抑癌基因Tg737在肝细胞癌发生发展中作用机制的研究
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摘要
【背景】
肝细胞癌(hepatocellular carcinoma, HCC)为原发性肝癌的一种临床分型，是我国常见的恶性肿瘤之一，且发病率逐年上升，其死亡率在恶性肿瘤中仅次于胃、食道居第三位。近年来HCC的诊断和治疗取得到了一定的发展，但预后仍然较差，阐明HCC的发生、发展机理，寻找新的、有效的杀伤HCC细胞方法是解决HCC诊治的根本途径。
Tg737基因是新近发现的一种HCC抑癌基因，最初进行研究时发现其与常染色体隐性遗传性多囊肾病（autosomal recessive polycystic kidney disease, ARPKD）orpk鼠突变模型研究中的目的基因相同。该基因编码824个氨基酸的多肽序列，该多肽链中包含有十个串行排列的三十四肽重复基序（tetratricopeptide repeat，TPR）。虽然Tg737基因已证实在肿瘤形成中起着重要作用，包括肝脏、肾脏、胰腺，但是针对其在HCC发病过程中如何发挥作用的研究仍处于起步阶段。对Tg737基因的生物学功能的深入研究将为HCC开辟新的研究领域，阐明HCC分子调控网络，为寻找新的HCC的防治靶点奠定基础。
【目的】
为更好验证Tg737在胎肝干/祖细胞（fetal liver stem/progenitor cells, FLSPCs）恶性转化中的作用机制，本课题首先完善了分化方案，进行脱细胞化肝脏生物衍生支架动态培养（dynamic cultured scaffold，DCS）诱导骨髓间充质干细胞（bonemesenchymal stem cells，BMSCs）、FLSPCs向肝样细胞分化的研究，进而将所建立的诱导分化方案用于下步相关研究；研究Tg737信号通路在HCC发生、发展中的作用，为HCC的防治提供新的理论依据及干预靶点。
【方法】
1.构建大鼠脱细胞肝支架；将富集的BMSCs、FLSPCs经鉴定后分别分为两组，一组接种于支架上，添加肝细胞生长因子（hepatocyte growth factor，HGF）进行DCS，另一组加HGF贴壁培养；指定时间点western blot检测甲胎蛋白（alpha fetoprotein，AFP）、白蛋白（albumin，ALB）、叉头框蛋白A1（forkhead box protein A1，FOXA1）、肝细胞核因子4α（hepatic nuclear factor4alpha，HNF4α），细胞色素氧化酶CYP1A2（cytochrome P4501A2，CYP1A2）的表达差异。
2.利用shRNA慢病毒技术对抑制FLSPCs中Tg737的表达，在嘌呤霉素压力下培养，得到稳定转染细胞克隆Tg737抑制FLSPCs（Tg737-silent FLSPCs，sFLSPCs）；利用PCGC再次纯化正常FLSPCs（normal FLSPCs，nFLSPCs）、sFLSPCs，并通过流式细胞检测分析纯化后两组细胞CD133的表达，确保用于下步实验细胞的干细胞特性；通过聚合酶链式反应（polymerase chain reaction，PCR）、western blot、免疫荧光技术检测两组细胞Tg737RNA和蛋白水平。通过细胞计数、噻唑蓝（3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide，MTT）比色法绘制nFLSPCs、sFLSPCs生长曲线；流式细胞技术检测细胞的增殖变化（包括增殖核抗原Ki67的表达、细胞周期和细胞凋亡）；HGF诱导分化后，通过酶联免疫吸附试验（enzyme linked immunosorbent assay，ELISA）在指定时间点检测细胞ALB和AFP表达变化，诱导分化后透射电镜观察Tg737抑制前后细胞的超微结构变化；进一步采用DCS+HGF诱导两组细胞分化，培养7天后收集两组细胞，western blot检测AFP、ALB、FOXA1、HNF4α、CYP1A2的表达差异；Transwell技术检测细胞的侵袭能力变化；裸鼠皮下成瘤实验，观察Tg737抑制前后细胞成瘤能力变化，以及细胞对动物肝脏的损害情况。Western blot检测细胞周期蛋白cyclin D1和cyclin B表达，验证细胞周期蛋白是否参加了Tg737调控FLSPCs增殖的变化；利用western blot技术检测Tg737抑制后，HNF4α表达的变化；利用激光共聚焦技术检测了Tg737与HNF4α在细胞内的定位情况，验证Tg737抑制导致FLSPCs分化受阻现象的可能分子机制是HNF4α作为Tg737的下游分子伴侣。
3.含10%胎牛血清（fetal bovine serum，FBS）的达氏修正依氏培养基（Dulbecco'sModified Eagle Medium，DMEM）常规培养HepG2和MHCC97-H细胞。除特别指出，以下实验均用含1%FBS的DMEM处理细胞。用浓度1％的氧处理细胞达指定时间建立低氧模型。采用Annexin V/碘化丙啶（propidium iodide，PI）流式检测法排出1%FBS、低氧对细胞凋亡的影响；用浓度为1％的氧分别处理细胞（1%FBS）达指定时间，以常氧培养细胞（1%FBS）为对照组，检测细胞黏附、侵袭、转移力。Western blot检测10%FBS常氧组、1%FBS常氧组、1%FBS低氧组Tg737表达的变化。pcDNA3.1-Tg737经脂质体瞬时转染HepG2、MHCC97-H，转染pcDNA3.1()组、脂质体孵育组、空白组为对照；转染6h后，在缺氧条件下检测Tg737表达上调对细胞黏附、侵袭、转移力的影响；Annexin V/PI排出凋亡对实验的影响。Western blot检测10%FBS常氧组、1%FBS常氧组、1%FBS低氧组polycystin-1表达的变化；ELISA检测polycystin-1,白细胞介素-8（interleukin-8，IL-8）、转化生长因子-β1（transforming growth factor，TGF-β1）分泌的变化；在缺氧条件下检测Tg737表达上调对polycystin-1表达/分泌，IL-8、TGF-β1分泌的影响。
