【作者】 刘鹏；

【导师】 巩鹏；

【作者基本信息】 大连医科大学 ， 外科学， 2019， 博士

【摘要】 一、目的肝细胞癌占原发性肝癌的80%左右,是恶性肿瘤相关死亡的主要原因之一。我国是全世界肝癌发生率最高的国家,发病率及死亡率均占全世界的50%以上。大部分肝癌患者被确诊时已属中晚期,临床上仅不足20%的患者能够接受根治性手术切除,术后复发和远处转移是导致肝癌死亡率高的主要因素。放疗和化疗对肝癌的治疗效果并不理想,靶向药物索拉菲尼,毒副作用较大,患者对其敏感性较低。因此,急需寻找新的治疗靶点及有效的治疗手段。甲胎蛋白(AFP)是目前应用最为广泛的肝癌诊断标志物,但在肝癌的患者中阳性率仍较低,目前仍缺乏新的有效的早期诊断及评估预后的标志物。而肿瘤睾丸抗原是一种在睾丸及肿瘤中特异性表达的抗原,可能通过调控细胞信号转导,促进肿瘤发生、发展。在睾丸中异常表达的抗原蛋白能够引起细胞和/或体液的免疫反应,被认为是免疫治疗的靶点锌指蛋白165(ZNF165)属于SCAN-C2H2锌指蛋白家族成员,因特异性表达于睾丸组织及肿瘤组织中,又被成为肿瘤-睾丸抗原。ZNF家族成员与多种恶性肿瘤的发生发展密切相关。已有研究报道,ZNF165可能作为一个促癌基因通过调控TGFβ信号通路影响乳腺癌细胞的增殖及迁移能力。此外,有研究者在肝癌组织中检测到ZNF165的异常表达,但ZNF165在肝癌发生发展中的作用及机制目前尚未见报道。为此,本研究将利用临床肝癌组织标本检测ZNF165在肝细胞癌中的表达情况以及与临床预后的关系。通过体内、体外实验探讨ZNF165在肝细胞癌发生发展中的作用机制,为肝癌的诊断及治疗提供新靶点和新思路。二、方法1.首先在临床上收集20例新鲜的肝癌组织及癌旁组织标本,用于RNA及蛋白水平检测。利用肝癌组织基因芯片(购买于上海芯超生物科技有限公司),临床资料完整,其中肝癌组织93例,癌旁正常组织87例,共取180个点进行检测。采用免疫组织化学法测定所有病理标本中ZNF165的表达情况,并对所有病例进行随访。分析ZNF165的表达与肝癌患者临床病理特征和预后的关系。数据分析应用SPSS19.0统计软,显著性差异标准用P<0.05表示。采用卡方检验比较组间差异;采用Kaplan-Meier法进行预后的生存分析,Log-Rank检验比较组间的差异;采用Logistic及COX回归风险模型进行预后的因素分析。2.利用脂质体转染法将重组慢病毒的ZNF165过表达质粒或ZNF165敲低质粒转染至人肝癌细胞系(Bel7402/HCCLM3)中。并通过Puromycin筛选稳定转染ZNF165过表达或敲低的Bel7402/HCCLM3细胞克隆。3.q-PCR法(两步法:预变性95℃,5分钟;变性95℃,30秒,退火和延伸60℃,60秒,40个循环)检测ZNF165及色氨酸通路相关基因IDO1、IDO2、TDO2、CYP1A1、CYP1B1、AhR 的表达。4.Western blot(聚丙烯酰氨凝胶电泳)法检测 ZNF165、p-Akt、IDO1、IDO2、TDO2、CYP1A1、AhR等蛋白的表达。5.双荧光素酶检测试剂盒(Dual-Luciferase Reporter Assay System)检测REPOTMAhR的转录活性。6.间接免疫荧光法(共聚焦显微镜)检测ZNF165、p-Akt、AhR在肝癌细胞内定位情况。7.利用Elisa试剂盒(酶标仪,波长590mm)检测Tryptophan、Kynurenine在细胞上清、组织匀浆及尿液中的含量。8.Phospho-Kinase Array试剂盒检测过表达ZNF165对多种磷酸化激酶的影响。9.MTT/Brdu法及克隆形成实验检测ZNF165/CYP1A1对肝癌细胞生长的影响,失巢凋亡/划痕/Transwell等实验检测ZNF165/CYP1A1对肝癌细胞的迁移/侵袭能力;Annexin-V/PI单染或双染法检测肝癌细胞周期和凋亡。10.建立肝癌皮下移植瘤裸鼠模型。取6周龄雄性无胸腺裸鼠饲养于SPF级独立隔离笼中。选取过表达/敲低ZNF165的肝癌细胞系(约5×106个),以150μL PBS混合后接种于鼠背部外侧皮下,进行后续实验。11.建立二乙基亚硝胺(DEN)诱导的SD大鼠原发性肝癌模型。取200g左右的雄性SD大鼠,DEN组首次给予腹腔注射DEN(150 mg/kg).随后将0.01%DEN(76ppm)加入饮用水中,持续8周;抑制剂组在含有DEN的饮水中加入LY294002(10mg/kg);空白对照组用灭菌后的PBS代替DEN,20周后进行相关实验。三、结果(一)ZNF165在肝细胞癌中的表达及对预后的影响1.ZNF165在肝癌组织中表达明显高于癌旁正常组织。IHC染色法和Western blot法在20对新鲜的肝细胞癌及癌旁正常组织中检测了 ZNF165的表达情况。ZNF165在肝细胞癌组织(13/20)和癌旁正常组织(2/20)中呈现不同程度的表达。ZNF165在年龄≥50、肿瘤≤5cm、临床分期Ⅲ+Ⅳ期的患者中高表达,在男性和女性中表达差异不明显。基因芯片结果显示,ZNF165主要定位于细胞核中,在癌组织中高表达,在癌旁正常组织中低表达。在配对的84例癌组织中,表达率为27.4%(23/84);而在癌旁正常组织中,ZNF165的表达率为3.6%(3/84)。利用TCGA数据库对369例肝癌组织和50例癌旁正常组织进行分析比较,结果显示:ZNF165在肝癌组织中表达明显高于癌旁正常组织。2.ZNF165的表达与肝癌患者的T分期分期呈正相关。对84例配对的肝癌患者进行分析比较,T1/T2期患者中ZNF165表达率为23.4%(10/43),T3/T4期患者中 ZNF165 表达率 43.9%(18/41),P<0.045。3.ZNF165对肝细胞癌患者预后的影响。本研究对84例随访资料完整的肝细胞癌患者进行了生存分析。Kaplan-Meier分析结果显示,ZNF165在肝癌组织中高表达与低表达组相比,总生存率显著降低,生存率分别为20.0%和43.9%,P<0.05。ZNF165高表达组的平均生存时间为28.44±5.426个月,中位生存时间17个月;ZNF165低表达组的平均生存时间为45.21 2±3.982个月,中位生存时间35个月。4.影响肝细胞癌患者预后的相关因素。单因素生存分析结果显示,ZNF165高表达(HR:2.519;95%CI:1.486-4.268;P=0.001)、组织分化(HR:1.975;95%CI:1.135-3.437;P=0.016)、T 分期(HR:2.053;95%CI:1.369-3.079;P=0.001)、M 分期(HR:41.166;95%CI:3.733-453.988;P=0.0.02)及 TNM 分期(HR:2.148;95%CI:1.392-3.313;P=0.001)与肝细胞癌患者的预后具有显著的相关性(见表3)。