法尼酯衍生物X受体对肝脏的保护作用及其对肝癌发生的影响
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摘要
肝癌的发生、发展是多因素、多基因和多步骤的复杂过程，学者们对其发生、发展的具体机制仍在不断探索之中。慢性肝炎病毒感染（HBV/HCV）是导致肝硬化发生的最主要原因，肝硬化是HCC发生的重要危险因素，近1/3的肝硬化患者最终发展至HCC。近年来，越来越多的证据显示机体代谢失调也与HCC的发生有关，FXR作为体内重要的核受体之一，对胆汁酸、糖类、脂类的稳态起到重要的调节作用，近年来的研究表明，FXR在肝脏再生、炎症甚至肝细胞癌的发生中均起到一定的调节作用。本研究明确了FXR在肝脏炎症、肝纤维化、肝细胞癌不同发生阶段的作用并探讨了部分作用机制，本研究的意义不仅在于进一步揭示FXR的功能，也为HCC的发生机制的研究提供了新的视角并为治疗及预防提供了新的靶点。本研究取得了如下的结果：
1.FXR对二硝基乙二胺（DEN）造成急性肝损伤的作用
本研究首先比较了在DEN急性作用下FXR缺陷小鼠(FXR-/-)与野生型小鼠（Wild-type）对其反应差异，结果证实在缺少FXR的保护作用下，小鼠肝脏表现出对DEN的敏感性增强，表现为转氨酶升高，肝细胞凋亡增加及细胞因子的释放增多，无论是炎症因子TNF-α、IL-6、IL-1β以及肝细胞再生相关因子TGF-α、HGF在FXR缺失肝脏中表达升高更明显。此外通过EMSA方法证实NF-κB转录活性也升高，但是在FXR-/-小鼠中变化更为明显。为了明确FXR对NF-κB的调节作用，在体外环境下，我们证实FXR激活后对肝癌细胞系及原代小鼠肝细胞中NF-κB介导的炎性因子（TNF-α、IL-6、COX2）表达具有显著的抑制作用，而在FXR缺陷肝细胞中，NF-κB介导的炎性因子的活化不能被抑制。结果说明FXR缺失小鼠肝脏对DEN造成肝损害敏感性增强的原因可能由于其缺乏对NF-κB的抑制作用，导致NF-κB过度激活及炎症反应的发生。
2. FXR在DEN造成小鼠HCC形成中的作用
本研究中，成功建立了DEN诱导的wild-type小鼠及FXR-/-小鼠HCC模型，FXR基因缺失可导致小鼠对DEN的敏感性增加，FXR缺陷的小鼠显示更高的成瘤率，表现出肿瘤结节更多、肿瘤体积更大的特点。在对组织细胞凋亡及增殖检测中，我们发现FXR-/-小鼠肝癌组织细胞凋亡及增殖活性均较wild-type小鼠明显，除此之外，肝内炎症反应也明显增强。在进一步针对组织标本检测中我们发现，细胞因子TNF-α、IL-6、IL-1β表达水平在癌组织中基因表达均高于周围正常组织，而TNF-α与IL-6在FXR-/-小鼠的癌及癌旁组织均较wild-type小鼠升高，尤以癌组织变化更为明显。通过EMSA实验证实NF-κB的转录活性无论是在癌组织还是癌旁组织较wild-type小鼠比较均明显增强。为了进一步研究DEN诱导wild-type和FXR-/-小鼠产生差异表型的分子学变化，我们检测了与肿瘤发生关系密切的因子表达情况，结果显示：在两组小鼠中，CyclinD1及CyclinE在肿瘤组织中的表达水平均高于相应的癌周组织。与wild-type小鼠比较，CyclinD1及CyclinE的表达无论在FXR-/-小鼠的肝癌组织还是癌周组织中均有所升高，尤以癌组织升高明显；P53蛋白在FXR-/-小鼠肿瘤及癌旁组织表达水平低于wild-type小鼠，尤其在肿瘤组织其表达受到明显抑制。进一步的体外研究结果提示上调FXR表达并不能影响UV诱导的P53表达。其机制还需要深入探讨。
3.FXR对肝纤维化形成的影响
在DEN慢性损伤小鼠模型中，FXR-/-小鼠中，TGF-β1及TIMP-1mRNA表达明显高于wild-type小鼠，说明在FXR缺失条件下，组织发生肝纤维化程度更加明显。为了进一步验证FXR与肝纤维化的关系，我们成功分离了wild-type及FXR-/-小鼠的肝脏星状细胞（HSC），加入FXR激活剂GW4064作用后，在TGF-β1作用下，检测TGFβ1靶基因PAI-1, α-SMA和Collagenα1的mRNA表达情况，同时我们也检测了FXR的靶基因SHP的表达情况。结果证明，HSC存在FXR的表达，激活FXR表达后能够显著抑制TGF-β1诱导的纤维化相关因子的表达，而在FXR缺失组未见到明显抑制效应。而在检测SHP表达中也意外发现TGF-β1能够下调SHP的表达。综合上述结果，FXR具有保护TGF-β1诱导肝纤维化的作用，同时在肝纤维化形成过程中TGF-β1分泌的增加还可抑制HSC中FXR的激活而加速肝纤维化的进程。
4.人HCC组织标本中FXR表达情况
