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Endocrine-Related CancerResearchY Liu et al.Role of CRHR1 in colitis-relatedcancer21:4642Quantitative real-time PCRPBMC isolationTotal RNA from the mouse colon was isolated using TRIzol(Invitrogen) according to the manufacturer’s protocol,and an equal amount of RNA (2 mg) was transcribed intocDNA using an RT reagent kit (iTaq, Hercules, CA, USA).Subsequently, quantitative real-time PCR was carried outwith SYBR Green oligonucleotides (iTaq) using a 7300Fast Real-Time PCR instrument (Applied Biosystems).The level of expression was calculated based on the PCRcycle number (CT) at which the exponential growth influorescence from the probe passed a certain thresholdvalue (CT). Relative gene expression was determined bydifferences in the CT values of the target genes afternormalization to the RNA input level, using the CT valueof ACTIN. Relative quantification was represented bystandard 2KDCT calculations. DCTZ(CT-target geneKCT-ACTIN).Each reaction was carried out in triplicate. Primersequences are given in Table 1.Mice were anesthetized in a diethyl ether atmosphere andblood was collected by cardiac paracentesis in a tubecontaining EDTA-2K buffer. Anti-coagulated blood waslayered onto mononuclear separating medium (Haoyang,Tianjin, China) and peripheral blood mononuclear cells(PBMC) were purified by gradient centrifugation (400 g,30 min) according to the manufacturer’s recommendations.Cell amount analysisBriefly, blood was sampled from retroorbital sinus in a tubecontaining EDTA-2K buffer, and stained for macrophagocyte cell markers CD11bC and CD11cK (e-Bioscience,San Diego, CA, USA) and then analyzed with flowcytometry (BD Biosciences, Franklin Lakes, NJ, USA).Table 1Immunoblotting analysisEqual amounts of protein from colon were subjected toSDS–PAGE analysis and immunoblotting using antiCRHR1 antibody (1:1000, overnight in 4 8C; NovusBiologicals), anti-p-NFkB antibody (1:1000, overnight in4 8C; Cell Signaling Technology, Danvers, MA, USA), antiNFkB antibody (1:1000, overnight in 4 8C; Cell SignalingTechnology), anti-p-STAT3 antibody (1:1000, overnight in4 8C; SAB), anti-STAT3 antibody (1:1000, overnight in4 8C; Cell Signaling Technology), anti-BCL2 antibody(1:1000, overnight in 4 8C; Cell Signaling Technology),anti-BAX antibody (1:1000, overnight in 4 8C; Abcam),anti-b-ACTIN antibody (1:5000, overnight in 4 8C, SAB),and anti-histone antibody (1:10 000, overnight in 4 8C;Abmart, Shanghai, China). Western blotting analysis wascarried out as described previously (Xu et al. 2009).Summary of qRT-PCR of primer sequencesNameSequencesGenBank accession numberCRHR1Sense: GGAACCTCATCTCGGCTTTCAAntisense: GTTACGTGGAAGTAGTTGTAGGCSense: TCTTGCTGTTAGCGGAGCGAntisense: TCGAATATGATGCGGTTCTGCSense: CCTCAGCCGGTTCTGATCCAntisense: GCGGAAAAAGTTAGCCGCAGSense: TGACCTGGGCTGTCCTGATGAntisense: GGTGCTCATGTCCTCATCCTGSense: CTGCAAGAGACTTCCATCCAGTTAntisense: GAAGTAGGGAAGGCCGTGGSense: GCTCTTACTGACTGGCATGAGAntisense: CGCAGCTCTAGGAGCATGTGSense: AGGCTGCCCCGACTACGTAntisense: GACTTTCTCCTGGTATGAGATAGCAAASense: GGGTGTCCCTTCACTTCTTTCAAntisense: TGGGAGGCACTTGCATTGASense: AAGCGTCATTGAATCACACCTGAntisense: TGACCTCAAACTTGGCAATACTCSense: ATGCTCCCCGGGCTGTATAntisense: CATAGGAGTCCTTCTGACCCATTCNM 007762.4UrocortinCRHIL1bIL6IL10TNFaCOX2IFNgACTINNM 021290.2NM 205769.2NM 008361.3NM 031168.1NM 010548.2NM 001278601.1NM 011198.3NM 008337.3NM 007393.3CRH, corticotropin-releasing hormone; CRHR, CRH receptor; TNFa, tumor necrosis factor alpha; IL, interleukin; IFNg, interferon gamma; COX2,cycloxygenase 2.http://erc.endocrinology-journals.orgDOI: 10.1530/ERC-14-0239q 2014 Society for EndocrinologyPrinted in Great BritainPublished by Bioscientifica Ltd.Downloaded from Bioscientifica.com at 02/14/2022 08:11:13PMvia free access

