cDNA was reversed using the HiScriptII Q Select RT SuperMix  with the manufacturer's guidelines. Real‐time PCR was performed using Real‐time PCR Master Mix  according to an Applied Biosystems Step Two Real‐Time PCR System . fl/flMst4 mice and fl/flVillin‐Cre;Mst4 mice. Tissues of the small intestines were dissected into 2–5 mm2 fractions and placed into 1.5 mL Eppendorf tubes. They were incubated in Ca2+‐free, Mg2+‐free HEPES buffer containing 20 × 10−3m EDTA and collagenase II at 37 °C for 30 min pulsed every 10 min with Vortex. The incubated tissues were subjected to low‐speed centrifugation, and IECs were collected from the centrifuged material and washed in cold HEPES (10 × 10−3m) buffer. RNA was extracted from three biological replicates.For RNA sequence, samples were prepared from small intestinal epithelia of RNA quality was assessed using an Agilent Bioanalyzer 2100  and sent for library preparation. Total RNA was amplified, labeled, and purified by RNAClean XP Kit  and RNase‐Free DNase Set  following the manufacturer's guidelines. Then, the purified RNA was sequenced by Illumina Hiseq X10 platform by Majorbio Genomics , followed by analyzing the sequence data using GRCm38.p5 genome database.The GEO accession number for the high‐throughput sequencing reported in this paper are GSE132565.−3m Tris·HCl, pH 8.0, 100 × 10−3m NaCl, 0.5% Triton X‐100, and 1 × 10−3m EDTA supplemented with Cocktail on shaking bed in 4 °C for 20 min. Then, the lysates were obtained by 4 °C centrifugation 13 000 rpm for 30 min and boiled with 5× SDS loading buffer. Finally, the proteins were separated by SDS‐PAGE, transfected to a PVDF membrane (Bio‐Rad), and blotted with indicated antibodies. Western blotting images were captured by the Mini Chemiluminescent Imaging and Analysis System .Whole cell contents were extracted from cells using western blotting buffer containing 20 × 10−3m Tris·HCl, pH 7.5, 100 × 10−3m NaCl, 0.5%NP‐40, and 1 × 10−3m EDTA supplemented with Cocktail. Then, the proteins were incubated with Flag‐beads (GenScript) or indicated antibodies together with protein A/G beads (Santa Cruz Biotechnologies) for overnight at 4 °C. And the beads were washed with IP buffer for three time and boiled with 1× SDS loading buffer. The interacted proteins were detected using indicated primary antibodies as stated above.For immunoprecipitation assay, cells lysates were obtained using immunoprecipitation (IP) buffer containing 20 × 10β‐catenin was defined following previous literature (see main text), and experimentally prepared by using ProteoExract Native Membrane Protein Extraction Kit  as previously described according to the manufacturer's instructions. Briefly, the cytoplasmic contents except for membrane proteins were obtained using Extraction Buffers I. Then, cytoplasmic proteins containing free β‐catenin was boiled with 5× SDS loading buffer, and free β‐catenin was detected using β‐catenin antibodies.Free 6‐tagged MST4 and β‐catenin or MBP‐tagged MST4 were cloned and expressed in pET28a vectors. Whole prokaryotic contents were induced and expressed by 0.5 × 10−3 m isopropyl‐β‐d‐thiogalactopyranoside (IPTG) in Terrific Broth medium at 16 °C in Escherichia coli BL21 (DE3) CodonPlus cells with the optical density of a sample at a wavelength of 600 nm (OD600) of 0.6–1.0. For His6‐tagged MST4 and β‐catenin proteins, the cells were collected by centrifugation after induction for 16 h and resuspended with lysis buffer A . Then, the cell lysates were obtained by a high‐pressure homogenizer and the supernatants were incubated with Ni Sepharose column  pre‐equilibrated with lysis buffer A. Finally, the proteins were eluted with lysis buffer A with 400 × 10−3m imidazole. For MBP‐tagged MST4, the cells were resuspended in lysis buffer B . And the contents were incubated to amylose resin (New England Biolabs) columns pre‐equilibrated with lysis buffer B. Then, the proteins were eluted with buffer B with 20 × 10−3m maltose. The proteins were finally purified with size‐exclusion chromatography in an SEC buffer containing 20 × 10−3m HEPES, pH 7.5, and 100 × 10−3m NaCl for further experiments.His−3 m HEPES pH 7.5, 100 × 10−3m NaCl, and 1 × 10−3m dithiothreitol) at 4 °C for 1 h. Then, the beads were washed and eluted with the buffer adding 20 × 10−3m maltose. The input and output samples were measured and analyzed with Coomassie Brilliant Blue staining or immunoblotting.MBP‐tagged proteins coupled onto amylose resin beads were incubated with the indicated proteins in the buffer  was added into the reaction system and incubated for 1 h at room temperature. The reaction mixture was detected using western blotting assay and the phosphorylated proteins were detected by a thiophosphate‐ester‐specific antibody . In addition, Flag‐tagged β‐catenin (WT and mutants), GSK3β, and CK1α proteins were immunoprecipitated and purified from HEK293FT cells after being transfected with the indicated plasmids for 48 h using Flag beads. For in vitro kinase assay, purified β‐catenin proteins were first dephosphorylated as described previously, and then mixed with GSK3β and CK1α proteins in an ATP‐containing buffer, and further incubated for 30 min at 30 °C.In Vitro kinase assay was performed according to the Kinase Reaction and Alkylation Protocol (abcam) as described previously.