【结果】
1.富集的BMSCs纯度较高，细胞活力较好，高表达干细胞表面标志物CD44，而低表达造血干细胞（haematopoietic stem cells，HSCs）表面标志物CD45。富集的FLSPCs个头较小，核/浆比较大，高表达干细胞标志CD133和CD49f，低表达成熟肝细胞标志，能分化为表达ALB的类成熟肝细胞。在两组均添加生长因子的条件下，与二维贴壁培养比，DCS可更好的诱导BMSCs向肝样细胞分化，体现在诱导后的细胞高表达内胚层和肝细胞相关基因和蛋白，超微结构与功能更贴近肝细胞；体内实验证实DCS+生长因子诱导后的BMSCs对肝衰竭有更好的治疗效果。同样，DCS+HGF培养较贴壁+HGF培养更好的诱导FLSPCs向肝样细胞分化。该方案可用于下步相关研究。
2.新纯化的nFLSPCs、sFLSPCs在形态学上无明显区别；在培养过程中，新纯化的sFLSPCs与nFLSPCs相比在第1天均高表达CD133，无统计学差别，随时间延长，两组细胞CD133表达均下降，nFLSPCs组下降更明显，在7d、14d sFLSPCs中CD133的表达均高于nFLSPCs； PCR、western blot、免疫荧光技术检测Tg737的mRNA与蛋白表达水平，抑制效果得到确认。排出了凋亡因素的影响后，我们发现由于Tg737的抑制通过促进细胞周期过程使细胞增殖速度明显加快；HGF诱导分化后，通过ELISA、透射电镜证实Tg737的抑制导致细胞分化受阻，DCS+HGF方案亦证实分化受阻；Transwell侵袭实验证实sFLSPCs获取了较强的侵袭能力；裸鼠成瘤实验显示Tg737抑制后sFLSPCs虽然在皮下未形成明显肿瘤，但是对肝脏造成了损伤、甚至形成了肿瘤。进一步分子机制的研究证实：shRNA抑制Tg737后，cyclinD1和cyclin B表达显著上升；HNF4α随着Tg737的抑制表达而极度下调，Tg737和HNF4α在细胞内具有共表达的趋势。
3.低血清浓度、低氧条件对HepG2和MHCC97-H的活性无影响；与对照组相比，低氧减弱了肝癌细胞黏附力、增强了细胞侵袭、转移力。随着低氧模型的建立，HepG2和MHCC97-H中Tg737表达降低，而低血清浓度对Tg737的表达并无影响；western blot发现pcDNA3.1-Tg737转染并经低氧处理后，与对照组相比，pcDNA3.1-Tg737转染组中Tg737的表达明显上调；进一步发现由缺氧造成的肝癌细胞黏附、侵袭和转移力改变也随低氧下上调Tg737表达而减弱；与对照组相比，低氧诱导可下调HepG2和MHCC97-H polycystin-1的表达/分泌，上调IL-8的分泌，同时上调总TGF-β1、活化TGF-β1水平，而低血清浓度对以上分子的表达并无影响；随着在缺氧条件下上调Tg737的表达，polycystin-1的表达/分泌上调、IL-8的分泌、总TGF-β1、活化TGF-β1水平也相应下调；细胞活性因素、空载体、脂质体均对实验结果无影响。
【结论】
1.成功建立了DCS诱导分化方案，改方案能更好的诱导BMSCs、FLSPCs向肝样细胞分化，可用于FLSPCs的相关研究。
2. Tg737的抑制表达能够促进FLSPCs发生恶性转化，该恶性转化过程可能通过调节其下游靶基因cyclin D1、cyclin B和HNF4α实现。
3.低氧条件下HepG2和MHCC97-H黏附力下降、侵袭、转移力增强；Tg737及其下游靶基因polycystin-1、IL-8和TGF-β1参与了这一生物学行为。
[Background]
Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, isamong the most common malignancies, with an increasing incidence in China. Itsmortality rate ranks the third place after stomach cancer and esophagus cancer. Despitethere are some improvements in diagnosis and therapy of HCC, it still remains bigproblem because the clarification of molecular mechanisms responsible for HCCpathogenesis is unclear. It is of paramount importance to elucidate the the occurrence andprogression of HCC, not only for an understanding of tumor biology but also to permit the development of specific therapies that effectively target HCC.