本研究进一步检测了FXR在人肝癌组织表达情况。共选取了41例确诊肝癌患者的术后组织标本（包括肝癌组织及癌周正常组织），通过定量PCR及免疫组化染色，评价了FXR相关基因及FXR蛋白在癌组织及癌旁组织的表达情况，检测结果提示FXR及FXR靶基因SHP在癌旁组织的表达明显低于癌周正常组织。进一步检测了前期Microarray结果中证实了在wild-type与FXR-/-小鼠肝脏表达差异最显著的基因，这些基因与细胞代谢、增殖及炎症相关。结果与我们预期相似，与正常肝组织比较，8个基因在人类HCC及FXR-/-小鼠肝脏表现出相似的上调或下调。在FXR-/-小鼠肝脏表达下调的基因CXCL1,DPYS, CD36, LIP1, SOCS3在人HCC中同样表达下调。其中FXR的靶基因SOCS3, CD36, LAGLS1已被证实与肿瘤的发生及发展密切相关。我们检测了FXR启动子的甲基化水平，在FXR的启动子区未发现典型的CpG岛，用去甲基化剂作用HepG2细胞72h，检测FXR基因表达情况，结果显示，FXR的表达在5-Aza-dC作用前后无明显变化。说明FXR下调与其启动子的高甲基化水平无明显相关。
5.炎性因子对FXR转录活性的影响
为了进一步探讨FXR下调的机制，小鼠肝脏原代肝细胞被分离培养，在培养体系中分别加入不同浓度的TNFα, IL-1β or IL-6。我们观察到在TNFα (10ng/ml), IL-1β (50ng/ml) and IL-6(1ng/ml)可以明显降低FXR mRNA的表达。为了明确FXR的表达是否与其启动子活性降低有关，将包含有HNF-1α结合位点(p150/+129)的人FXR启动子序列插入含有荧光素酶（luciferase）报告基因的pGL-3载体中，构建重组质粒报告质粒转染HepG2细胞及Hep3B细胞，转染24h后，分别在培养体系中加入TNFα, IL-1β, or IL-6（10ng/ml,50ng/ml）作用24h，应用双荧光素酶报告系统检测荧光素酶活性，结果显示，在HepG2细胞中，底物中的luciferase活性明显降低，说明FXR启动子的活性受到抑制，在Hep3B中重复上述实验我们得出了相同的结果，尤其在高浓度的IL-1β、TNF-α作用下，抑制作用更明显。如将突变序列的FXR启动子报告质粒(pGL3mut)转染HepG2细胞，在同样的条件下，底物中的luciferase活性无明显变化，FXR启动子的活性没有受到抑制。结果说明炎性因子可以通过HNF1α使FXR的转录活性下降。进一步检测了炎性因子对HNF1α基因蛋白表达的影响，结果显示细胞因子对HNF1α的基因及蛋白表达无明显的抑制作用。
为了进一步明确炎性因子对FXR的下调机制，应用染色质免疫共沉淀(CHIP)实验检测了炎性因子TNFα对HNF1α DNA结合活性的影响。结果显示：加入IgG抗体的对照组在TNFα的刺激作用下，对FXR结合活性没有影响，而加入HNF-1α抗体组在TNFα刺激后与未处理组比较，HNF-1α与FXR启动子的结合活性明显下降，差异有显著性。为了进一步确定其对肝癌细胞的作用，在另一个肝癌细胞系Hun-7中重复了上述实验，得到了同样的结果。上述结果提示炎性因子可以通过降低HNF1α与FXR启动子的结合活性从而使FXR的转录活性下降，导致FXR表达降低。
HCC development is multi-factorial, complex multi-gene andmulti-step process, HCC occurrence, the specific mechanisms ofdevelopment continues to be explored. Chronic hepatitis C virus infection(HBV/HCV) is the most important reason leading to cirrhosis, livercirrhosis is an important risk factor of HCC and nearly1/3patients withcirrhosis developed to HCC eventually. In recent years, more and moreevidence demonstrated that metabolism disorders also associated with theoccurrence of HCC, nuclear receptor FXR as an important regulator ofbile acids, carbohydrates, lipids play an important role in regulatinghomeostasis, and recent studies showed, that FXR also related with liverregeneration, inflammation and even hepatocellular carcinoma. In thisstudy, the important roles of FXR in hepatic inflammation, fibrosis, andhepatocellular carcinoma were further investigated in mice model andhuman HCC patients. Our study is not only to reveal the function of FXR,furthermore, it provides a new perspectives for the treatment and a newtarget of HCC.