β‐catenin proteins were co‐incubated to induce phosphorylation of β‐catenin in the buffer containing 50 × 10−3m Tris pH 7.5, 1 × 10−3m ATP, 10 × 10−3m MgAc2, and 0.1 × 10−3m EGTA for 30 min at 30 °C. The reaction mixture was precipitated with TCA solution and subsequently dissolved in 8 m urea and 100 × 10−3m Tris, pH 8.5. Then, mass spectrometry analysis was carried out in National Center for Protein Science Shanghai . For mass spectrometry (MS) assay of β‐catenin mutants, HEK293FT cells were transfected with the indicated plasmids and the cell lysates were immunoprecipitated using Flag‐beads. Then, the beads were washed for three times and resuspended in 8 m urea and 100 × 10−3m Tris, pH 8.5. Finally, the mixture was measured and analyzed in Shanghai Majorbio Bio‐Pharm Technology Co., Ltd. .Purified MST4 and β‐catenin mutants were coexpressed in HEK293FT cells for 48 h. Then, cytoplasmic contents were extracted from cells using IP buffer and incubated with HA antibodies and protein A/G beads (Santa Cruz Biotechnologies) for overnight at 4 ℃. Subsequently, the ubiquitination of β‐catenin was detected using western blotting assay with indicated primary antibodies.The proteins of HA‐Ub (K48) and Flag‐Tissue microarray sections were prepared by Outdo Biotech . All samples used were derived from 90 CRC patients approved by the Ethics Committee of Taizhou Hospital of Zhejiang province, China. Detailed clinical information was collected from all patients except 6, whose pathological records for TNM stage were lost. The median follow‐up period was 52 months .β‐catenin (Thr40) antibody . Evaluation of immunohistochemical staining was carried out by two independent pathologists blinded to the medical records of patients. Results were assessed based on the extent  and intensity (0–3) of staining protein in 200 examined cells. Subsequently, the IHC result was calculated as 0–1, negative (−); 2–4, weakly positive (+); 5–8, moderately positive (++), and 9–12, strongly positive (+++).Tumor microarray sections were incubated with an anti‐MST4 antibody (1:100 dilution) or anti‐p‐α = 0.1), and then two‐tailed unpaired Student's t‐test was used for comparing between two groups. One‐way ANOVA and post hoc Bonferroni t‐test were performed for multiple comparisons. The n.s., *, **, and *** represent no significance, p > 0.05, p < 0.05, p < 0.01, and p < 0.001, respectively. Data represent means ± SD from two to three independent experiments. For transcription analysis, the data were acquired from at least three repeats. For quantitative analysis of immunofluorescence, the data were randomly captured and analyzed from murine intestinal tissues of at least four samples. For sequencing analysis, the mice were selected at random for three repeats to acquire RNA sequence data. The mice were selected at random for at least four samples to perform tumor number and size analysis of AOM/DSS‐induced mice model. The values of sample size were shown in figure legends and had no statistical methods to preprocess the exact values. The Fisher's Exact test was used to determine the interaction between two categorical variables. The correlation between two continuous variables was calculated by the Spearman's rank test. The Kaplan–Meier method and the log‐rank test were performed to analyze patients’ overall survival.Statistical analysis was performed using GraphPad Prism 8  and SPSS statistical software ver. 13.0.1 . Normal distribution of continuous data was evaluated by the Kolmogorov–Smirnov test. The values were subjected to a standard normal distribution (The authors declare no conflict of interest.β‐catenin. M.C., F.W., J.J., L.H., and W.B. carried out biochemical analysis. M.L. and C.D. provided the CRC patient samples and performed related analyses. F.C. and M.W. carried out mice genotyping. D.W., L.L., Y.Z., S.X.H., X.L., J.L., and L.A. discussed and analyzed the data. H.Z., S.J., and Z.Z. designed the experiments and wrote the manuscript. Z.Z. and S.J. supervised the project.H.Z., M.L., and C.D. contributed equally to this work. H.Z. and S.J. performed most of the cellular experiments and in vivo analyses. M.C., Y.T., and W.W. made constructs for Supporting InformationClick here for additional data file.