Tg737, a new kind of tumor suppressor gene in HCC, was originally identified byinsertional mutagenesis and positional cloning as the gene which is altered in autosomalrecessive polycystic kidney disease in the TgN737Rpw transgenic mouse line. The Tg737gene encodes an824amino acid protein which includes10copies of a34-amino acidtetratrico-peptide repeat motif. Some previous studies provided evidence that Tg737mayplay an important role in carcinogenesis of kidney and pancreas. However, the role oftumor suppressor gene Tg737in the pathogenesis of HCC and the underlying molecularmechanisms remain poorly understood.
[Aims]
In order to effectively investigate whether Tg737may function in the malignanttransformation of fetal liver stem/progenitor cells (FLSPCs) and underlying molecularmechanisms, we first improved the differention method. To induce hepatic differentiation,bone mesenchymal stem cells (BMSCs) and FLSPCs were cultured using dynamiccultured scaffold (DCS), furthermore, this improved differention method was used insubsequent related experiments. Then, to investigate the role of Tg737signaling pathwayin in the pathogenesis of HCC and the underlying cellular and molecular mechanisms. Toexplore new melecular mechanisms regulating HCC pathogenesis and provide newtheoretical basis for HCC prevention and therapy.
[Methods]
1. A modified protocol previously reported was used to obtaine the decellularized liver.In this study, the identified murine BMSCs and FLSPCs were randomized into2groupsrespecticely. We compared the hepatic differentiation of BMSCs and FLSPCs in abiomatrix scaffold from rat liver (dynamic cultured scaffold (DCS) and in the presence ofhepatocyte growth factor (HGF)) with a two-dimensional substrate(adherent culture and inthe presence of HGF). At the indictaed time of cell growth, cells were harvested by trypsindigestion and then the expression of hepatocyte-specific genes alpha fetoprotein (AFP),albumin (ALB), forkhead box protein A1(FOXA1), hepatic nuclear factor4alpha (HNF4α)and cytochrome P4501A2(CYP1A2) were measured by western blot.
2. Tg737inhibition was achieved by short hairpin RNA (shRNA). StableshRNA-expressing clones (Tg737-silent FLSPCs, sFLSPCs) were selected by puromycindihydrochloride. To select cells expressing high levels of stem cell marker CD133for usein the next study, first step in this part was purification of Tg737normal FLSPCs (nFLSPCs) and sFLSPCs by PCGC. Then, CD133were analyzed by flow cytometry at1,7and14days during the culture period. Following RNAi of Tg737, the mRNA andprotein levels of sFLSPCs were measured by PCR, western blot andimmunocytochemistry analyses. The celluar growth curve was determined using cellcounting and3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.The proliferative capacity studies were determined by flow cytometry, including thecellular proliferation marker Ki-67expression analysis, cell cycle and apoptosis. Atindicated time of cell growth in the presence of HGF, enzyme linked immunosorbent assay(ELISA) was used for quantifying the ALB and AFP secreted by nFLSPCs and sFLSPCs.After incubation of nFLSPCs and sFLSPCs in complete medium supplemented with HGFfor7days, cells were harvested for fine structure analysis. nFLSPCs and sFLSPCs werealso seeded into scaffold for DCS, after7days in culture with complete mediumsupplemented with HGF, western blot was performed to measure AFP, ALB, FOXA1,HNF4α and CYP1A2expression. The invasion ability was evaluated by transwell assay.The in vivo tumorigenesis was detected by injection nFLSPCs and sFLSPCs intosubcutaneous of athymic mice. In order to investigate whether whether cyclin D1andcyclin B were involved in the effects of Tg737on cell proliferation and cell cycleprogression, western blot analysis was performed. Using western blot andimmunocytochemistry, we also analyzed the synergy of Tg737and HNF4α in thedifferentiation of FLSPCs.