多因素生存分析显示,ZNF165高表达(HR:2.000;95%CI:1.113-3.592:P=0.001)、M 分期(HR:21.085:95%CI:1.043-426.125;P=0.047)是肝细胞癌患者患者的独立预后因素。(二)ZNF165通过Akt调控IDO-Kyn-AhR通路靶向CYP1A1影响肝癌细胞的生长的机制研究1.ZNF165对肝癌细胞的影响。利用慢病毒构建稳定敲低/高表达ZNF165的肝癌细胞(Bel7402/HCCLM3),敲低ZNF165能够抑制细胞增殖、迁移、侵袭的能力,高表达ZNF165能够促进细胞增殖、迁移、侵袭的能力。2.ZNF165调控色氨酸通路。RNAseq测序结果显示,在敲低ZNF165的细胞后显著下调了 Tryptophan代谢通路的活性以及通路中CYP1A1的表达。qPCR及Western blot检测证实敲低/过表达ZNF165减少/增加肝癌细胞中IDO、AhR、CYP1A1的表达及AhR的转录活性;利用Elisa试剂盒检测发现敲低/过表达ZNF165减少/增加细胞上清中Kynurenine/Tryptophan的含量。3.ZNF165通过AhR影响CYP1A1的表达。过表达ZNF165能够增加AhR的转录活性,而敲低ZNF165能够降低AhR的转录活性;共聚焦结果显示,过表达ZNF165能够增加AhR入核,敲低ZNF165减少AhR入核;核质分离实验检测到过表达ZNF165后,细胞核内的AhR表达增加,细胞质内减少,敲低ZNF165后,细胞核内的AhR减少,细胞质内的AhR增加。在敲低AhR或AhR抑制剂处理的细胞中,过表达ZNF165不能增加CYP1A1的表达。4.ZNF165 通过 IDO1 调控 Kyn-AhR-CYP1A1 通路。Kynurenine 能够增加 AhR的转录活性并且增加CYP1A1的表达;在应用IDO1抑制剂或敲低IDO1的细胞中,过表达ZNF165不能够增加细胞上清中Kynurenine的含量。应用IDO1、AhR的抑制剂或敲低AhR、IDO1均能阻断ZNF165增加CYP1A1的表达。5.ZNF165 通过 Akt 促进 IDO1-Kyn-AhR 通路。过表达 ZNF165 增加了 p-Akt的蛋白水平,并且促进p-Akt在细胞膜上定位。利用共聚焦显微镜观察到ZNF165与Akt在细胞核表面有明显的共定位。Co-IP实验检测到ZNF165与Akt存在外源性和内源性相互作用。ZNF165促进FGF9分泌,进而间接增加p-Akt的表达。过表达活化的Akt及Akt激活剂处理后,IDO1、AhR;应用Akt、IDO1、AhR的抑制剂均能阻断ZNF165对CYP1A1表达的影响。6.PI3K抑制剂LY294002能够抑制ZNF165的表达。应用PI3K抑制剂处理后,抑制剂LY294002能够明显减少ZNF165的表达,并且呈剂量和时间依赖性。活化的Akt及Akt激活剂SC79能够回复LY294002引起的ZNF165表达的减少。IP结果显示,LY294002可能通过泛素化降解ZNF165,主要作用于K48Ub。7.LY294002抑制DEN诱导肝癌形成。建立DEN诱导的SD大鼠肝癌模型,采用IHC及Western blot法检测到肝癌组织中ZNF165、p-Akt、IDO、CYP1A1表达明显升高。DEN诱导的SD大鼠的肝脏组织匀浆及尿液中Kynurenine的含量与对照组相比明显增加。DEN能够诱导正常肝细胞L02表达ZNF165,能够被LY294002 阻断。四、结论1.ZNF165在肝细胞癌中高表达并与其预后不良相关:2.ZNF165靶向调控CYP1A1影响肝癌细胞生长;3.ZNF165通过活化Akt调控IDO-Kyn-AhR通路:4.DEN诱导肝癌形成及ZNF165表达;5.LY294002通过泛素化降解ZNF165并且抑制DEN诱导肝癌形成。更多还原

【Abstract】 Ⅰ.ObjectiveHepatocellular carcinoma(HCC)accounts for 80%of primary hepatocellular carcinoma,which is the main cause of cancer-related death.China has a large number of pateients of liver cancer,where new cases and deaths account for more than 50%of the world.Less than 20%of HCC patients can receive radical resection clinically.Recurrence and metastasis are important factors affecting the prognosis of HCC.At present,there is still a lack of specific therapeutic drugs for the patients after liver carcinomectomy.HCC is devoid of sensitivity to radiotherapy and/or chemotherapy,and the therapeutic effect is not ideal.HCC patients with liver cirrhosis have poor tolerance to drugs.Sorafenib as a representative targeted drug is expected to improve the prognosis of HCC patients,but only suitable for non-surgical patients.Furthermore,the long-term efficacy of Sorafenib needs to be further verified by a large number of clinical research data.Therefore,there is an urgent need to find effective novel treatment targets for liver cancer.Alpha fetoprotein(AFP)is currently