Thermally conductive and electrically insulating materials have attracted much attention due to their applications in the field of microelectronics, but through-plane thermal conductivity of materials is still low at present. In this paper, a simple and environmentally friendly strategy is proposed to improve the through-plane thermal conductivity of epoxy composites using a 3D boron nitride (3D-BN) framework. In addition, the effect of filler sizes in 3D-BN skeletons on thermal conductivity was investigated. The epoxy composite with larger BN in lateral size showed a higher through-plane thermal conductivity of 2.01 W/m·K and maintained a low dielectric constant of 3.7 and a dielectric loss of 0.006 at 50 Hz, making it desirable for the application in microelectronic devices. With the rapid development of 5G, artificial intelligence and the Internet of Things, there is a significant demand for thermally conductive and electrically insulating materials for microelectronic devices, which are suffering from elevated operation temperature ,2,3,4. T2O3 [3N4 [The composite strategy has been deemed as the most effective method to enhance thermal conductivity by incorporating ceramic fillers, such as AlN ,9,10, Al2O3 ,12,13, S2O3 [3N4  and BN [2O3 [3N4 ,16. Espe2O3 [3N4 ,18,19,202O3 [3N4 ,22,23. F2O3 [3N4  used nan2O3 [3N4 ,27,28,292O3 [3N4 ,31. UnfoIn this work, we prepared a 3D network skeleton with aligned BN in an epoxy-based composite and investigated the correlation between the size and aspect ratio of BN and thermal conductivity. Additionally, we proposed a technique for permeating the epoxy resin into the 3D-BN skeleton under vacuum conditions. This method can ensure that the 3D-BN network frame is fully filled with the epoxy solution to reduce the voids in the composites. Another important preparation process is to observe the sample along the Z-axis to obtain directional BN across the plane. The epoxy composite with a bigger BN size showed a higher through-plane thermal conductivity of 2.01 W/m·K and lower dielectric constant and dielectric loss, which is suitable for the application of microelectronic devices. Moreover, the preparation of the polymer composites with 3D-BN is simple, eco-friendly and scalable, and the TIM is promising for practical applications in microelectronic packaging in the next generation of electronic devices.Bisphenol A-epoxy resin  was purchased from HEXION. Methyl tetrahydrophthalic anhydride (MTHPA) and N,N-dimethylbenzylamine (BDMA) were supplied from Aladdin Industrial Corporation , which were employed as curing agent and accelerator, respectively. Isopropyl alcohol (99%) was purchased from Aladdin Industrial Corporation . BN platelets in various sizes were supplied by Dandong Chemical Research Institute Limited Company , and the larger ones, middle ones and smaller ones were abbreviated as LBN, MBN and SBN, respectively.The 3D-BN framework was prepared by a typical vacuum filter self-assembly process . In brieα) and specific heat (Cp) were tested via laser flash analyzer ). Densities (ρ) of specimens were estimated by measuring their volume and mass. TC values of specimens were obtained by a typical equation: TC = α × Cp × ρ. The specimen was sliced into a square sheet with 10 mm in side length and 1 mm in thickness. Subsequently, graphite coating was deposited onto the surface of samples. For dielectric measurements, permittivity and dielectric loss were measured via dielectric analyzer ). Gold coating was deposited onto the surface of samples via an auto sputtering device. Thermal gravimetric analyzer and differential scanning calorimeter ) were performed at the heat rate of 10 °C/min in air and nitrogen atmosphere, respectively. Surface morphology and microstructure of fillers and their composites were investigated by a field emission SEM  and X-ray diffractometer . The surface temperature images of the samples were obtained by an infrared thermal imager . The dynamic mechanical analysis was performed by a dynamic mechanical analyzer  [Thermal diffusivity  . SpecimeZ-axis direction. Finally, the epoxy composites with vertically-oriented BN platelets (perpendicular to the planar direction of sample) were obtained. In these kinds of epoxy composites, the BN skeleton via vacuum filtration self-assembly has a three-dimensional network with interconnected structures. Three kinds of BN platelets  were used. The morphology and size of the BN platelets are exhibited in Z-axis (through-plane) direction. In addition, during the vacuum casting process, few voids were observed in these epoxy composites due to the complete removal of bubbles in the vacuum environment, as shown in The surface morphology and structure of V-3D-BN/epoxy composites prepared by various methods were compared and studied. As revealed by the SEM images in In general, the thermal conductivity of inorganic–organic composites is basically determined by the structure, dispersion, loading capacity, surface properties of inorganic materials and the thermal resistance between the interfacial filler and the polymer matrix ,39,40,41The 3D network with oriented and interconnected structure can form more thermal transport channels and increase phonon transport and reduce interface thermal resistance. Compared with V-3D-LBN/epoxy, A-3D-LBN/epoxy showed lower TC due to the existence of voids and bubbles, of which the void percentage was calculated from the TGA results in To investigate the effect of sizes of BN on its orientation, the directions and angles of BN in the composite were demarcated to quantitatively analyze the orientation degree of BN. The 90° and 0° represented perpendicular and parallel to sample surface, respectively. Based on some previous references ,44, the Based on the importance of dielectric properties, glass transition temperature and mechanical characteristics for microelectronic packaging, these properties were investigated and evaluated for their practical applications. In this work, we presented a simple and eco-friendly strategy via filtrating self-assembly and vacuum infiltration to obtain the 3D-BN network skeleton with alignment in the through-plane direction, and we investigated the effect of BN sizes on through-plane TC in the epoxy matrix. Specifically, the through-plane TC values of A-3D-SBN/epoxy, A-3D-MBN/epoxy and A-3D-LBN/epoxy were 1.10, 1.24 and 1.47 W/m·K, respectively. BN with a bigger lateral size in the epoxy matrix was easier to connect to each other, leading to less phonon scatter. More importantly, a technique of epoxy pouring in a vacuum condition was developed to make bulk BN be fully filled the with epoxy solution. The V-3D-LBN/epoxy composite of 15.4 vol.% BN had a through-plane TC of 2.01 W/m·K, comparatively low permittivity of 3.7 and a loss tangent of 0.006 at 50 Hz, which is suitable for the application of microelectronic devices. This work shows that BN with larger transverse dimensions is more likely to improve thermal conductivity, so the transverse dimensions of two-dimensional materials should be emphasized and pursued in subsequent studies, rather than focusing only on obtaining thinner two-dimensional materials. It is significantly important for practical application in the next generation of microelectronic devices to study the effect of size of two-dimensional materials on thermal conductivity because it is not just related to performance, but also related to the cost of materials.