3. HepG2and MHCC97-H cells were routinely cultured in Dulbecco's Modified EagleMedium (DMEM) supplemented with10%fetal bovine serum (FBS). In all subsequentrelated experiments, the cells were treated with medium supplemented with1%FBS,unless otherwise noted. For the incubation of cells under hypoxic conditions, the cellswere exposed to1%O2with5%CO2at37°C for the indicated times. To exclude thepossibility of apoptosis-related effects caused by low-serum medium under normoxic orhypoxic conditions in subsequent experiments, Annexin V/propidium iodide (PI) assayswere performed. HepG2and MHCC97-H cells were subjected to normoxic or hypoxicconditions, and the cell adhesion, invasion and migration capabilities were evaluated. Theexpression of Tg737under normoxia or hypoxia was detected using western blot.Furthermore, we created HepG2and MHCC97-H cells that overexpressed Tg737prior toincubation under hypoxia and investigated their metastatic characteristics. Finally, weanalyzed the involvement of critical molecular events (polycystin-1, interleukin-8(IL-8) and transforming growth factor β1(TGF-β1)) known to regulate invasion and migration.
[Results]
1. A systematic method for BMSCs enrichment used in this study can effectively enrichand purify BMSCs. The harvested BMSCs are in a good state, highly express stem cellsmarkers CD44, low expression of hematopoietic stem cell markers CD45. The enrichedFLSPCs were with small size, larger nuclear/plasma, highly express stem cells markersCD49f and CD133, low expression of mature liver markers. The isolated FLSPCs couldgenerate mature ALB expressing hepatocytes. In the presence of HGF, DCS could betterstimulate the BMSCs to express endodermal and hepatocyte-specific genes and proteinsassociated with improved functions, and the cells exhibited the ultrastructuralcharacteristics of mature hepatocytes. When transplanted into acute liver injured mice,DCS plus GF cultured cells exhibited increased liver function, survival, engraftment intothe host liver and further hepatic differentiation. Compared to the adherent with HGFcultured FLSPCs, the combination of DCS and HGF could better better induce FLSPCs todifferentiation into hepatocyte-like cells. This improved differentiation method can beused in subsequent related experiments.
2. The microscopic observations of freshly purified nFLSPCs and sFLSPCs revealed nosignificant morphological changes in sFLSPCs. Over the course of cell culture, on day1nFLSPCs and sFLSPCs showed high expression levels of CD133and there was nosignificant difference between the two groups; with time, the expression of CD133gradually decreased in both groups. CD133expression was significantly higher insFLSPCs than in nFLSPCs at all time points sampled except day1. Following Tg737inhibition, the mRNA and protein levels of sFLSPCs decreased as shown by PCR, westernblot and immunocytochemistry analyses. Excluding apoptosis-related effects, we foundthat Tg737inhibition resulted in enhanced cell proliferation through promoting cell-cycleprogression. Tg737inhibition also resulted in significant arrest of cell differentiation,stable expression of both ALB and AFP and quiescent ultrastructure. The improveddifferentiation method DCS plus HGF also confirmed differentiation arrest. Furthermore,the FLSPCs with Tg737inhibition got strong invasion and hepatocarcinogenesis ability.Cyclin D1and cyclin B are important for cell growth and are key signaling proteins in cellcycle progression. Silencing of Tg737induced increases in cyclin D1and cyclin B proteinlevels. Following downregulation of Tg737, the level of HNF4α decreased; these twoproteins co-localized in FLSPCs.
3. The treatment of HepG2and MHCC97-H cells with low-serum medium undernormoxic or hypoxic conditions did not significantly affect cell viability in vitro. Exposureof these two HCC cell lines to hypoxic conditions reduced HCC cell adhesion andfacilitated invasion and migration. In this study, Tg737expression was significantlyinhibited in HepG2and MHCC97-H cells following exposure to hypoxia. Thedownregulation of Tg737expression corresponded to significantly decreased adhesion andincreased invasion and migration. The treatment of cells with low-serum medium undernormoxia did not significantly affect Tg737expression. Hypoxia also decreased theexpression/secretion of polycystin-1, increased the IL-8, and increased the levels of activeand total TGF-β1. Moreover, the decrease in adhesiveness and the increase in the invasiveand migratory capacities of hypoxia-treated hepatoma cells were attenuated bypcDNA3.1-Tg737transfection prior to hypoxia. Finally, following the upregulation ofTg737, the expression/secretion of polycystin-1increased, and the secretion of IL-8andthe levels of active and total TGF-β1decreased correspondingly. Cell viability,liposome/pcDNA3.1(-) had no effects in our study.
[Conclusions]
1. An effective method for hepatic differentiation of BMSCs and FLSPCs issuccessfully constructed. The combination of DCS and HGF could better promote theBMSCs and FLSPCs to mature into functional HNCs. This improved differentiationmethod can be used in related experiments of FLSPCs.
2. Tg737inhibition by shRNA in FLSPCs leads to malignant transformation throughincreasing proliferation by accelerating cell-cycle progression and concomitantdifferentiation arrest, furthermore, cyclin D1, cyclin B and HNF4α signaling pathway maybe an important intermediary in this process.
3. Tg737contributes to hypoxia-induced invasion and migration, partially through thepolycystin-1, IL-8, and TGF-β1pathway.