ResearchY Liu et al.In situ intestinal proliferation assayThe number of proliferating cells in intestinal epitheliumwas determined using the immunoperoxidase stainingprotocol with the thymidine analog 5-bromo-2-deoxyuridine (BrdU, Sigma). In brief, a working solution of BrdUin PBS at 1 mg/ml was prepared and injected mice i.p. with1 ml (1 mg) of BrdU solution. Twenty-four hours later,colon tissues were collected, fixed in 10% neutral bufferedformalin, and embedded in paraffin. Immunohistochemistry was carried out using the BrdU In situ DetectionKit (BD Pharmingen, San Jose, CA, USA) as describedearlier (Zaki et al. 2010).Role of CRHR1 in colitis-relatedcancer21:4643also displayed enhancement of CRHR1 expression inthe carcinomatous tissues compared with normal andpericarcinomatous tissues (Fig. 1D). We further analyzedthe localizations of CRHR1 using immunohistochemistry.Similarly, CRHR1 and its ligands were less expressed inthe mucosal inflammatory cells of normal mice, whiletheir expression were all upregulated in the CAC mice.Interestingly, CRHR1 was mainly located in inflammatorycells of pericarcinomatous area, while CRHR1 were mostlyexpressed in the pejorative epithelial cells in the tumorarea (Fig. 1E). These results indicated that increasedCRHR1 might have an important biological functionduring the development of CAC.Endocrine-Related CancerTUNEL stainingThe TUNEL procedure was performed using the in situ CellDeath Detection Kit (Roche Applied Science). Proteinase K(20 mg/ml, Sigma) was applied to the tissue sections for15 min at room temperature. Endogenous peroxidasewas blocked by incubation of the sections with 2% H2O2for 5 min. The sections were incubated with the TdTenzyme for 2 h at 37 8C. Antidigoxin-peroxidase solutionwas applied for 30 min, followed by exposure to diaminobenzidine (DAB) substrate for 3–5 min and counterstaining with hematoxylin.Statistical analysisData were represented as meanGS.E.M. The differencein survival was shown by Kaplan–Meier plot. The log-ranktest was used to compare significant survival difference.Statistical significance was determined by Student’st-test. A P value !0.05 was considered statisticallysignificant.ResultsExpression of CRHR1 in the colon of mice and CRC patientsTo explore the role of CRHR1 in CAC development,Crhr1C/C mice were injected with a single dose of theDNA-methylating agent AOM followed by three rounds oforal administration of DSS to trigger chronic CAC (Fig. 1A)and the mice were fed with tap water as the controls. Thequantitative real-time PCR showed that the expression ofCRHR1 and its ligands (UCN1 and CRH) were obviouslyincreased in the colon of CAC mice compared withcontrols (Fig. 1B) and similar results were observed at theprotein level of CRHR1 by western blotting (Fig. 1C).As in the model of CAC mice, slices from CRC patientshttp://erc.endocrinology-journals.orgDOI: 10.1530/ERC-14-0239q 2014 Society for EndocrinologyPrinted in Great BritainCRHR1 deficiency contributed to the alleviation ofcolitis-associated tumorigenesisTo dissect the exact effect of CRHR1 on CAC, Crhr1K/Kmice were generated and we induced colon tumorigenesiswith AOM and DSS as mentioned above. Changes in bodyweight were monitored daily throughout the studyduration and colonic tumor burden was determined80 days after AOM and DSS treatment. Mortality andweight loss were