1.The role of FXR on DEN inducing acute liver injury
In the first study, the different response to the acute effects of DENin FXR deficient mice and wild-type mice were compared, the resultsshowed livers of mice without the protection of FXR exhibited enhancedsensitivity to DEN, performance of higer transaminase levels, increasedapoptosis of hepatocytes and cytokines secreting. The expression mRNAslevels of inflammatory cytokines either TNF-α, IL-6, IL-1β or regeneration-associated factor TGF-α, HGF were elevated moreobviously in the livers absence of FXR in24h timepoints. NF-κBtranscriptional activity was further confirmed to be increased in FXR-/-mice by EMSA compared to wild-type mice. In order to clarify the role ofFXR in regμl ating NF-κB activity, in vitro study was further investigated.The resμl ts showed NF-κB-mediated inflammatory cytokines (TNF-α,IL-6, COX2) expression has significantly inhibit after activation of FXRin both primary hepatocellular carcinoma cell lines and mouse liver cells,but the same suppressive effect coμl d not seen in FXR deficient livercells. These results suggested that increased sensitivity to DEN inFXR-deficient mice may due to their lack of inhibition of NF-κB by FXR,resulting in NF-κB activation and the occurrence of excessiveinflammatory response.
2.The role of FXR on formation of HCC mice model induced by DEN.
In this study, DEN-induced HCC model in wild-type and FXRdeficient mice were successful established. With FXR gene deletion, themice showed increased susceptibility to DEN, which displayed a highertumor formation rate (100%VS.80%), more tumor nodules and greatertumor volume characteristics. In apoptosis and proliferation assays, wefound that apoptosis and proliferation were significantly increased inFXR-/-mice cancer cells compared with wild-type liver cells, in addition,liver inflammation was also significantly enhanced. For further tissuespecimens, we found that the cytokine TNF-α, IL-6, IL-1β expressionlevel of gene expression in cancer tissues were higher than thesurrounding normal tissue, and expressions of TNF-α and IL-6werehigher in both FXR-/-adjacent tissue and cancer tissue compared withwild-type mice, especially in cancer tissues. Increased transcriptionalactivity of NF-κB by EMSA was confirmed in either adjacent tissues or cancer tissue of FXR-/-mice compared with wild-type mice. To furtherinvestigate molecular phenotype differences in hepatocarcinogenesis, weexamined the expression of factors related to tumor occurrence, theresults showed that CyclinD1and CyclinE mRNA levels in tumor tissueswere higher than the corresponding peritumoral tissues in the two groupsof mice compared with wild-type mice, expression of cyclinE andcyclinD1in HCC tissue and peritumoral tissues were elevated in FXR-/-mice, especially in cancer tissues; P53protein levels were much lower inFXR-/-mice tumors and adjacent tissues than in wild-type mice,particularly its expression in tumor tissues was significantly inhibited.But the further results in vitro showed FXR can not affect P53expression after UV irridation. The mechanism needs to be futherinvestigated.