肝细胞癌(hepatocellular carcinoma, HCC)为原发性肝癌的一种临床分型，是我国常见的恶性肿瘤之一，且发病率逐年上升，其死亡率在恶性肿瘤中仅次于胃、食道居第三位。近年来HCC的诊断和治疗取得到了一定的发展，但预后仍然较差，阐明HCC的发生、发展机理，寻找新的、有效的杀伤HCC细胞方法是解决HCC诊治的根本途径。
Tg737基因是新近发现的一种HCC抑癌基因，最初进行研究时发现其与常染色体隐性遗传性多囊肾病（autosomal recessive polycystic kidney disease, ARPKD）orpk鼠突变模型研究中的目的基因相同。该基因编码824个氨基酸的多肽序列，该多肽链中包含有十个串行排列的三十四肽重复基序（tetratricopeptide repeat，TPR）。虽然Tg737基因已证实在肿瘤形成中起着重要作用，包括肝脏、肾脏、胰腺，但是针对其在HCC发病过程中如何发挥作用的研究仍处于起步阶段。对Tg737基因的生物学功能的深入研究将为HCC开辟新的研究领域，阐明HCC分子调控网络，为寻找新的HCC的防治靶点奠定基础。
【目的】
为更好验证Tg737在胎肝干/祖细胞（fetal liver stem/progenitor cells, FLSPCs）恶性转化中的作用机制，本课题首先完善了分化方案，进行脱细胞化肝脏生物衍生支架动态培养（dynamic cultured scaffold，DCS）诱导骨髓间充质干细胞（bonemesenchymal stem cells，BMSCs）、FLSPCs向肝样细胞分化的研究，进而将所建立的诱导分化方案用于下步相关研究；研究Tg737信号通路在HCC发生、发展中的作用，为HCC的防治提供新的理论依据及干预靶点。
【方法】
1.构建大鼠脱细胞肝支架；将富集的BMSCs、FLSPCs经鉴定后分别分为两组，一组接种于支架上，添加肝细胞生长因子（hepatocyte growth factor，HGF）进行DCS，另一组加HGF贴壁培养；指定时间点western blot检测甲胎蛋白（alpha fetoprotein，AFP）、白蛋白（albumin，ALB）、叉头框蛋白A1（forkhead box protein A1，FOXA1）、肝细胞核因子4α（hepatic nuclear factor4alpha，HNF4α），细胞色素氧化酶CYP1A2（cytochrome P4501A2，CYP1A2）的表达差异。
2.利用shRNA慢病毒技术对抑制FLSPCs中Tg737的表达，在嘌呤霉素压力下培养，得到稳定转染细胞克隆Tg737抑制FLSPCs（Tg737-silent FLSPCs，sFLSPCs）；利用PCGC再次纯化正常FLSPCs（normal FLSPCs，nFLSPCs）、sFLSPCs，并通过流式细胞检测分析纯化后两组细胞CD133的表达，确保用于下步实验细胞的干细胞特性；通过聚合酶链式反应（polymerase chain reaction，PCR）、western blot、免疫荧光技术检测两组细胞Tg737RNA和蛋白水平。通过细胞计数、噻唑蓝（3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide，MTT）比色法绘制nFLSPCs、sFLSPCs生长曲线；流式细胞技术检测细胞的增殖变化（包括增殖核抗原Ki67的表达、细胞周期和细胞凋亡）；HGF诱导分化后，通过酶联免疫吸附试验（enzyme linked immunosorbent assay，ELISA）在指定时间点检测细胞ALB和AFP表达变化，诱导分化后透射电镜观察Tg737抑制前后细胞的超微结构变化；进一步采用DCS+HGF诱导两组细胞分化，培养7天后收集两组细胞，western blot检测AFP、ALB、FOXA1、HNF4α、CYP1A2的表达差异；Transwell技术检测细胞的侵袭能力变化；裸鼠皮下成瘤实验，观察Tg737抑制前后细胞成瘤能力变化，以及细胞对动物肝脏的损害情况。Western blot检测细胞周期蛋白cyclin D1和cyclin B表达，验证细胞周期蛋白是否参加了Tg737调控FLSPCs增殖的变化；利用western blot技术检测Tg737抑制后，HNF4α表达的变化；利用激光共聚焦技术检测了Tg737与HNF4α在细胞内的定位情况，验证Tg737抑制导致FLSPCs分化受阻现象的可能分子机制是HNF4α作为Tg737的下游分子伴侣。
3.含10%胎牛血清（fetal bovine serum，FBS）的达氏修正依氏培养基（Dulbecco'sModified Eagle Medium，DMEM）常规培养HepG2和MHCC97-H细胞。除特别指出，以下实验均用含1%FBS的DMEM处理细胞。用浓度1％的氧处理细胞达指定时间建立低氧模型。采用Annexin V/碘化丙啶（propidium iodide，PI）流式检测法排出1%FBS、低氧对细胞凋亡的影响；用浓度为1％的氧分别处理细胞（1%FBS）达指定时间，以常氧培养细胞（1%FBS）为对照组，检测细胞黏附、侵袭、转移力。Western blot检测10%FBS常氧组、1%FBS常氧组、1%FBS低氧组Tg737表达的变化。pcDNA3.1-Tg737经脂质体瞬时转染HepG2、MHCC97-H，转染pcDNA3.1()组、脂质体孵育组、空白组为对照；转染6h后，在缺氧条件下检测Tg737表达上调对细胞黏附、侵袭、转移力的影响；Annexin V/PI排出凋亡对实验的影响。Western blot检测10%FBS常氧组、1%FBS常氧组、1%FBS低氧组polycystin-1表达的变化；ELISA检测polycystin-1,白细胞介素-8（interleukin-8，IL-8）、转化生长因子-β1（transforming growth factor，TGF-β1）分泌的变化；在缺氧条件下检测Tg737表达上调对polycystin-1表达/分泌，IL-8、TGF-β1分泌的影响。
