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The Effect And Mechanism Of Apoptosis Signal-Regulating Kinase 1 On Hepatocellular Carcinoma

Posted on:2017-02-27Degree:DoctorType:Dissertation
Country:ChinaCandidate:C F JiangFull Text:PDF
GTID:1314330536966997Subject:Internal Medicine
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【Background and Objective】Hepatocellular carcinoma(HCC)is one of the most common type of malignant tumors worldwide,which seriously affects people’s health and lives.Albeit great progress have been made in the field of diagnosis and treatment with the development of medical technology,the long-term survival remains unsatisfied,mainly due to postoperative recurrence or metastasis.Recently differentiation therapy has become a new direction in treatment of solid tumors.Hepatocyte nuclear factor4α(HNF4α),a principal member of the hepatic transcription factor network,plays an indispensable role in the regulation of hepatic lineage differentiation and function maintenance.Our previous studies have indicated that HNF4α could induce the differentiation of malignant cells into mature hepatocytes and dramatically suppress the proliferation of hepatoma cells.Additionally,overexpresssion of HNF4α showed a strong anti-tumor effect on HCC in vivo.However,its molecular mechanisms are yet to be fully determined.Apoptosis signal-regulating kinase 1(ASK1)is a member of the mitogenactivated protein kinase kinase kinase(MAP3K)family that activates downstream MAP kinases(MAPKs),c-Jun N-terminal kinases(JNKs)and p38 MAPKs,in response to various stresses.It plays a pivotal role in various stress responses,including cell death,differentiation,and production of inflammatory cytokines.In addition,accumulating evidence suggests that the diverse functions of ASK1 are involved in many tumorigenesis.Nevertheless,to date,how ASK1 is involved in HCC is still not fully understood.Our previous studies have found that HNF4α can upregulate the expression of ASK1 in hepatoma cells by c DNA microarray screening.Meanwhile the ASK1 m RNA and protein expression and its downstream phosphorylation of JNK and P38 protein expression levels were significantly increased when HNF4α was overexpressed.These findings suggest that ASK1 may mediate the differentiation of HNF4ɑ on HCC.Based on the background and our previous experimental results,in the current study,we will investigate the role of ASK1 on HCC by in vitro and in vivo assays and elucidate its role in the differentiation of HNF4α on HCC,which will shed new light on the treatment of HCC.【Methods】1.The expression and the clinical significance of ASK1 in HCC patients(1)The mRNA levels of HNF4α and ASK1 in clinical samples were analyzed by Real-time PCR(RT-PCR).(2)The relationship between ASK1 and HCC malignant phenotype such as AFP level,tumor size,tumor stage,vascular invasion was analyzed.(3)HCC tissue microarray block was performed to detect ASK1 protein level.The relationship between ASK1 and HCC prognosis was analyzed.2.The effect of ASK1 on the biological characteristics of HCC cells(1)Effect of ASK1 on cell apoptosisHep3B and Huh7 cells were infected by AdASK1 or AdRFP.The apoptosis rate of hepatoma cell lines was detected by flow cytometry.(2)Effect of ASK1 on proliferation and colony formationHep3B or Huh7 were infected with Ad ASK1 or Ad RFP or transfected with si ASK1 or si NC to examine the ability of proliferation,colonies formation.To evaluate the inhibitory effect of ASK1 on cell proliferation,transfected or infected HCC cells were plated into 24-well plates overnight and then seeded into triplicate wells of a 96-well plate.Cell proliferation was assessed using Cell Counting Kit-8(CCK-8).For colony formation assay,the treated cells were seeded onto cell dish at low density and cultured for 2-4 weeks.Colonies were then fixed with 4% paraformaldehyde and visualized by crystal violet staining.(3)Effect of ASK1 on the migration and invasion in vitroFor the migration and invasion assays,transfected or infected cells were seeded at 3×104per transwell into the upper chamber without or with a matrigel coated membrane,After 24 or 48 hours of incubation,cells adhering to the underside were stained with crystal violet and photographed under inverted microscope.