the most widely used diagnostic marker for liver cancer,but the positive rate in patients with liver cancer is still low,and there is still a lack of new and effective early diagnosis and prognostic markers.Tumor testis antigen is an antigen specifically expressed in testis and tumor,which may promote tumorigenesis and development by regulating cell signal transduction.Abnormal expression of antigen protein in testis can induce cellular and/or humoral immune response and is considered as the target of immunotherapyZinc finger protein 165(ZNF165)is a tumor-testicular antigen,which belongs to the SCAN family and is specifically expressed in testicular and tumor tissues.It has been reported that ZNF165 may be an oncogene to affect the proliferation and migration of breast cancer cells by regulating TGF signaling pathway.In addition,some researchers have detected abnormal expression of ZNF165 in liver cancer tissues,but the effect of ZNF165 on liver cancer and the specific mechanism of action have not been reported.This study aims to reveal the correlation between ZNF165 and the clinical prognosis of HCC,and to explore the role of ZNF165 in HCC tumorigenesis and development.Our study will provide important theoretical basis and novel strategies for the early diagnosis and targeted treatment of HCC.Ⅱ.Methods1.There were 20 tumor tissue samples and 20 paracancerous tissue samples for RNA and protein detection.Gene chip of liver cancer tissue was provided by Shanghai xinchao biotechnology co.,LTD.,with complete clinical data,including 93 cases of liver cancer tissue and 87 cases of adjacent normal tissue.A total of 180 points were detected and analyzed.The expression of ZNF165 in all pathological specimens was determined by immunohistochemistry,and all cases were followed up.The correlation was analyzed between the expression of ZNF165 and the clinicopathological features and prognosis of HCC patients.The SPSS 19.0 statistical software was used for data analysis,with p<0.05 was considered significant.The correlation between the two indicators and the major clinical characteristics of HCC was analyzed by chi-square test.Kaplan-meier curve was used for univariate analysis of prognosis,and log-rank test was used for comparison between groups.Multivariate analysis of prognosis was performed using COX regression risk model.2.The recombinant lentiviruses expressing ZNF165 were transfected into HCC cell lines(Be17402/HCCLM3)by liposome transfection reagent.Stable transfectants were selected with 1～3μg/ml puromycin,and individual clones were isolated.3.The mRNA levels of ZNF165 and tryptophan pathway related genes(IDO1,IDO2,TDO2,AhR.CYP1A1,CYP1B1)were detected by RT-PCR(two step:degeneration within 95℃.5 minutes;degeneration within 95℃.30 seconds:annealing within 60℃.60 seconds;40 