However, the specific link between them in SCI is unclear. In addition, targeting posttraumatic astrocyte apoptosis is crucial for improving neural degeneration and locomotor function. Therefore, in this article, we studied the relationship of β-catenin and SIRT1 using in the SCI rat model and primary astrocyte treated with hydrogen peroxide (H2O2) or lithium chloride (LiCl). Results showed that after SCI, SCI area and motor function recover over time, and β-catenin is gradually increased to the seventh day and then in turn decreases until 4 weeks, positively correlated with cell apoptosis. The expression of SIRT1 and downstream FOXO4 gradually increased, and β-catenin is negatively correlated with SIRT1 expression. Moreover, treatment with H2O2 in primary cultured astrocyte significantly increased β-catenin and Caspase-3 expression, while decreased SIRT1 and Forkhead box O- (FOXO-) 4. The immunofluorescence results are consistent with this. Administration of LiCl further aggravates the above results. These findings suggest that SIRT1 is negatively correlated with β-catenin in SCI, which promotes the apoptosis of motor neuron cells, which may be related to the participation of FOXO4.Sirtuin-1 (SIRT1) has anti-inflammatory and antioxidant effects and has been reported to be involved in spinal cord injury (SCI). Wnt/ SCI -catenin  and Sirt-catenin  are invoin vivo and in vitro models of SCI through miR-494. There are also studies show that resveratrol can protect SCI by activating SIRT1/AMPK signaling pathway-mediated autophagy and inhibiting apoptosis [SIRT1 is a protein deacetylase of class III that relies on nicotinamide adenine dinucleotide (NAD) . SIRT1 ppoptosis .β-catenin signaling pathway is implicated in the growth of the nervous system, affecting cell patterning and proliferation, neuronal connectivity and survival, cell adhesion, cell motility and polarity, and axon guidance [β-catenin is negatively correlated with SIRT1 during neuronal apoptosis. And they found that upregulation of SIRT1 can inhibit hydrogen peroxide- (H2O2-) induced osteoblast apoptosis through the Forkhead box O (FOXO) 1/β-catenin pathway [β-catenin signal regulates apoptosis and extracellular matrix degradation of chondrocytes in osteoarthritis treated with resveratrol [β-catenin and SIRT1 as well as its role and underlying mechanism in regulating apoptosis after SCI, which may help to develop effective treatment plan for SCI.The Wnt/guidance , 16. Rec pathway . SIRT1 cveratrol . This reA total of 90 mature male Sprague–Dawley (SD) rats (180-220 g) were acquired from the Experimental Animal Centre of Liaoning Medical University. And all rats were kept in a room controlled temperature with a cycle of 12 h light/dark cycle exposure. All experiment processes abode by the Care and Use of Laboratory Animals released by the US National Institutes of Health.N = 16 per group): sham group, 3 days after SCI group (3dSCI), 7 days after SCI group (7dSCI), 14 days after SCI group (14dSCI), 28 days after SCI group (28dSCI), sham group for laminectomy, and the other group using a modified rat model hammer method .Rats were separated to 5 groups randomly . Spinal cords of neonatal rat were ejected from the vertebral column, treated with 0.25% trypsin  for 15 min, followed by mechanical trituration in DMEM +10% heat-inactivated fetal bovine serum . After centrifugation at 800 rpm for 5 min, the cells were suspended in DMEM, containing FBS and 1% penicillin/streptomycin . The culWe used locomotor rating scales of Basso-Beattie-Bresnahan (BBB)  to evaluμg) were transferred onto polyvinylidene fluoride (PVDF) membrane. At room temperature, the membrane was blocked with 5% skimmed milk in the Tris-buffered saline with TBST for 2 h, and then, it was incubated at 4°C with the primary antibody overnight. The following primary antibody was contained in the hybridization solution: anti-β-catenin , anti-FOXO4 , anti-Caspase-3 , and anti-SIRT1 . TBST was used to rinse the membrane three times, which was then incubated with the secondary antibody  for 2 h at room temperature. ChemiDoc-ItTMTS2 Imager  was employed to develop immunoreactive band, and the ImageJ software  was applied for analyzing. The above steps were repeated three times.RIPA lysis buffer  was used for dissolving the prepared tissue, followed by the quantification of the final protein concentration (2 g/L) by BCA kit . After being separated by SDS-PAGE, the proteins  for 5 seconds for differentiation, which had been stained by eosin after washing with deionized water for half of an hour. Again, the slices were rinsed and dehydrated in gradient alcohol. In the final, the slices were crystal clear, fixed with xylene to the upper neutral balsam.Following staining with hematoxylin for 10 s, we quickly washed portrait sections of 5 μm into the solution of chloroform and ethanol (1 : 1) and rehydrated the slices through decreasing the concentration of alcohol  in order to acknowledge the numbers of neurons and notice the changes of morphology in different groups. Cresyl violet solution of 0.1% was used to rinse immerse the slices for 40 minutes at 40°C, and then, the slices were rinsed with deionized water and applied with 95% alcohol for differentiation, 100% alcohol for dehydration, and xylene for 5 minutes for being transparent. In the final, cover glass was covered on the slices using DPX.We immersed the coronal slices of 5 μm were dried in air for 2 h at the room temperature, followed by the incubation with normal goat serum of 5% for 2 h at the room temperature, and the cells were also treated. The primary antibodies were as follows: anti-β-catenin  and anti-SIRT1 , and the secondary antibodies were as follows: FITC goat anti-mouse IgG  and Cy3 goat anti-rabbit IgG . In the final, DAPI solution (1 : 800) was used to restain nucleus, and all the images above were captured by a fluorescence microscope .At the room temperature, primary neurons pretreated were fixed by PFA of 4% for 30 minutes. The transverse slices of 5 T test was used for comparison between the two groups, and one-way ANOVA was used for comparison between multiple