(4)Effect of ASK1 on HCC cell tumorigenicity in vivoAd ASK1 infected Hep3 B or Huh7 cells were inoculated subcutaneously into both flanks of each BALB/c nude mouse(n=6).Tumor formation and size(volume = 0.5 × width2×length)were assessed periodically for 6 weeks and the final tumor size and weight were estimated.3.Effect of ASK1 on HCC xenografts in vivo(1)Effect of ASK1 on the subcutaneous tumorThe subcutaneously implanted HCC model was established by injecting Huh7 cells into the armpits of nude mice.When tumor vovlme reached about 200 mm3,mice were randomly assigned to treatment groups.Ad ASK1 and Ad RFP by intratumoral injection was administered twice a week for up to 3 weeks.Kinetics of tumor formation was estimated.At the end,tumors were removed for further analysis.(2)Effect of ASK1 on the transplanted orthophoric tumorHuh7 cells with Luciferase gene were used to establish a subcutaneous tumor as described previously.Once tumor reached to 500 mm3,tumor were removed from the tumor-bearing mice and cut to 1mm3 tumor block.NOD/SCID mice were anesthetized,and tumor block was planted into the liver.One week later,either Ad ASK1 or Ad RFP was injected via the tail vein twice a week for up to 3 weeks.At the time of euthanasia,mice were killed and livers were removed for analysis.(3)Mechanism of anti-tumor effect of ASK1In order to clarify the molecular mechanisms of ASK1,Western Blot was used to detect the expression of ASK1 and downstream p38 and JNK pathways.4.The role of ASK1 in the suppression of HNF4α on HCC(1)ASK1 is regulated by HNF4αRT-PCR and Western Blot were used to detect the m RNA and protein expression levels of ASK1 and downstream p38 and JNK pathways in Hep3 B cells infected with Ad HNF4α.(2)The interaction between HNF4α and ASK1The Jasper Database was used to predict the putative binding sites between HNF4α and ASK1.The chromatin immunoprecipitation assay was performed to confirm the interaction of HNF4α with the promoter of ASK1.PGL3-promoter-ASK1 luciferase reporter plasmid was constructed.The point mutation of HNF4α binding site on ASK1 promoter reporter vector were performed using overlap extension by PCR.Reporter assay was performed to detect the effect of HNF4α on the wild type PGL3-promoter-ASK1 luciferase reporter plasmid and the mutated luciferase reporter vector to prove the direct binding of HNF4α to the ASK1 promoter.(3)The reversal role of ASK1 to HNF4αThe cell proliferation and apoptosis of Hep3 B cells infected with AdHNF4α or AdGFP and transfected with si ASK1 or si NC were detected to evaluate the role of ASK1 on the malignant phenotypes reversion by HNF4α.In addition,a cluster of hepatocyte marker genes were detected by Real-time PCR in Hep3 B cells infected with Ad HNF4α or Ad GFP and transfected with si ASK1 or si NC.5.Statistical analysesStatistical analysis was performed using SPSS software(16.0 version),with a P value<0.05 considered significant.Student’s t test was used to analyze the data of experiments involving only two groups.Statistical comparisons of more than two groups were evaluated using one-way analysis of variance.Unequal variances pairing were performed using the Wilcoxon signed rank test or the Mann-Whitney U test.The chi-square test was used to compare two sample rates.The Kaplan-Meier method was used to calculate the survival time,and its significance was determined with log-rank test.【Results】1.The clinical significance of differentially expression of ASK1 in HCC patients(1)ASK1 and HNF4α were downregulated in HCC tissuesWe examined the mRNA expression of HNF4α and ASK1 in human HCC samples(n =60).The data showed that HNF4α and ASK1 were decreased in more than 70% of the HCC tissues,respectively,as compared with their surrounding noncancerous tissues.Moreover,HNF4 a expression was positively correlated with the levels of ASK1 in HCCs of patients.(2)ASK1 was closely related to clinical malignant phenotype of HCCThe low level expression of ASK1 in HCCs was associated with more aggressive pathologic features including big tumor size(P=0.034),late tumor stage(P=0.009)and absence of tumor encapsulation(P= 0.013).(3)Reduced ASK1 expression is associated with poor prognosis for human HCCASK1 protein levels was downregulated in 53 of 86 cases(61.63%)compared with noncancerous tissues.Patients with low ASK1 expression exhibited a much lower overall survival(OS)compared with those with high ASK1 expression(median OS 22 and 47 months,respectively;P=0.0116).2.The inhibition of ASK1 on malignant properties of HCC cells(1)ASK1 promotes apoptosis of hepatoma cells in vitroASK1 overexpression induced cell death in Hep3 B and Huh7,which was assessed by annexin V staining.(2)ASK1 inhibited proliferation and colony formation of HCC cellsProliferation assay and colony formation assay were performed to assess the effect of ASK1 on cell proliferation.Compared to the control cells infected by Ad RFP,Hep3 B and Huh7 cell lines infected by Ad ASK1 showed lower proliferation rate up to more than 50%.Meanwhile,Ad ASK1 could also significantly reduced the ability of colon formation.