3. Effect of FXR on liver fibrosis
In DEN chronic injury mouse model, TGF-β1and TIMP-1mRNAlevels in FXR-/-mice livers were significantly higher than wild-type mice,indicating that the degree of liver fibrosis is more obvious in the absenceof FXR conditions. To further investigate the relationship between FXRand liver fibrosis, we successfully separated the hepatic stellate cells fromthe livers of wild-type and FXR-/-mice, the mRNA expression of TGFβ1target gene PAI-1, α-SMA and Collagenα1were examined with TGF-β1stimμl ating after adding FXR activators--GW4064in HSCs, and theexpression of FXR target genes SHP has also been checked. The resultsdemonstrated that there FXR activation of hepatic stellate cells cansignificantly inhibit the expression of TGF-β1-induced expression offibrosis-related factors, and the significant inhibition can not be seen inthe absence of FXR group. Intrestingly, the expression of SHP can bedownregμl ated by TGF-β1inversely. Based on the results, FXR can protect TGF-β1-induced hepatic fibrosis, liver fibrosis and meanwhile thesecretion of TGF-β1after HSC activation can inhibit FXR activationwhich accelerate the process of the formation of hepatic fibrosis.
4. FXR expression in Human HCC tissue specimens
The expression of FXR in human HCC tissues were furtherexamined. In the study,41specimens (including hepatocellular carcinomaand adjacent normal tissue) were selected according to differentpathological grade, both the mRNA expression of FXR and related genesand the protein levels of FXR in human HCC tissue were significantlydecreased compared to adjacent normal tissues by quantitative PCR andimmunohistochemical staining. We further checked some genes whichhad been confirmed to be significant differentially expressed genes inwild-type and FXR-/-mice liver using microarray assay previously, thesegenes related with cellular metabolism, proliferation and inflammation.Similar results were obtained in human HCC tissus compared withnormal liver tissue, eight genes in human HCC and FXR-/-mouse liversshowed similar upregulated or downregμl ated. CXCL1, DPYS, CD36,LIP1, and SOCS3which were decreased in FXR-/-mice liver also showeddownregμl ated in human HCC. FXR target gene SOCS3, CD36andLAGLS1has been confirmed closely associated with tumorigenesis andtumor development. Then we examined the level of methylation of FXRpromoter region, no typical CpG island is found in FXR promoter region.FXR gene expression is detected after methylation agent was added toHepG2cells for72h, the results showed that no significant change ofFXR mRMA expression with5-Aza-dC. The resμl ts demonstrated thatdownregulation of FXR expession was no significant correlated to thehypermethylation of FXR promoter.
5. Inflammatory factors regulating the transcriptional activity of FXR
To further investigate the mechanism of reduction of FXR in humorHCC tissues, primary hepatocytes were isolated and differentconcentrations of TNFα, IL-1β or IL-6were added to the cμl ture system.We observed that TNFα (10ng/ml), IL-1β (50ng/ml) and IL-6(1ng/ml) can significantly reduce the expression of FXR mRNA levels. Tofurther investigate whether the downregμl ated of FXR expression is dueto decreased activity of FXR promoter, the sequence of HNF-1α bindingsites (p-150/+129) in promoter of human FXR was inserted intorecombinant plasmid vector pGL-3which contain luciferase (luciferase)reporter gene. The reporter plasmids were transfected into HepG2cellsfor24h, respectively, in the culture system with TNFα, IL-1β, or IL-6(10ng/ml,50ng/ml) for24h, we used double fluorescence applicationsluciferase reporter system to detect luciferase activity, and the resultsshowed that the substrate of luciferase activity was significantly reducedwhich demonstrated the FXR promoter activity was inhibited bycytokines. The experiment was repeated in Hep3B cell line and the sameresults were obtained, the effect was more obvious at high concentrationsof IL-1β, TNF-α. Furthermore,, the substrate of luciferase activity was nosignificant change and FXR promoter activity was not inhibited if themutant sequence of FXR were inserted to promoter reporter plasmid(pGL3mut) then transfected HepG2cells under the same conditions. Theresults indicated that inflammatory cytokines can decrease FXRtranscriptional activity by HNF-1α. Then we examined the effect on geneexpression of HNF1α with inflammatory factors, the results show thatcytokines can not regulate the gene and protein expression of HNF1α.