【结果】
1.富集的BMSCs纯度较高，细胞活力较好，高表达干细胞表面标志物CD44，而低表达造血干细胞（haematopoietic stem cells，HSCs）表面标志物CD45。富集的FLSPCs个头较小，核/浆比较大，高表达干细胞标志CD133和CD49f，低表达成熟肝细胞标志，能分化为表达ALB的类成熟肝细胞。在两组均添加生长因子的条件下，与二维贴壁培养比，DCS可更好的诱导BMSCs向肝样细胞分化，体现在诱导后的细胞高表达内胚层和肝细胞相关基因和蛋白，超微结构与功能更贴近肝细胞；体内实验证实DCS+生长因子诱导后的BMSCs对肝衰竭有更好的治疗效果。同样，DCS+HGF培养较贴壁+HGF培养更好的诱导FLSPCs向肝样细胞分化。该方案可用于下步相关研究。
2.新纯化的nFLSPCs、sFLSPCs在形态学上无明显区别；在培养过程中，新纯化的sFLSPCs与nFLSPCs相比在第1天均高表达CD133，无统计学差别，随时间延长，两组细胞CD133表达均下降，nFLSPCs组下降更明显，在7d、14d sFLSPCs中CD133的表达均高于nFLSPCs； PCR、western blot、免疫荧光技术检测Tg737的mRNA与蛋白表达水平，抑制效果得到确认。排出了凋亡因素的影响后，我们发现由于Tg737的抑制通过促进细胞周期过程使细胞增殖速度明显加快；HGF诱导分化后，通过ELISA、透射电镜证实Tg737的抑制导致细胞分化受阻，DCS+HGF方案亦证实分化受阻；Transwell侵袭实验证实sFLSPCs获取了较强的侵袭能力；裸鼠成瘤实验显示Tg737抑制后sFLSPCs虽然在皮下未形成明显肿瘤，但是对肝脏造成了损伤、甚至形成了肿瘤。进一步分子机制的研究证实：shRNA抑制Tg737后，cyclinD1和cyclin B表达显著上升；HNF4α随着Tg737的抑制表达而极度下调，Tg737和HNF4α在细胞内具有共表达的趋势。
3.低血清浓度、低氧条件对HepG2和MHCC97-H的活性无影响；与对照组相比，低氧减弱了肝癌细胞黏附力、增强了细胞侵袭、转移力。随着低氧模型的建立，HepG2和MHCC97-H中Tg737表达降低，而低血清浓度对Tg737的表达并无影响；western blot发现pcDNA3.1-Tg737转染并经低氧处理后，与对照组相比，pcDNA3.1-Tg737转染组中Tg737的表达明显上调；进一步发现由缺氧造成的肝癌细胞黏附、侵袭和转移力改变也随低氧下上调Tg737表达而减弱；与对照组相比，低氧诱导可下调HepG2和MHCC97-H polycystin-1的表达/分泌，上调IL-8的分泌，同时上调总TGF-β1、活化TGF-β1水平，而低血清浓度对以上分子的表达并无影响；随着在缺氧条件下上调Tg737的表达，polycystin-1的表达/分泌上调、IL-8的分泌、总TGF-β1、活化TGF-β1水平也相应下调；细胞活性因素、空载体、脂质体均对实验结果无影响。
【结论】
1.成功建立了DCS诱导分化方案，改方案能更好的诱导BMSCs、FLSPCs向肝样细胞分化，可用于FLSPCs的相关研究。
2. Tg737的抑制表达能够促进FLSPCs发生恶性转化，该恶性转化过程可能通过调节其下游靶基因cyclin D1、cyclin B和HNF4α实现。
3.低氧条件下HepG2和MHCC97-H黏附力下降、侵袭、转移力增强；Tg737及其下游靶基因polycystin-1、IL-8和TGF-β1参与了这一生物学行为。
[Background]
Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, isamong the most common malignancies, with an increasing incidence in China. Itsmortality rate ranks the third place after stomach cancer and esophagus cancer. Despitethere are some improvements in diagnosis and therapy of HCC, it still remains bigproblem because the clarification of molecular mechanisms responsible for HCCpathogenesis is unclear. It is of paramount importance to elucidate the the occurrence andprogression of HCC, not only for an understanding of tumor biology but also to permit the development of specific therapies that effectively target HCC.