groups. Correlations between the expression of β-catenin and SIRT1 or Caspase-3 were performed using the Spearman correlation analysis. The data were expressed as mean ± standard deviation (SD), and P < 0.05 is considered as a significant difference.Data were all analyzed by the SPSS 22.0 software. H&E and Nissl staining were accomplished at 3, 7, 14, 21, and 28 days after SCI to assess the neurological self-recovery. The results showed that the spinal cord of rats was structurally intact with normal cell morphology in the sham group. At 3 days after SCI, the central gray matter and dorsal white matter were obviously damaged, necrosis and large-area cavities appeared, some nerve cells had nuclear pyknosis, and a large number of inflammatory cells infiltrated. But the damaged area recovered over time . The numAfter SCI, we measured BBB scores at 0, 1, 3, 7, 14, 21, and 28 days to evaluate functional recovery. The results in β-catenin and SIRT1 at different time points after SCI and its relationship with astrocyte apoptosis, we used Western blot and immunofluorescence to measure the expression changes of β-catenin, SIRT1, FOXO4, and Caspase-3 protein. The results of Figures β-catenin protein increased first and then decreased, while the protein SIRT1 showed a gradual upward trend  to clarify the relationship between the Wnt/β-catenin pathway and SIRT1 and FOXO4. The results showed that apoptosis induced by H2O2 increased proapoptotic protein Caspase-3 expression, and LiCl significantly increased the level of β-catenin protein expression. In contrast, SIRT1 protein and its downstream FOXO4 protein significantly downregulated after the addition of LiCl along with H2O2 induction (Furthermore, we built an injury and apoptosis model by exposing glial cells to Hnduction . The immnduction .β-catenin and protein SIRT1 were negatively correlated in the SCI rat model of secondary injury. LiCl, the Wnt pathway activator, increased the expression of β-catenin and inhibited the expression of SIRT1 and FOXO4.This research primarily showed that the protein β-catenin expression after SCI. Consistent with these studies, in the present study, we found that β-catenin was negatively correlated with SIRT1 expression in SCI rats and that β-catenin showed a positive correlation with the expression of the apoptotic protein Caspase-3.Sirtuins are class III of histone deacetylases . Among tβ-catenin pathway, involving in human disease, tumor, and embryonic development [β-catenin. When it is activated, the β-catenin accumulated in the cytoplasm will move into the nucleus to activate the TCF transcription factor participating in the DNA transcription [β-catenin is the key regulator of the Wnt pathway. More recently, lithium has been demonstrated as a Wnt signaling pathway activator in animal and cell models of neurodegenerative diseases and tumors [β-catenin was significantly upregulated compared with the decrease in SIRT1 expression downstream of the protein FOXO4 after lithium-treated glial cells. In line with this observation, there was a negative correlation between β-catenin and SIRT1 in the regulation of neuronal apoptosis in rats. Some reports have reported that activated SIRT1 reduces the β-catenin expression through phosphorylation of mesenchymal stem cells [The canonical Wnt pathway exerts a significant influence on cell development and differentiation . Apoptoselopment . Howevercription , 28. Thed tumors –31. In oem cells , 33. It β-catenin showed an inverse relationship in apoptosis in SCI rats, and there was a regulatory relationship between them. These results suggested that we offered a new molecular mechanism for the potential clinical application of SIRT1 to treat SCI by activating or inhibiting β-catenin, although the complex interregulatory mechanism of SIRT1 and β-catenin is not yet clear to us. However, it lays the foundation for our next study.In this paper, we found SIRT1 and


Biomaterials hold great promise for vaccines and immunotherapy. One emerging biomaterials technology is microneedle (MNs) delivery. MNs are arrays of micrometer-sized needles that are painless and efficiently deliver cargo to the specialized immunological niche of the skin. MNs typically do not require cold storage and eliminate medical sharps. Nearly all materials exhibit intrinsic properties that can bias immune responses toward either pro-immune or inhibitory effects. Thus, because MNs are fabricated from degradable polymers to enable cargo loading and release, understanding the immunological profiles of these matrices is essential to enable new MN vaccines and immunotherapies. Additionally, understanding the mechanical properties is important because MNs must penetrate the skin and conform to a variety of skin or tissue geometries. Here we fabricated MNs from important polymer classes – including extracellular matrix biopolymers, naturally-derived polymers, and synthetic polymers – with both high- and low-molecular-weights (MW). We then characterized the mechanical properties and intrinsic immunological properties of these designs. The library of polymer MNs exhibited diverse mechanical properties, while causing only modest changes in innate signaling and antigen-specific T cell proliferation. These data help inform the selection of MN substrates based on the mechanical and immunological requirements needed for a specific vaccine or immunotherapy application. Existing pathogens and diseases continue to create challenges for current vaccine and immunotherapy technologies. The challenges span not only efficacy and selectivity, but also distribution, storage, and compliance , 2. MostBiomaterials – including polymer and lipid nanoparticles, engineered scaffolds, and biodegradable materials – are being intensely investigated for vaccines and immunotherapies across infectious disease , 6, cancAs alluded to earlier, one key ability of biomaterials is targeting specific tissue niches. In this context, one emerging approach particularly relevant for vaccines and immunotherapies are microneedles (MNs). MNs are small arrays of micrometer-sized needles made of synthetic or natural