In order to further investigate the mechanism of inflammatorycytokines on FXR expression, chromatin immunoprecipitation (CHIP)experiments were applied to examined the effect of inflammatory cytokines TNFα on HNF1α DNA binding activity with FXR. The resultsshowed: FXR binding activity was not affected with TNFα stimulation inIgG antibody control group, but the binding activity of HNF-1promoterand FXR was significant decreased after TNFα stimulation in HNF-1antibody group compared with the untreated group. In order to determineits effect on liver cancer cells, we repeated the experiment above inanother tumro cell line Huh-7, and got the same result. These resultssuggest that inflammatory cytokines can impair the binding activity ofHNF1α promoter and FXR, which led to the transcriptional activity ofFXR decreased, resulting in decreased expression of FXR.
1.FXR对二硝基乙二胺（DEN）造成急性肝损伤的作用
本研究首先比较了在DEN急性作用下FXR缺陷小鼠(FXR-/-)与野生型小鼠（Wild-type）对其反应差异，结果证实在缺少FXR的保护作用下，小鼠肝脏表现出对DEN的敏感性增强，表现为转氨酶升高，肝细胞凋亡增加及细胞因子的释放增多，无论是炎症因子TNF-α、IL-6、IL-1β以及肝细胞再生相关因子TGF-α、HGF在FXR缺失肝脏中表达升高更明显。此外通过EMSA方法证实NF-κB转录活性也升高，但是在FXR-/-小鼠中变化更为明显。为了明确FXR对NF-κB的调节作用，在体外环境下，我们证实FXR激活后对肝癌细胞系及原代小鼠肝细胞中NF-κB介导的炎性因子（TNF-α、IL-6、COX2）表达具有显著的抑制作用，而在FXR缺陷肝细胞中，NF-κB介导的炎性因子的活化不能被抑制。结果说明FXR缺失小鼠肝脏对DEN造成肝损害敏感性增强的原因可能由于其缺乏对NF-κB的抑制作用，导致NF-κB过度激活及炎症反应的发生。
2. FXR在DEN造成小鼠HCC形成中的作用
本研究中，成功建立了DEN诱导的wild-type小鼠及FXR-/-小鼠HCC模型，FXR基因缺失可导致小鼠对DEN的敏感性增加，FXR缺陷的小鼠显示更高的成瘤率，表现出肿瘤结节更多、肿瘤体积更大的特点。在对组织细胞凋亡及增殖检测中，我们发现FXR-/-小鼠肝癌组织细胞凋亡及增殖活性均较wild-type小鼠明显，除此之外，肝内炎症反应也明显增强。在进一步针对组织标本检测中我们发现，细胞因子TNF-α、IL-6、IL-1β表达水平在癌组织中基因表达均高于周围正常组织，而TNF-α与IL-6在FXR-/-小鼠的癌及癌旁组织均较wild-type小鼠升高，尤以癌组织变化更为明显。通过EMSA实验证实NF-κB的转录活性无论是在癌组织还是癌旁组织较wild-type小鼠比较均明显增强。为了进一步研究DEN诱导wild-type和FXR-/-小鼠产生差异表型的分子学变化，我们检测了与肿瘤发生关系密切的因子表达情况，结果显示：在两组小鼠中，CyclinD1及CyclinE在肿瘤组织中的表达水平均高于相应的癌周组织。与wild-type小鼠比较，CyclinD1及CyclinE的表达无论在FXR-/-小鼠的肝癌组织还是癌周组织中均有所升高，尤以癌组织升高明显；P53蛋白在FXR-/-小鼠肿瘤及癌旁组织表达水平低于wild-type小鼠，尤其在肿瘤组织其表达受到明显抑制。进一步的体外研究结果提示上调FXR表达并不能影响UV诱导的P53表达。其机制还需要深入探讨。
3.FXR对肝纤维化形成的影响
在DEN慢性损伤小鼠模型中，FXR-/-小鼠中，TGF-β1及TIMP-1mRNA表达明显高于wild-type小鼠，说明在FXR缺失条件下，组织发生肝纤维化程度更加明显。为了进一步验证FXR与肝纤维化的关系，我们成功分离了wild-type及FXR-/-小鼠的肝脏星状细胞（HSC），加入FXR激活剂GW4064作用后，在TGF-β1作用下，检测TGFβ1靶基因PAI-1, α-SMA和Collagenα1的mRNA表达情况，同时我们也检测了FXR的靶基因SHP的表达情况。结果证明，HSC存在FXR的表达，激活FXR表达后能够显著抑制TGF-β1诱导的纤维化相关因子的表达，而在FXR缺失组未见到明显抑制效应。而在检测SHP表达中也意外发现TGF-β1能够下调SHP的表达。综合上述结果，FXR具有保护TGF-β1诱导肝纤维化的作用，同时在肝纤维化形成过程中TGF-β1分泌的增加还可抑制HSC中FXR的激活而加速肝纤维化的进程。
4.人HCC组织标本中FXR表达情况