Tg737, a new kind of tumor suppressor gene in HCC, was originally identified byinsertional mutagenesis and positional cloning as the gene which is altered in autosomalrecessive polycystic kidney disease in the TgN737Rpw transgenic mouse line. The Tg737gene encodes an824amino acid protein which includes10copies of a34-amino acidtetratrico-peptide repeat motif. Some previous studies provided evidence that Tg737mayplay an important role in carcinogenesis of kidney and pancreas. However, the role oftumor suppressor gene Tg737in the pathogenesis of HCC and the underlying molecularmechanisms remain poorly understood.
[Aims]
In order to effectively investigate whether Tg737may function in the malignanttransformation of fetal liver stem/progenitor cells (FLSPCs) and underlying molecularmechanisms, we first improved the differention method. To induce hepatic differentiation,bone mesenchymal stem cells (BMSCs) and FLSPCs were cultured using dynamiccultured scaffold (DCS), furthermore, this improved differention method was used insubsequent related experiments. Then, to investigate the role of Tg737signaling pathwayin in the pathogenesis of HCC and the underlying cellular and molecular mechanisms. Toexplore new melecular mechanisms regulating HCC pathogenesis and provide newtheoretical basis for HCC prevention and therapy.
[Methods]
1. A modified protocol previously reported was used to obtaine the decellularized liver.In this study, the identified murine BMSCs and FLSPCs were randomized into2groupsrespecticely. We compared the hepatic differentiation of BMSCs and FLSPCs in abiomatrix scaffold from rat liver (dynamic cultured scaffold (DCS) and in the presence ofhepatocyte growth factor (HGF)) with a two-dimensional substrate(adherent culture and inthe presence of HGF). At the indictaed time of cell growth, cells were harvested by trypsindigestion and then the expression of hepatocyte-specific genes alpha fetoprotein (AFP),albumin (ALB), forkhead box protein A1(FOXA1), hepatic nuclear factor4alpha (HNF4α)and cytochrome P4501A2(CYP1A2) were measured by western blot.
2. Tg737inhibition was achieved by short hairpin RNA (shRNA). StableshRNA-expressing clones (Tg737-silent FLSPCs, sFLSPCs) were selected by puromycindihydrochloride. To select cells expressing high levels of stem cell marker CD133for usein the next study, first step in this part was purification of Tg737normal FLSPCs (nFLSPCs) and sFLSPCs by PCGC. Then, CD133were analyzed by flow cytometry at1,7and14days during the culture period. Following RNAi of Tg737, the mRNA andprotein levels of sFLSPCs were measured by PCR, western blot andimmunocytochemistry analyses. The celluar growth curve was determined using cellcounting and3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.The proliferative capacity studies were determined by flow cytometry, including thecellular proliferation marker Ki-67expression analysis, cell cycle and apoptosis. Atindicated time of cell growth in the presence of HGF, enzyme linked immunosorbent assay(ELISA) was used for quantifying the ALB and AFP secreted by nFLSPCs and sFLSPCs.After incubation of nFLSPCs and sFLSPCs in complete medium supplemented with HGFfor7days, cells were harvested for fine structure analysis. nFLSPCs and sFLSPCs werealso seeded into scaffold for DCS, after7days in culture with complete mediumsupplemented with HGF, western blot was performed to measure AFP, ALB, FOXA1,HNF4α and CYP1A2expression. The invasion ability was evaluated by transwell assay.The in vivo tumorigenesis was detected by injection nFLSPCs and sFLSPCs intosubcutaneous of athymic mice. In order to investigate whether whether cyclin D1andcyclin B were involved in the effects of Tg737on cell proliferation and cell cycleprogression, western blot analysis was performed. Using western blot andimmunocytochemistry, we also analyzed the synergy of Tg737and HNF4α in thedifferentiation of FLSPCs.