cycles).4.Western blot assay was used to detect the expression levels of ZNF165 and tryptophan-related proteins(IDO1.IDO2,TDO2,AhR,CYP1A1,CYP1B1).5.A double luciferase assay kit was used to detect the transcriptional activity of AhR.6.The intracellular localization of ZNF165,p-akt and AhR was detected by indirect immunofluorescence assay.7.The consentrations of tryptophan and kynurenine in cell supernatant,tissue homogenate and urine were detected by Elisa kit.8.The effect of ZNF165 on the activation of kinases was detected using a phospho-kinase Array kit.9.The effect of ZNF165/CYP1A1 on the growth of HCC cells was detected by MTT/Brdu and clone formation assays,and the migration/invasion ability of HCC cells was analyzed by anoikis/scratch/transwell assays.Cell cycle and apoptosis were detected by flow cytometry.10.The subcutaneously implanted tumor model was established.Nude mice aged 6 weeks without thymus were reared in SPF independent cages.5×106 cells were inoculated under the dorsal lateral skin of nude mice with a volume of 150μl.The experiments were conducted.11.Establishment of a diethyl nitrosamine(DEN)-induced primary liver cancer model.Male SD rats with 200g around were selected,and the DEN group was given an intraperitoneal injection of DEN(150 mg/kg)for the first time,and 0.01%DEN was added to drinking water for the following 8 weeks.After intraperitoneal injection of DEN(150 mg/kg)for the first time in the inhibitor group,LY294002(10mg/kg,)was added into the water containing 0.01%DEN for 12 weeks.DEN was replaced by sterilized PBS in the blank control group and relevant experiments were conducted 20 weeks later.Ⅲ.Results(Ⅰ)The correlation between the expression of ZNF165 and prognosis in HCC1.The expression of ZNF165 in liver cancer tissues was significantly higher than that in adjacent normal tissues.The expression of ZNF165 was detected by IHC staining and Western blot in 20 pairs of fresh hepatocellular carcinoma and adjacent normal tissues.ZNF165 was expressed to varying degrees in hepatocellular carcinoma(13/20)and adjacent normal tissues(2/20).ZNF165 is highly expressed in patients with age 50,tumor 5cm,and clinical stage Ⅲ+Ⅳ,with no significant difference between male and female.The results of gene microarray showed that ZNF165 was mainly located in the nucleus,highly expressed in the cancer tissues,and low expressed in the adjacent normal tissues.In the paired 84 cancer tissues,the expression rate was 27.4%(23/84).The expression rate of ZNF165 was 3.6%(3/84)in para-cancer normal tissues.TCGA database was used to analyze and compare 369 cases of liver cancer tissues and 50 cases of adjacent normal tissues.The results showed that the expression of ZNF 165 in liver cancer tissues was significantly higher than that in adjacent normal tissues.2.The