本研究进一步检测了FXR在人肝癌组织表达情况。共选取了41例确诊肝癌患者的术后组织标本（包括肝癌组织及癌周正常组织），通过定量PCR及免疫组化染色，评价了FXR相关基因及FXR蛋白在癌组织及癌旁组织的表达情况，检测结果提示FXR及FXR靶基因SHP在癌旁组织的表达明显低于癌周正常组织。进一步检测了前期Microarray结果中证实了在wild-type与FXR-/-小鼠肝脏表达差异最显著的基因，这些基因与细胞代谢、增殖及炎症相关。结果与我们预期相似，与正常肝组织比较，8个基因在人类HCC及FXR-/-小鼠肝脏表现出相似的上调或下调。在FXR-/-小鼠肝脏表达下调的基因CXCL1,DPYS, CD36, LIP1, SOCS3在人HCC中同样表达下调。其中FXR的靶基因SOCS3, CD36, LAGLS1已被证实与肿瘤的发生及发展密切相关。我们检测了FXR启动子的甲基化水平，在FXR的启动子区未发现典型的CpG岛，用去甲基化剂作用HepG2细胞72h，检测FXR基因表达情况，结果显示，FXR的表达在5-Aza-dC作用前后无明显变化。说明FXR下调与其启动子的高甲基化水平无明显相关。
5.炎性因子对FXR转录活性的影响
为了进一步探讨FXR下调的机制，小鼠肝脏原代肝细胞被分离培养，在培养体系中分别加入不同浓度的TNFα, IL-1β or IL-6。我们观察到在TNFα (10ng/ml), IL-1β (50ng/ml) and IL-6(1ng/ml)可以明显降低FXR mRNA的表达。为了明确FXR的表达是否与其启动子活性降低有关，将包含有HNF-1α结合位点(p150/+129)的人FXR启动子序列插入含有荧光素酶（luciferase）报告基因的pGL-3载体中，构建重组质粒报告质粒转染HepG2细胞及Hep3B细胞，转染24h后，分别在培养体系中加入TNFα, IL-1β, or IL-6（10ng/ml,50ng/ml）作用24h，应用双荧光素酶报告系统检测荧光素酶活性，结果显示，在HepG2细胞中，底物中的luciferase活性明显降低，说明FXR启动子的活性受到抑制，在Hep3B中重复上述实验我们得出了相同的结果，尤其在高浓度的IL-1β、TNF-α作用下，抑制作用更明显。如将突变序列的FXR启动子报告质粒(pGL3mut)转染HepG2细胞，在同样的条件下，底物中的luciferase活性无明显变化，FXR启动子的活性没有受到抑制。结果说明炎性因子可以通过HNF1α使FXR的转录活性下降。进一步检测了炎性因子对HNF1α基因蛋白表达的影响，结果显示细胞因子对HNF1α的基因及蛋白表达无明显的抑制作用。
为了进一步明确炎性因子对FXR的下调机制，应用染色质免疫共沉淀(CHIP)实验检测了炎性因子TNFα对HNF1α DNA结合活性的影响。结果显示：加入IgG抗体的对照组在TNFα的刺激作用下，对FXR结合活性没有影响，而加入HNF-1α抗体组在TNFα刺激后与未处理组比较，HNF-1α与FXR启动子的结合活性明显下降，差异有显著性。为了进一步确定其对肝癌细胞的作用，在另一个肝癌细胞系Hun-7中重复了上述实验，得到了同样的结果。上述结果提示炎性因子可以通过降低HNF1α与FXR启动子的结合活性从而使FXR的转录活性下降，导致FXR表达降低。
HCC development is multi-factorial, complex multi-gene andmulti-step process, HCC occurrence, the specific mechanisms ofdevelopment continues to be explored. Chronic hepatitis C virus infection(HBV/HCV) is the most important reason leading to cirrhosis, livercirrhosis is an important risk factor of HCC and nearly1/3patients withcirrhosis developed to HCC eventually. In recent years, more and moreevidence demonstrated that metabolism disorders also associated with theoccurrence of HCC, nuclear receptor FXR as an important regulator ofbile acids, carbohydrates, lipids play an important role in regulatinghomeostasis, and recent studies showed, that FXR also related with liverregeneration, inflammation and even hepatocellular carcinoma. In thisstudy, the important roles of FXR in hepatic inflammation, fibrosis, andhepatocellular carcinoma were further investigated in mice model andhuman HCC patients. Our study is not only to reveal the function of FXR,furthermore, it provides a new perspectives for the treatment and a newtarget of HCC.