3. HepG2and MHCC97-H cells were routinely cultured in Dulbecco's Modified EagleMedium (DMEM) supplemented with10%fetal bovine serum (FBS). In all subsequentrelated experiments, the cells were treated with medium supplemented with1%FBS,unless otherwise noted. For the incubation of cells under hypoxic conditions, the cellswere exposed to1%O2with5%CO2at37°C for the indicated times. To exclude thepossibility of apoptosis-related effects caused by low-serum medium under normoxic orhypoxic conditions in subsequent experiments, Annexin V/propidium iodide (PI) assayswere performed. HepG2and MHCC97-H cells were subjected to normoxic or hypoxicconditions, and the cell adhesion, invasion and migration capabilities were evaluated. Theexpression of Tg737under normoxia or hypoxia was detected using western blot.Furthermore, we created HepG2and MHCC97-H cells that overexpressed Tg737prior toincubation under hypoxia and investigated their metastatic characteristics. Finally, weanalyzed the involvement of critical molecular events (polycystin-1, interleukin-8(IL-8) and transforming growth factor β1(TGF-β1)) known to regulate invasion and migration.
[Results]
1. A systematic method for BMSCs enrichment used in this study can effectively enrichand purify BMSCs. The harvested BMSCs are in a good state, highly express stem cellsmarkers CD44, low expression of hematopoietic stem cell markers CD45. The enrichedFLSPCs were with small size, larger nuclear/plasma, highly express stem cells markersCD49f and CD133, low expression of mature liver markers. The isolated FLSPCs couldgenerate mature ALB expressing hepatocytes. In the presence of HGF, DCS could betterstimulate the BMSCs to express endodermal and hepatocyte-specific genes and proteinsassociated with improved functions, and the cells exhibited the ultrastructuralcharacteristics of mature hepatocytes. When transplanted into acute liver injured mice,DCS plus GF cultured cells exhibited increased liver function, survival, engraftment intothe host liver and further hepatic differentiation. Compared to the adherent with HGFcultured FLSPCs, the combination of DCS and HGF could better better induce FLSPCs todifferentiation into hepatocyte-like cells. This improved differentiation method can beused in subsequent related experiments.
2. The microscopic observations of freshly purified nFLSPCs and sFLSPCs revealed nosignificant morphological changes in sFLSPCs. Over the course of cell culture, on day1nFLSPCs and sFLSPCs showed high expression levels of CD133and there was nosignificant difference between the two groups; with time, the expression of CD133gradually decreased in both groups. CD133expression was significantly higher insFLSPCs than in nFLSPCs at all time points sampled except day1. Following Tg737inhibition, the mRNA and protein levels of sFLSPCs decreased as shown by PCR, westernblot and immunocytochemistry analyses. Excluding apoptosis-related effects, we foundthat Tg737inhibition resulted in enhanced cell proliferation through promoting cell-cycleprogression. Tg737inhibition also resulted in significant arrest of cell differentiation,stable expression of both ALB and AFP and quiescent ultrastructure. The improveddifferentiation method DCS plus HGF also confirmed differentiation arrest. Furthermore,the FLSPCs with Tg737inhibition got strong invasion and hepatocarcinogenesis ability.Cyclin D1and cyclin B are important for cell growth and are key signaling proteins in cellcycle progression. Silencing of Tg737induced increases in cyclin D1and cyclin B proteinlevels. Following downregulation of Tg737, the level of HNF4α decreased; these twoproteins co-localized in FLSPCs.
3. The treatment of HepG2and MHCC97-H cells with low-serum medium undernormoxic or hypoxic conditions did not significantly affect cell viability in vitro. Exposureof these two HCC cell lines to hypoxic conditions reduced HCC cell adhesion andfacilitated invasion and migration. In this study, Tg737expression was significantlyinhibited in HepG2and MHCC97-H cells following exposure to hypoxia. Thedownregulation of Tg737expression corresponded to significantly decreased adhesion andincreased invasion and migration. The treatment of cells with low-serum medium undernormoxia did not significantly affect Tg737expression. Hypoxia also decreased theexpression/secretion of polycystin-1, increased the IL-8, and increased the levels of activeand total TGF-β1. Moreover, the decrease in adhesiveness and the increase in the invasiveand migratory capacities of hypoxia-treated hepatoma cells were attenuated bypcDNA3.1-Tg737transfection prior to hypoxia. Finally, following the upregulation ofTg737, the expression/secretion of polycystin-1increased, and the secretion of IL-8andthe levels of active and total TGF-β1decreased correspondingly. Cell viability,liposome/pcDNA3.1(-) had no effects in our study.
[Conclusions]
1. An effective method for hepatic differentiation of BMSCs and FLSPCs issuccessfully constructed. The combination of DCS and HGF could better promote theBMSCs and FLSPCs to mature into functional HNCs. This improved differentiationmethod can be used in related experiments of FLSPCs.
2. Tg737inhibition by shRNA in FLSPCs leads to malignant transformation throughincreasing proliferation by accelerating cell-cycle progression and concomitantdifferentiation arrest, furthermore, cyclin D1, cyclin B and HNF4α signaling pathway maybe an important intermediary in this process.
3. Tg737contributes to hypoxia-induced invasion and migration, partially through thepolycystin-1, IL-8, and TGF-β1pathway.
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