significantly reduced in Crhr1K/K micecompared with Crhr1C/C during the early stage of tumorinduction (day 10 after AOM injection) (Fig. 2A and B).Consistently, Crhr1K/K mice were markedly protectedfrom AOM and DSS-induced tumorigenesis (Fig. 2C, D, E,and F).Decreased tumor burdens were associated withlower incidence of dysplasia and less inflammatorycytokines expressionHistological examination revealed that 70% of Crhr1C/Cmice developed high-grade dysplasia, of which 30% wereclassified as adenocarcinoma with invasive growth. Bycontrast, only 40% of Crhr1K/K mice displayed high-gradedysplasia and no adenocarcinomas were observed in anyof Crhr1K/K (Fig. 3A and B). Histological scores from thequantifications of ulceration, inflammation, and hyperplasia of the mucosa were significantly decreased inCrhr1K/K mice (Fig. 3C and D). Besides, both mRNA andprotein expression of proinflammatory cytokines, such asIL1b, IL6, and TNFa, were markedly lower, whereas theanti-inflammatory factor, IL10 was significantly higher inCrhr1K/K than that in Crhr1C/C mice (Fig. 3E). We nextanalyzed the expressions of multiple cytokines andtumorigenic factors cycloxygenase 2 (COX2) and interferon gamma (IFNg) in colon tissue as these factors oftenPublished by Bioscientifica Ltd.Downloaded from Bioscientifica.com at 02/14/2022 08:11:13PMvia free access

Y Liu et al.ResearchBCrhr1 / Crhr1 –/–6040200CCrhr1 / Crhr1 –/–1.21.51.11.00.9**0.8Crhr1 / Crhr1 –/–1.00.5*0.001020304050607080Days (d)Days (d)DEF20Percentage of tumor size(diameter)Number of tumors in per colon1001510504020r1 –/– 2 mm2–5 mm 5 mmrhCrhr1 –/–60CCrhr1 / r1 / Crhr1 –/–rh / 800CCrhr1Crhr1 / Crhr1 –/–**25Endocrine-Related 7880Percent survival8021:4Tumor incidence (%)100Body weight change (%)ARole of CRHR1 in colitis-relatedcancerFigure 2Decreased CAC tumorigenesis in Crhr1-deficient (Crhr1K/K) mice.(A) Survival rate of Crhr1C/C mice (27.27%) and Crhr1K/K mice (47.62%)were shown by the Kaplan–Meier plot. The log-rank test indicated therewere significant survival differences between Crhr1K/K (nZ21 mice pergroup) and WT (Crhr1C/C) (nZ33 mice per group) PZ0.001. (B) Bodyweight loss was shown. Day 15 (Crhr1C/C: 79.77%G0.389%; Crhr1K/K:96.13%G0.775%) and day 18 (Crhr1C/C: 78.6%G1.22%; nZ11; Crhr1K/K:103.96%G0.60%; nZ17). *P!0.05 vs Crhr1C/C. (C) The incidence of tumorwas analyzed (Crhr1K/K: nZ17 mice per group; Crhr1C/C: nZ11 mice pergroup). (D) Mice were killed to determine tumor development in the colonon day 80. (E) Numbers of tumors in the whole colon were counted (nZ6mice per group) Crhr1C/C: 16.4G0.35; Crhr1K/K: 6.3G0.36). (F) Diameter ofthe tumors was measured at the end of the study (nZ6 mice per group).Data represented meanGS.E.M.; *P!0.05, **P!0.01 vs Crhr1C/C.drive tumorigenesis. Similarly, mRNA levels of Cox2 andIfng were markedly reduced in Crhr1K/K mice than thosein Crhr1C/C mice (Fig. 3F). These results suggested thatCRHR1 activation increased the risk of tumorigenesis withenhanced inflammation and hyperplasia, and a higherincidence of dysplasia.analysis revealed more severely damaged colonic mucosawith extensive loss of crypt structures and epithelial celldenudation, larger areas of ulceration, and more extensiveinfiltration of inflammatory cells in Crhr1C/C group(Fig. 4C), which was also reflected in the pathologicalassessment of colitis severity scores (Fig. 4D and E). Takentogether, these results suggested that CRHR1 played acritical role during the early stage of colitis-associatedtumorigenesis, because from day 25 onwards, occurrenceof tumor formation was observed as