matrices. The design and length scale of these technologies ensure delivery of cargo across the skin barrier, and efficient access to the unique immunological niche within the skin . BecauseBeyond skin targeting, MNs provide other advantages including painless delivery – since the needles are too short to reach pain receptors, elimination of medical sharps, and incorporation and release of multiple immune cargos. Importantly, because the synthetic or natural polymer matrices used to synthesize MNs typically stabilize biological cargo, MNs often eliminate the need for refrigeration or cold-chain distribution . MNs canToward this need, here we focused on understanding the mechanical and immunological profiles of key classes of polymer matrices used to form degradable MNs. Degradable or dissolvable designs are particularly relevant for immune applications because this strategy enables the encapsulation of cargo and degradation or dissolution to deliver cargo with controlled-release kinetics –24. SomeLow Bloom and High Bloom Gelatin, Sodium Carboxymethyl Cellulose (90kDa and 700kDa), Dextran (9-11kDa and 150kDa), PVA (13-23kDa and 85-124kDa), and PVP (10kDa and 1300kDa) were purchased from Sigma Aldrich, U.S.A. HA (<10kDa and 100-150kDa) was purchased from Lifecore Biomedical, USA.MNs were fabricated using a solvent casting process using an MN master and polydimethylsiloxane (PDMS)  molds. This method was adapted with modifications from our lab’s previous work on MNs , includiAll MNs were imaged using a Phenom XL G2 Desktop SEM at 180x magnification, high vacuum, accelerating voltage of 15kV, and using a backscattered electron detector (BSD). To get a complete picture of the tines, the MNs were mounted on a 45° stud.To characterize the mechanical properties (stiffness and fracture force) of the MNs, a Dynamic Mechanical Analyzer (DMA) TA Q800 was used in the static stress-controlled mode using a compression clamp. In brief, the MN array was fixed on the lower plate, and the upper plate was moved towards the MN array in the strain ramp mode of the instrument at the speed of 0.01mm/s. The compression was stopped once the loading displacement reached 300µm. For approximating the stiffness of the MNs, blocks of polymers  were used. Qualitatively, stiffness was also measured by compressing the MNs using a tweezer and taking images before and after compression.For DC Activation studies, primary DCs were isolated from spleens of naïve C57BL/6 mice using CD11c+ magnetic isolation beads . Spleens were isolated, minced, and incubated in Spleen Dissociation Media , dissociated using a 16G needle, passed through a 40μm strainer, resuspended in MACS buffer containing CD11c+ magnetic isolation beads, and passed through an LS column in a magnet, with CD11c+ cells being collected in a final wash. Isolated DCs were plated at a density of 100,000 cells per 200μL in wells of a 96-well plate. These CD11c+ DCs were stimulated with MN tines dissolved in DC Media. Cells treated with LPS (1µg/mL), and cells treated with PBS were used as a positive and negative control, respectively. Polymer concentrations ranging from 0.0001-100µg per well for each lower and higher MW polymer were used for the treatment groups. After incubation with the groups for 24h, the DCs were washed twice with FACS buffer and then blocked using Fc Block  for 10min at room temperature. The cells were then stained with antibodies for CD80, CD86, CD40, and viability. All antibodies were fluorescent conjugates and were used by staining for 20min at a 1:100 dilution in FACS buffer  and 1:200 dilution for Viability dye. Cells were then washed twice with FACS buffer for analysis by flow cytometry. Flow cytometry was performed on a FACS Celesta (BD Biosciences) and CytoFLEX flow cytometer (Beckman Coulter), and analyzed using FlowJo. For gene expression analysis, DCs were cultured for 24h with LPS and MN substrate or MN substrates alone prior to isolation and analysis.5 T cells per well (making the ratio 1:3 DC to T cells). T cell proliferation was determined by the mean fluorescence intensity of the eFluor 670 signal and compared with the positive and negative controls.To see the effect of the MN matrices on T cell proliferation and expansion, CD11c+ DCs isolated as previously mentioned were treated with the MN solution (0.0001-100µg), LPS, and PBS. Soluble SIINFEKL (5µg/mL) was also pulsed into the wells along with the MN substrates and controls. After 48h, T cells isolated from OT-1 mice using CD8+ T cell negative selection kits  were stained with cell proliferation dye eFluor 670 (0.5µM/well) during a 5 min incubation at room temperature. T cells were then co-cultured with each DC sample by adding 3x 10Gapdh), Mm99999915_g1; actin beta (Actb), Mm00607939_s1; 18s rRNA (18s), Mm00434228_m1; interleukin 6 (Il-6), Mm00446190_m1; interleukin 10 (Il-10), Mm99999067_m1; interferon-gamma (Ifn-γ), and Mm00441891_m1; tumor necrosis factor (Tnf-α). qPCR was performed in a MicroAmp Optical 384-well reaction plate  with optical adhesive film  on a QuantStudio 7 Flex Real-Time PCR System .For gene expression analysis, RNA was isolated using the Quick-RNA Microprep Kit , where cells were lysed in their wells using a lysis buffer, genomic material was captured in a silica-based matrix, and DNA was degraded with DNase I. RNA was diluted to 20ng/μL in RT-qPCR grade water . cDNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit . The qPCR reaction mix was made using TaqMan Gene Expression Assay probes in TaqMan Gene Expression Master Mix . Taqman probes utilized were: glyceraldehyde 3-phosphate dehydrogenase .All characterization studies were replicated at least three times, and all data points, along with mean ± standard deviation, were reported. Cellular analyses were replicated at least twice to ensure reproducibility of biological effects. For DC activation, T cell studies, and RT-qPCR studies, one-way ANOVA with Tukey post-test corrections for multiple comparisons were used to compare groups. Analysis and hierarchal clustering were done in MATLAB v.R2019b using the clustergram function, where the data were