1.The role of FXR on DEN inducing acute liver injury
In the first study, the different response to the acute effects of DENin FXR deficient mice and wild-type mice were compared, the resultsshowed livers of mice without the protection of FXR exhibited enhancedsensitivity to DEN, performance of higer transaminase levels, increasedapoptosis of hepatocytes and cytokines secreting. The expression mRNAslevels of inflammatory cytokines either TNF-α, IL-6, IL-1β or regeneration-associated factor TGF-α, HGF were elevated moreobviously in the livers absence of FXR in24h timepoints. NF-κBtranscriptional activity was further confirmed to be increased in FXR-/-mice by EMSA compared to wild-type mice. In order to clarify the role ofFXR in regμl ating NF-κB activity, in vitro study was further investigated.The resμl ts showed NF-κB-mediated inflammatory cytokines (TNF-α,IL-6, COX2) expression has significantly inhibit after activation of FXRin both primary hepatocellular carcinoma cell lines and mouse liver cells,but the same suppressive effect coμl d not seen in FXR deficient livercells. These results suggested that increased sensitivity to DEN inFXR-deficient mice may due to their lack of inhibition of NF-κB by FXR,resulting in NF-κB activation and the occurrence of excessiveinflammatory response.
2.The role of FXR on formation of HCC mice model induced by DEN.
In this study, DEN-induced HCC model in wild-type and FXRdeficient mice were successful established. With FXR gene deletion, themice showed increased susceptibility to DEN, which displayed a highertumor formation rate (100%VS.80%), more tumor nodules and greatertumor volume characteristics. In apoptosis and proliferation assays, wefound that apoptosis and proliferation were significantly increased inFXR-/-mice cancer cells compared with wild-type liver cells, in addition,liver inflammation was also significantly enhanced. For further tissuespecimens, we found that the cytokine TNF-α, IL-6, IL-1β expressionlevel of gene expression in cancer tissues were higher than thesurrounding normal tissue, and expressions of TNF-α and IL-6werehigher in both FXR-/-adjacent tissue and cancer tissue compared withwild-type mice, especially in cancer tissues. Increased transcriptionalactivity of NF-κB by EMSA was confirmed in either adjacent tissues or cancer tissue of FXR-/-mice compared with wild-type mice. To furtherinvestigate molecular phenotype differences in hepatocarcinogenesis, weexamined the expression of factors related to tumor occurrence, theresults showed that CyclinD1and CyclinE mRNA levels in tumor tissueswere higher than the corresponding peritumoral tissues in the two groupsof mice compared with wild-type mice, expression of cyclinE andcyclinD1in HCC tissue and peritumoral tissues were elevated in FXR-/-mice, especially in cancer tissues; P53protein levels were much lower inFXR-/-mice tumors and adjacent tissues than in wild-type mice,particularly its expression in tumor tissues was significantly inhibited.But the further results in vitro showed FXR can not affect P53expression after UV irridation. The mechanism needs to be futherinvestigated.
3. Effect of FXR on liver fibrosis
In DEN chronic injury mouse model, TGF-β1and TIMP-1mRNAlevels in FXR-/-mice livers were significantly higher than wild-type mice,indicating that the degree of liver fibrosis is more obvious in the absenceof FXR conditions. To further investigate the relationship between FXRand liver fibrosis, we successfully separated the hepatic stellate cells fromthe livers of wild-type and FXR-/-mice, the mRNA expression of TGFβ1target gene PAI-1, α-SMA and Collagenα1were examined with TGF-β1stimμl ating after adding FXR activators--GW4064in HSCs, and theexpression of FXR target genes SHP has also been checked. The resultsdemonstrated that there FXR activation of hepatic stellate cells cansignificantly inhibit the expression of TGF-β1-induced expression offibrosis-related factors, and the significant inhibition can not be seen inthe absence of FXR group. Intrestingly, the expression of SHP can bedownregμl ated by TGF-β1inversely. Based on the results, FXR can protect TGF-β1-induced hepatic fibrosis, liver fibrosis and meanwhile thesecretion of TGF-β1after HSC activation can inhibit FXR activationwhich accelerate the process of the formation of hepatic fibrosis.