expression of ZNF165 in HCC tissues was significantly higher than that in adjacent normal tissues(P<0.01).The expression rate of ZNF 165 in T1/T2 stage patients was 23.4%(10/43),while that in T3/T4 stage patients was 43.9%(18/41),P<0.045.3.The expression level of ZNF 165 was significantly correlated with T stage in HCC patients.In this study,84 patients with hepatocellular carcinoma with complete follow-up data were analyzed for survival.Kaplan-meier analysis showed that compared with the low expression group,the overall survival rate of ZNF 165 in liver cancer tissues with high expression was significantly reduced,and the survival rates were 20.0%and 43.9%,respectively,P<0.05.The mean survival time of the ZNF165 high-expression group was 28.44±5.426 months,and the median survival time was 17 months.The mean survival time of the low expression group of ZNF 165 was 45.212±3.982 months,and the median survival time was 35 months.4.Related factors affecting prognosis of patients with hepatocellular carcinoma.Single factor survival analysis showed that ZNF 165 was highly expressed(HR:2.519;95%CI:1.486-4.268;P=0.001),tissue differentiation(HR:1.975;95%CI:1.135-3.437;P=0.016),T staging(HR:2.053;95%CI:1.369-3.079;P=0.001),M staging(HR:41.166;95%ci:3.733-453.988;P=0.002)and TNM staging(HR:2.148;95%CI:1.392-3.313;P=0.001)was significantly correlated with the prognosis of patients with hepatocellular carcinoma(see table 3).Multivariate survival analysis showed that ZNF165 was highly expressed(HR:2.000;95%CI:1.113-3.592;P=0.001),M staging(HR:21.085;95%CI:1.043-426.125;P=0.047)is an independent prognostic factor for patients with HCC.(Ⅱ)ZNF165 regulates IDO-Kyn-AhR pathway targeting CYP1A1 and promotes the growth of HCC cells via Akt1.Knockdown/overexpression of ZNF165 in HCC cells(Bel7402/HCCLM3)can inhibit/promote proliferation,migration and invasion,and resistance of Sorafenib,respectively.2.RNAseq array results showed that Tryptophan pathway and expression of CYP1A1 were significantly down-regulated in ZNF165 knockdown cells.qPCR and Western blot assays confirmed that knockdown/overexpression of ZNF165 reduced/increased the expression of IDO,AhR,CYP1A1 and the transcriptional activity of AhR in HCC cells,respectively.Elisa assay showed that knockdown/overexpression of ZNF165 reduced/increased the content of Kynurenine/Tryptophan in the supernatant of cells,respectively.3.ZNF165 affects the expression of CYP1A1 through AhR.Overexpression of ZNF165 increased the transcriptional activity of AhR,while knockdown of ZNF165 decreased the transcriptional activity of AhR.Confocal results showed that overexpression of ZNF165 could increase AhR enucleation,while knockdown of ZNF165 could decrease AhR enucleation.After ZNF165 overexpression,AhR expression increased in the nucleus and decreased