described above.CRHR1 was critical in tumor induction during theearly stage of CACAs described earlier, the weight loss and survival ratedifferences between Crhr1C/C and Crhr1K/K mice weredetected at the early stage of tumorigenesis. These resultspromoted us to hypothesize that CRHR1 influencing ontumorigenesis occur early from the onset of tumorigenesis. To characterize this possibility in detail, we collectedthe colons from mice killed at day 10, 15, and 25 afterAOM injection. Interestingly, colons from Crhr1K/K micewere significantly longer than those from Crhr1C/C miceafter day 15 (Fig. 4A and B). In addition, .orgDOI: 10.1530/ERC-14-0239q 2014 Society for EndocrinologyPrinted in Great BritainInflammatory cell activity was decreased in Crhr1K/K micePrevious studies suggested that CRHR1 was a keyinflammation regulator (Xu et al. 2009, Zocco et al.2010). To further confirm the effect of CRHR1 oninflammation in terms of CAC, inflammatory cytokineproduction was analyzed during day 0 to day 25. Notsurprisingly, the mRNA expression for Il1b, Il6, and Tnfawas decreased, while Il10 was increased in Crhr1K/K micePublished by Bioscientifica Ltd.Downloaded from Bioscientifica.com at 02/14/2022 08:11:13PMvia free access

Y Liu et al.ResearchRole of CRHR1 in colitis-relatedcancerCrhr1 / AdenocarcinomaCrhr1–/–High gradeLow gradeRelative mRNAEA1.5100 Low gradeHigh gradeAdenocarcinoma1.0Crhr1 / Crhr1–/–Crhr1–/–IL1β31 kDaActin43 kDa***TNFαCrhr1 / 1.0Crhr1–/–TNFα23 kDaActin43 kDa0.5***0.00.5Relative mRNA1.50.0Crhr1 / CHistology scoreCrhr1–/–Crhr1 / Crhr1–/–4*Crhr1 / 1.00.5***Crhr1–/–IL626 kDaActin43 ative mRNA*2flaIL60.030**Crhr1 / Crhr1–/–40IL1017 kDa20Actin43 kDa0DF121.586***420Crhr1 / Crhr1–/–1.00.50.01.5IFNγ***Relative mRNA10Relative mRNATotal histology scoreEndocrine-Related CancerPercentage of micewith dysplasia1.5Relative mRNABCrhr1 / 0.51.5200 645IL1β1.00.021:4COX2Crhr1 / Crhr1–/–1.00.5***0.0Figure 3Alleviated CAC severity in Crhr1K/K mice attributed to reduced inflammationresponse. (A) Representative images of H&E staining of colon sectionsshowing dysplasia. (B) Overall grading of dysplasia in each genotype. (C) H&Estained sections were scored for inflammation, ulceration, hyperplasia, andinflamed area, and determined in a double-blind manner. (D) Total histologyhttp://erc.endocrinology-journals.orgDOI: 10.1530/ERC-14-0239q 2014 Society for EndocrinologyPrinted in Great Britainscores from Crhr1C/C mice (scoresZ10G0.20) and Crhr1K/K mice(scoresZ4.2G0.33). (E) Expression of IL1b, IL6, IL10, and TNFa in colon wereanalyzed. (F) RNA for expression analysis of IFNg and COX2 by real-time PCR.Data represented meanGS.E.M.; nZ5–7 mice per group; *P!0.05, **P!0.01,***P!0.001 vs Crhr1C/C littermates.Published by Bioscientifica Ltd.Downloaded from Bioscientifica.com at 02/14/2022 08:11:13PMvia free access

Y Liu et al.ResearchADay 0Day 10Role of CRHR1 in colitis-relatedcancerDay 15Day 25CDay 0Day 1021:4Day 15646Day 25Crhr1 / 200 Crhr1 –/–200 Histology E***1ype–r1 –/rhCCrhr1 / r1 –/–CCrhr1 /Crhr18Crhr1 / Crhr1 –/–3Infla**Crhr1 / –/–Totalhistology scoreColon length (cm)10Endocrine-Related Cancer4nBrh – r1 –/r1 /rhCCrhrhCCrhr1 / r1 –/–D***Crhr1 / Crhr1 –/–Figure 4CRHR1 induced tumor at the early stage of tumorigenesis. (A, B, and C)Mice were killed at day 0, 10, 15, and 25 after AOM injection. (A and B)Changes in colon length was monitored. (C) Mucosal histology wasexamined by H&E staining. (D and E) Colitis severity scores were determinedat day 25 in a double-blind manner. Data represented meanGS.E.M.; nZ9 pergroup; *P!0.05, **P! 