4. FXR expression in Human HCC tissue specimens
The expression of FXR in human HCC tissues were furtherexamined. In the study,41specimens (including hepatocellular carcinomaand adjacent normal tissue) were selected according to differentpathological grade, both the mRNA expression of FXR and related genesand the protein levels of FXR in human HCC tissue were significantlydecreased compared to adjacent normal tissues by quantitative PCR andimmunohistochemical staining. We further checked some genes whichhad been confirmed to be significant differentially expressed genes inwild-type and FXR-/-mice liver using microarray assay previously, thesegenes related with cellular metabolism, proliferation and inflammation.Similar results were obtained in human HCC tissus compared withnormal liver tissue, eight genes in human HCC and FXR-/-mouse liversshowed similar upregulated or downregμl ated. CXCL1, DPYS, CD36,LIP1, and SOCS3which were decreased in FXR-/-mice liver also showeddownregμl ated in human HCC. FXR target gene SOCS3, CD36andLAGLS1has been confirmed closely associated with tumorigenesis andtumor development. Then we examined the level of methylation of FXRpromoter region, no typical CpG island is found in FXR promoter region.FXR gene expression is detected after methylation agent was added toHepG2cells for72h, the results showed that no significant change ofFXR mRMA expression with5-Aza-dC. The resμl ts demonstrated thatdownregulation of FXR expession was no significant correlated to thehypermethylation of FXR promoter.
5. Inflammatory factors regulating the transcriptional activity of FXR
To further investigate the mechanism of reduction of FXR in humorHCC tissues, primary hepatocytes were isolated and differentconcentrations of TNFα, IL-1β or IL-6were added to the cμl ture system.We observed that TNFα (10ng/ml), IL-1β (50ng/ml) and IL-6(1ng/ml) can significantly reduce the expression of FXR mRNA levels. Tofurther investigate whether the downregμl ated of FXR expression is dueto decreased activity of FXR promoter, the sequence of HNF-1α bindingsites (p-150/+129) in promoter of human FXR was inserted intorecombinant plasmid vector pGL-3which contain luciferase (luciferase)reporter gene. The reporter plasmids were transfected into HepG2cellsfor24h, respectively, in the culture system with TNFα, IL-1β, or IL-6(10ng/ml,50ng/ml) for24h, we used double fluorescence applicationsluciferase reporter system to detect luciferase activity, and the resultsshowed that the substrate of luciferase activity was significantly reducedwhich demonstrated the FXR promoter activity was inhibited bycytokines. The experiment was repeated in Hep3B cell line and the sameresults were obtained, the effect was more obvious at high concentrationsof IL-1β, TNF-α. Furthermore,, the substrate of luciferase activity was nosignificant change and FXR promoter activity was not inhibited if themutant sequence of FXR were inserted to promoter reporter plasmid(pGL3mut) then transfected HepG2cells under the same conditions. Theresults indicated that inflammatory cytokines can decrease FXRtranscriptional activity by HNF-1α. Then we examined the effect on geneexpression of HNF1α with inflammatory factors, the results show thatcytokines can not regulate the gene and protein expression of HNF1α.
In order to further investigate the mechanism of inflammatorycytokines on FXR expression, chromatin immunoprecipitation (CHIP)experiments were applied to examined the effect of inflammatory cytokines TNFα on HNF1α DNA binding activity with FXR. The resultsshowed: FXR binding activity was not affected with TNFα stimulation inIgG antibody control group, but the binding activity of HNF-1promoterand FXR was significant decreased after TNFα stimulation in HNF-1antibody group compared with the untreated group. In order to determineits effect on liver cancer cells, we repeated the experiment above inanother tumro cell line Huh-7, and got the same result. These resultssuggest that inflammatory cytokines can impair the binding activity ofHNF1α promoter and FXR, which led to the transcriptional activity ofFXR decreased, resulting in decreased expression of FXR.
引文
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