standardized for each gene to compare across multiple groups, and clustering was performed using a single linkage (nearest neighbor). Statistical calculations were performed using GraphPad Prism v.9.1.0.We began by assessing polymers with three different origins 1) those derived from the extracellular matrix - gelatin and hyaluronic acid (HA), 2) naturally-derived polymers - carboxymethyl cellulose (CMC) and dextran, and 3) synthetic polymers - polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP). MNs were prepared from each material class by a solvent casting fabrication method involving a MN master and PDMS mold  . One exception was a modest increase observed during treatment with low MW versions of the natural polymers, CMC and dextran. For CD86, nearly all polymers led to a small, but statistically significant increase in activation relative to PBS . In contrast, CD40 expression was significantly higher for all the MN matrices when compared to the PBS group . However, in all cases the LPS positive control led to much higher activation than relative to the MN matrices , the magnitude was less than that observed for the low dose treatments . This may indicate engagement of feedback mechanisms during encounter of high or persistent polymer doses  of MN matrices. Generally we found that the matrices did not increase CD80 relative to PBS, for either low or high MW polymers .CJ and RO are employees of the VA Maryland Health Care System. CJ has an equity position with Avidea Technologies. The views reported in this paper do not reflect the views of the Department of Veterans Affairs or the United States Government.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Gallbladder cancer (GBC) is the most malignant cancer of the biliary tract cancer and presents poor prognosis. CircRNAs have been identified as critical regulators of multiple stages in tumor progression. In the study, we first demonstrated that circular RNA circβ-catenin expression was upregulated in GBC tissues when compared to adjacent normal tissues and associated with advanced clinical stage and poor prognosis in GBC patients. Silencing of circβ-catenin obviously suppressed GBC cell proliferation and cell cycle progression in vitro, but circβ-catenin overexpression had the opposite effects. In vivo, silencing of circβ-catenin inhibited tumor growth. Furthermore, we also found that circβ-catenin promoted GBC cell lactate production, pyruvate production, ATP quantity, and extracellular acidification rate (ECAR), which suggested that circβ-catenin regulated Warburg effect in GBC. Mechanistic analysis further highlighted that circβ-catenin promoted Stathmin 1 (STMN1) expression through sponging miR-223 in GBC progression. In addition, knockdown of STMN1 inhibited cell growth and Warburg effect in GBC. In summary, our findings indicated that circβ-catenin/miR-223/STMN1 axis could regulate cell growth and Warburg effect in GBC. Targeting circβ-catenin might be a potential therapeutic strategy for GBC. Gallbladder cancer (GBC) is the fifth most common cancer among gastrointestinal cancers and the most common cancer of the biliary tract worldwide . Due to Circular RNAs are a novel group of endogenous noncoding RNAs and are characterized by their covalently closed-loop structures without a 5′ cap or a 3′ Poly A tail . CurrentCircβ-catenin originated from β-catenin gene locus is predominantly localized in the cytoplasm and has been revealed to promote liver cancer cell growth through activation of the WNT pathway . In our P = 0.021), but no association with other clinicopathological characteristics (Table P > 0.05). Moreover, Kaplan–Meier survival curve and log-rank test showed that GBC patients with higher expression of circβ-catenin have significantly shorter overall survival (OS) than those with the lower expression of circβ-catenin . Multivariate Cox survival analysis also showed that higher circβ-catenin expression  and advanced TNM stage  were independent prognostic factors for poor survival of GBC patients  and found that miR-223 could form complementary base pairing with circβ-catenin . Each tissue sample was snap-frozen in liquid nitrogen for further analysis. All clinicopathological diagnoses were confirmed by two pathologists. All of tumor tissues received no radiotherapy or chemotherapy before surgery. This study was approved by the ethics committee of Xinhua Hospital. Written informed consents were acquired from patients.2.Four human GBC cell lines SGC-996, NOZ, GBC-SD, and OCUG-1 and a human intrahepatic biliary epithelial cell line H69 were used in the present study. GBC-SD, SGC-996, and OCUG-1 cell lines were obtained from Cell Bank of the Chinese Academy of Science . The NOZ cell line was purchased from the Health Science Research Resources Bank . All of the cell lines were cultured in Dulbecco’s modified Eagle’s medium  contained 10% fetal bovine serum . Cells were cultured in a humidified incubator at an atmosphere with 5% COTwo shRNAs against circβ-catenin were designed and bought from RiboBio Co., Ltd. . Two shRNAs (shRNA-1 or shRNA-2) or shRNA-NC (sh-NC) were transfected into GBC cells using Lipofectamine 3000 (Invitrogen) following the manufacturer’s instructions. The pLCDH-circβ-catenin (circβ-catenin) was synthesized using full-length circβ-catenin and subcloned into a pLCDH-vector (vector) with the cloning sites BamHI/EcoRI . MiR-223 mimic (5′-UGUCAGUUUGUCAAAUACCCCA-3′), miR-223 inhibitor (5′-UGGGGUAUUUGACAAACUGACA-3′), and miRNA control (5′-CAGUACUUUUGUGUAGUACAA-3′) were designed and bought from RiboBio Co., Ltd. . Transfection of STMN1 siRNA (5′-AAGAGAAACUGACCCACAA-dTdT-3′) and scramble control (5′-UUCUCCGAACGUGUCCGU-dTdT-3′)  were performed using Lipofectamine 2000 Transfection Reagent (Invitrogen).