in the cytoplasm.After ZNF165 knockdown,AhR decreased in the nucleus and increased in the cytoplasm.In cells treated with knockdown AhR or AhR inhibitor,overexpression of ZNF165 could not increase the expression of CYP1A1.4.ZNF165 regulates the kyn-ahr-cyplal pathway through IDO1.Kynurenine can increase the transcriptional activity of AhR and increase the expression of CYP1A1.Overexpression of ZNF165 did not increase the content of Kynurenine in the supernatant of cells treated with IDO1 inhibitors or knockdown IDO1.The application of inhibitors of IDO1 and AhR or knockdown of AhR and IDO1 can block ZNF165 and increase the expression of CYP1A1.5.ZNF165 promotes the idol-kyn-ahr pathway through Akt.Overexpression of ZNF165 increased p-akt protein levels and promoted p-akt localization on cell membranes.The co-localization of ZNF165 and Akt on the nuclear surface was observed by confocal microscopy.Exogenous and endogenous interactions between ZNF165 and Akt were detected by co-ip experiment.ZNF165 promotes the secretion of FGF9 and indirectly increases the expression of p-akt.IDO1 and AhR were treated with overexpression of activated Akt and Akt activator.The effect of ZNF165 on CYP1A1 expression was blocked by the inhibitors of Akt,IDO1 and AhR.DEN induced HCC model was established in SD rats.The expressions of ZNF165,p-akt,IDO and CYP1A1 in tumor tissues were detected by IHC and Western blot and the expression levels of these molecules were significantly increased and positively correlated.6.PI3K inhibitor LY294002 inhibited the expression of ZNF165 After treatment with PI3K inhibitor,inhibitor LY294002 can significantly reduce the expression of ZNF165 in a dose-dependent and time-dependent manner.Activated Akt and Akt activator SC79 reverted to ly294002-induced reduction in ZNF165 expression.IP results showed that LY294002 could degrade ZNF165 by ubiquitination,mainly acting on K48Ub.7.LY294002 inhibited DEN to induce hepatocellular carcinoma.DEN induced liver cancer model in SD rats was established,and the expressions of ZNF165,p-akt,IDO and CYP1A1 in liver cancer tissues were detected to be significantly increased by IHC and Western blot.The contents of Kynurenine in liver homogenates and urine of den-induced SD rats were significantly increased compared with the control group.DEN can induce the expression of ZNF165 in normal hepatocytes.which can be blocked by LY294002.Ⅳ.Conclution1.Overexpression of ZNF165 predicts poor prognosis in HCC.2.ZNF165 promotes the proliferation,migration,and invasion of HCC cells through targeted regulate CYP1A1.3.ZNF165 up-regulates IDO-Kyn-AhR pathway via activates Akt.4.DEN induces HCC tumorigenesis and promotes the expression of ZNF165.5.LY294002 degrades ZNF165 through ubiquitination and inhibits DEN-induced HCC tumorigenesis.更多还原