(3)ASK1 suppressed the migration and invasion ability of HCC cellsHepatoma cells up-regulating ASK1 exerted significant inhibition on migration and invasion.In contrast,inhibition of endogenous ASK1 by si ASK1 not only mildly promoted cell growth and colony formation but also enhanced migration and invasion of HCC cells.(4)ASK1 decreased the tumorigenicity of hepatoma cells in miceHep3B or Huh7 cells infected with Ad ASK1 or Ad RFP were subcutaneously transplanted into the flanks of Balb/c nude mice.After 6 weeks,xenografts were detected in 80%(4 of 5)of mice in the Ad RFP group of Huh7 cells.The tumors of the RFP group were detected in 100% of Hep3B-inoculated mice.However,no xenografts were observed until 6 weeks in either ASK1-infected hepatoma cell-inoculated mice.3.ASK1 revealed substantial anti-tumor effect in vivo(1)Intratumoral injection of AdASK1 inhibited tumor growthIntratumoral injection of Ad ASK1 significantly reduced the growth and weight of Huh7 cell xenografts compared to the control Ad RFP group.Real time PCR revealed that expression of ASK1 was striking increased in Ad ASK1-treated tumor nodules.Furthermore,treatment with Ad ASK1 displayed decreased Ki-67 expression and contained more TUNEL-positive cells,which were verified by H&E staining and immunohistochemistry.(2)Systemic injection of Ad ASK1 suppressed the transplanted orthophoric tumor of liverAfter intravenous injection of Ad ASK1,fluorescence in Ad ASK1 injection group was significantly weaker than that in the control group.When mice were sacrificed 1week after last injection,it showed that 8/8 mice treated with Ad RFP developed larger tumor formation in the liver.However,1/8 mice treated with Ad ASK1 showed no tumor nodule and 2/8 mice displayed very small tumor formation.The remaining 5/8 tumor blocks were also significantly smaller than the control group.Histological analysis further confirmed the result.(3)The mechanism of anti-tumor effect of ASK1We detected the protein expression of downstream p38 and JNK pathways by Western Blot.The result showed that p38 pathway not JNK was activated in vitro and in vivo when ASK1 over-expressed.Furthermore,p38 inhibitor SB202190 could partially block ASK1 suppression on HCC cell proliferation,demonstrating that ASK1 suppresses the malignant properties of HCC via up-regulating the phosphorylation level of p38.4.HNF4α suppresses HCC partially through upregulation of ASK1(1)ASK1 is positive regulated by HNF4αThe mRNA and protein expression levels of ASK1 appeared to be sensitive to the level of HNF4α,which was increased by HNF4α overexpression and decreased by HNF4αknockdown.Moreover,the phosphorylation level of JNK and p38 were higher in Ad HNF4α than in Ad GFP.(2)HNF4α bind directly to the promoter of ASK1CHIP assay and the followed RT-PCR revealed that HNF4α could bind directly to the promoter of ASK1.Reporter assay revealed that ectopic expression of HNF4α increased the luciferase activity of the wild-type promoter of ASK1,which was impaired by mutation of ASK1,indicating that ASK1 is a direct downstream target for HNF4α.(3)ASK1 inhibition reversed the suppressive effects of HNF4α on HCCTo determine whether HNF4α exerts its function via ASK1,si ASK1 was transfected into HCC cells overexpressing HNF4α.As expected,silenced the expression of ASK1 blocked the inhibition of HNF4α on hepatoma cell proliferation.Similarly,flow cytometry analysis showed that the effect of HNF4α on promoting cell apoptosis was partially reversed by si ASK1 in Hep3 B cells.In addition,RT-PCR confirmed that the re-expression of some hepatocyte marker genes by ectopic HNF4α was also blocked by si ASK1 in Hep3 B cells.Conversely,ectopic expression of ASK1 mimicked the function of HNF4α resulting in increased re-expression of these hepatocyte marker genes.Consistently,the effect of HNF4α induced phosphorylation of p38 was antagonized by silencing of ASK1.【Conclusions】1.The expression of ASK1 is positively correlated with HNF4α in HCC tissues and low expression of ASK1 is associated with aggressive behaviors of human HCC.2.ASK1 suppresses malignant properties of hepatoma cells.3.ASK1 exhibites substantial anti-tumor effect in vivo,which may proposed as an effective therapeutic target for HCC.4.Inhibition of the endogenous ASK1 partially reversed the suppressive effects of HNF4α on HCC.
Keywords/Search Tags:apoptosis signal-regulating kinase 1(ASK1), hepatocellular carcinoma(HCC), molecular therapy, hepatocyte nuclear factor 4α(HNF4α)
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