−ΔΔCt methods were used to analyze the quantitate mRNA expression.Total RNAs were extracted from GBC tissues and cell lines using TRIzol reagent  according to the manufacturer’s instructions. The cDNAs were synthesized from total RNA using Prime-Script RT reagent kit . U6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were utilized as controls. All primers were designed and purchased from Sangon Biotech . The primer for circβ-catenin forward: 5′-AGTGCTGAAGGTGCTATCTGT-3′, the primer for circβ-catenin reverse: 5′-AGGTAAGACTGTTGCTGCCA-3′; the primer for STMN1 forward: 5′-GCCTGTCGCTTGTCTTCT-3′, the primer for STMN1 reverse: 5′-TCATGGGACTTGCGTCTT-3′; the primer for GAPDH forward: 5′-CAACAGCCTCAAGATCATCAGC-3′, the primer for GAPDH reverse: 5′-TTCTAGACGGCAGGTCAGGTC-3′; the primer for U6 forward: 5′-CTCGCTTCGGCAGCACA-3′, the primer for U6 reverse: 5′-AACGCTTCACGAATTTGCGT-3′; the 2Cytoplasmic and nuclear RNAs were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents  following all manufacturer protocols. We followed this experiment with qRT-PCR analysis.GBC cells were collected and divided into groups, respectively. Cells were treated with or without RNase R  at 37 °C for 20 min. Then, RNA was purified with phenol, chloroform, and isoamyl alcohol. Circβ-catenin stability was determined by detecting circβ-catenin expression by qRT-PCR.Cells were fixed in 4% paraformaldehyde and washed with PBS for 30 min. After treating with FISH probes specific for biotin-labeled RNA probe (circβ-catenin: ACT + TCAAA + TACCCAGCT + TC + TACAAT)  in hybridization buffer overnight, cells were washed with PBS three times and then blocked with HRP blocker. Cells were hybridized in hybridization buffer and 4 ng/µl of RNA probe in 2 × SSC plus 50% formamide 60 °C overnight. Finally, signals were detected using a tyramide-conjugated Alexa 488 fluorochrome TSA kit (Life Technologies).3) were seeded into 96-well plates before transfection. After transfection at 1, 2, 3, 4, and 5 days after cell transfection, a CCK8 solution (10 μl) was added to each well. Then, after 2 h of incubation at 37 °C, absorbance at 450 nm was determined using Spectra Max 250 spectrophotometer .Cell viability was measured using cell counting kit-8  following the manufacturer’s instructions. In brief, cells  was used to remove RNA at a concentration of 2 mg/mL for 30 min at 37 °C. 20 mg/mL propidium iodide  was added to mark the DNA content for 20 min at 37 °C. The percentages of GBC cells in different phases of the cell cycle were analyzed by the flow cytometer.Cells were harvested and lysed using lysis buffer . The proteins were separated by 8–10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes , which were blocked in 5% nonfat milk and then incubated with primary antibodies against STMN1 , β-catenin , or GAPDH  at 4 °C overnight. Then, the horseradish peroxidase-conjectured goat anti-rabbit secondary antibody  was incubated with the membranes. Finally, the protein bands were visualized by using an electrochemiluminescence (ECL) system and the gray values were measured by Image J software.The biotin-labeled miR-223 probe (UGUCAGUUUGUCAAAUACCCCA) and the negative control probe were designed and synthesized by Gene Pharma Biotech . Briefly, NOZ and SGC-996 cells were fixed, lysed, and centrifuged. Then, Streptavidin-coupled Dynabeads (Invitrogen) were washed and resuspended in the buffer. Then, an equal volume of the biotin-labeled miRNAs was added in the buffer. After incubating at room temperature for 10 min, the coated beads were separated with a magnet for 2 min and washed three times. The isolated RNAs were then subjected to qRT-PCR analysis.http://www.circbase.org/). The target miRNAs of circβ-catenin were predicted with circular RNA interactome (https://circinteractome.nia.nih.gov).circβ-catenin sequence data were obtained from circBase . The circβ-catenin-WT/MUT plasmids were co-transfected with miR-NC or miR-223 mimic into NOZ and SGC-996 cells. STMN1 3′UTR or corresponding MUT were designed and was inserted into psicheck2 vector . The STMN1-WT/MUT plasmids were co-transfected with vector, circβ-catenin, miR-223 mimic or circβ-catenin + miR-223 mimic in NOZ or SGC-996 cells. The luciferase activity normalized to Renilla luciferase activity was analyzed via a Dual-Luciferase reporter assay system .6) stably expressing circβ-catenin shRNA or control group were subcutaneously injected into either side of the flank area of 3-week-old BALB/c athymic nude mice. Tumor size or volume was measured every week. All mice were euthanized after 4 weeks and subcutaneous tumors were dissected and collected. Tumor volume (V) was measured weekly and calculated using the equation V = (a × b2)/2, Final tumor weights were measured and tumor samples were subjected to IHC staining. All animal experiments were performed in the animal laboratory center at Xinhua Hospital and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .BALB/c nude mice (3 weeks old) were purchased from Vital River Laboratory Animal Technology (China). NOZ cells , treated with secondary antibody for 30 min, and stained with diaminobenzidine (DAB) until brown granules appeared.4 cells/well was cultured in a 96-well XF microplate (Seahorse Biosciences) overnight. Cells were washed before incubation with assay medium (0.5 mL) for 1 h at 37 °C. Then, cartridge ports A, B, and C were loaded with 75 μL of glucose (80 mM), oligomycin (9 μM), and 2-DG (1 M), respectively. The ECAR after glucose treatment indicated the glycolysis rate. The ECAR after oligomycin treatment indicated the glycolysis capacity.The extracellular acidification rate (ECAR) or oxygen consumption rate (OCR) was measured using a Seahorse XF24 analyzer (Seahorse Biosciences). Briefly, after cell transfection, 1 × 10t-test. Differences between multiple cell lines were assessed using one-way ANOVA followed by the least significant difference (LSD) test. Correlations between circβ-catenin expression level and clinicopathological characteristics were analyzed by Pearson’s correlation test. Multivariate Cox proportional hazards regression models were used to identify factors associated with survival. The survival analysis was conducted with the Kaplan–Meier plots and the log-rank test. P values < 0.05 were considered as statistical significance.The data was analyzed by SPSS 20.0  and GraphPad Prism 6.0  and are shown the mean ± standard deviation (SD). The differences between the two unpaired groups were assessed using unpaired Student’s