0.01, ***P! 0.001 vs Crhr1C/C littermates.after day 10 (Fig. 5A). We therefore hypothesized that theloss of CRHR1 may protect from colitis-associated tumorigenesis by attenuating immune cell activation andinflammatory responses in response to DSS treatment. Totestify this possibility, immunohistochemical analysis wasused to characterize the immune cell associated with theinduction of hyperinflammatory responses in the colon.During inflammation response, more lymphocytes andmacrophages infiltrated into the colon mucosa of Crhr1C/Cmice (Fig. 5B). To determine the inflammatory cell typeregulated by CRHR1, immunostained mucosa of largeintestine with the markers of macrophage, B cells, T cells,and anti-CRHR1 was examined. The results showed thatCRHR1 was mostly detected in macrophages but not in Bcells and T cells (Fig. 5C). Flow cytometry analysis showedthat lower levels of macrophages in the whole peripheralblood of Crhr1K/K mice than in those of Crhr1C/C micefrom day 10 (Fig. 5D). To further characterize whetherCRHR1 deficiency could affect the secretion of inflammatory cytokines, mononuclear cells (macrophage precursors)collected from the whole peripheral blood of Crhr1C/Cand Crhr1K/K mice were equally seeded into plate andstimulated with LPS for 6 h, and the inflammatory factorsin the supernatant were monitored using ELISA. The ELISAanalysis showed significantly less IL6 and TNFa releasedfrom mononuclear cells in Crhr1K/K mice, but nodifference in the levels of IL1b and IL10 between Crhr1K/Kand Crhr1C/C mice (Fig. 5E).Thus, these results suggested that less susceptibletumorigenesis of Crhr1K/K was partly owing to weakerinflammatory response in inflammatory cells, especiallythe OI: 10.1530/ERC-14-0239q 2014 Society for EndocrinologyPrinted in Great BritainCRHR1 regulated colonic cells survival andproliferation through NFkB and STAT3 signalsThe expression of tumorigenic and proinflammatorygenes is modulated by signal transduction pathwaysmediated by NFkB and STAT proteins. To understandwhether these pathways and molecules were deregulatedin the presence of CRHR1, we examined the activation ofNFkB and STAT signaling pathways by western blotting.Indeed, after AOM/DSS treatment, significantly higheractivation levels of NFkB (p65 phosphorylation) andphosphorylated STAT3 were observed in the colon tissueof Crhr1C/C mice at day 25 after AOM injection (Fig. 6A).Published by Bioscientifica Ltd.Downloaded from Bioscientifica.com at 02/14/2022 08:11:13PMvia free access

Y Liu et al.ResearchCRHR12520ayDDay1510ayayDDRelative mRNA***10***52520400 Day15ayDCD20Day25100DF4/80MacsCrhr1 / 400 Crhr1 –/–Cell number ( 105)80Endocrine-Related Cancer400 NFα1.5Crhr1 / Crhr1 –/–1.5ay******0.010Relative mRNA***Relative mRNA**10Relative mRNA1.00.5CIL6IL1β1.5DARole of CRHR1 in colitis-relatedcancerCrhr1 / Crhr1 –/–60*4020**Day 15Day 250Day 10400 Figure 5The presence of CRHR1 led to increased inflammatory cell activation incolon tissue. (A) Real-time PCR was carried out to detect the expression ofIL1b, IL6, IL10, and TNFa in the colons collected from day 0, 10, 15, and 25after AOM injection. (B) Colon tissue collected at day 25 was immunostained for the macrophage marker F4/80, B cell marker CD20, and T cellmarker CD3. (C) Mucosa of large intestines (collected at day 25) was stainedwith markers of macrophage, B cells, T cells, and ant

Crhr1 C/ mice during treatment with AOM and DSS. Tumorigenesis was significantly reduced inCrhr1K/K mice, . (Shacter & Weitzman 2002, Karin et al. 2006, Lin & Karin 2007). . Another key player in apoptosis is the