【作者】 刘磊；

【导师】 史本康； 邹永新；

【作者基本信息】 山东大学 ， 外科学（泌尿外科）（专业学位）， 2020， 博士

【摘要】 第一部分KDM6A对膀肢癌发生发展的影响及其机制的研究研究背景膀胱癌(bladdercancer)是泌尿系最常见的恶性肿瘤,2018年全球统计数据显示其发病率在所有恶性肿瘤中位列第10位。根据病理类型,膀胱癌可分为非肌层浸润性膀胱癌(non-muscle invasive bladder cancer,NMIBC)和肌层浸润性膀胱癌(muscle invasive bladder cancer,MIBC)。NMIBC(临床分期为 Ta 或 T1)约占所有膀胱癌的75%,预后较好,5年生存率达90%;MIBC(T2-T4)约占25%,5年生存率约为60%,若出现淋巴结转移或远处转移预后更差。是否发生肌层浸润和远处转移对于膀胱癌病人的预后至关重要。因此,探究膀胱癌浸润转移的机制和寻找治疗靶点具有重要的临床价值。表观遗传学(epigenetics)指基因的表达水平发生变化而DNA碱基序列不变,最终发生表型改变,并且这种现象可以稳定遗传。其研究对象为表观遗传修饰,主要包括DNA甲基化、组蛋白修饰、染色质重塑等。基因组全外显子测序显示染色质重塑基因表达异常在膀胱癌中普遍存在,表明表观遗传学紊乱为膀胱癌的重要特征。在所有染色质重塑基因中,赖氨酸去甲基化酶6A(lysine demethylase 6A,KDM6A)在膀胱癌中突变率最高。KDM6A,又称为X染色体普遍转录的四肽重复序列(ubiquitously transcribed tetratricopeptiderepeat on X chromosome,UTX),是一种组蛋白去甲基化酶,可特异性催化组蛋白H3第27位赖氨酸(H3K27)脱甲基。其功能与zeste基因增强子同源物2(enhancer of zeste homolog 2,EZH2)相反,后者负责催化H3K27发生三甲基化(H3K27me3),而启动子组蛋白形成H3K27me3为转录抑制的标志。此外,KDM6A可通过招募组蛋白乙酰化转移酶促使H3K27发生乙酰化,此过程不依赖其去甲基化酶活性。据报道,KDM6A在多数实体肿瘤中具有抑癌作用,如直肠癌、肾癌等。在膀胱癌中,KDM6A突变率高达30%,但其对膀胱癌的肌层浸润和远处转移的影响尚无详细研究。方法1.探究KDM6A在膀胱癌细胞和组织中的表达水平(1)细胞水平:利用Western blot检测KDM6A在不同分化程度的膀胱癌细胞系(RT4,T24,5637)和永生化膀胱上皮细胞系(SV-HUC-1)中的表达水平。(2)组织水平:利用免疫组织化学染色检测KDM6A在膀胱癌组织芯片中的表达水平,结合病人的临床病理参数进行分析,并探究其与病人预后的关系。2.KDM6A对于膀胱癌细胞增殖能力的影响(1)利用慢病毒感染和嘌呤霉素筛选,在3种膀肮癌细胞中分别过表达或敲除KDM6A,利用Western blot和RT-qPCR验证其表达效率。(2)通过MTT、克隆形成实验,检测KDM6A对于3种膀胱癌细胞增殖能力的影响。3.KDM6A对于膀胱癌细胞迁移浸润能力的影响(1)利用划痕实验和Transwell迁移实验,检测KDM6A对于T24、5637细胞迁移能力的影响;通过Transwell浸润实验,检测KDM6A对于膀胱癌细胞侵袭浸润能力的影响。(2)为了探究KDM6A在体内对于膀胱癌细胞浸润转移的影响,我们构建了裸鼠肿瘤肺转移模型。对裸鼠尾静脉注射稳筛的KDM6A过表达或敲除的膀胱癌细胞,4周后处死并解剖裸鼠,观察KDM6A对于肿瘤细胞肺转移的影响。(3)为探究KDM6A在膀胱癌中对上皮-间质转化过程的影响,利用RT-qPCR检测E-Cad、N-Cad、Snail、Zebl、Vimentin在KDM6A过表达或敲除膀胱癌细胞中的表达水平。4.探究KDM6A的下游调控机制(1)将KDM6A过表达和敲除的膀胱癌细胞及其各自对照细胞进行转录组测序,对表达变化的基因进行GO功能富集分析。(2)进一步分析转录组测序数据,根据表达倍数变化、基因功能和文献报道选择合适的靶基因进行研究。(3)通过Western blot和RT-qPCR,在膀胱癌细胞中检测KDM6A对于靶基因表达水平的影响。(4)利用免疫组织化学染色,检测靶蛋白在膀胱癌组织芯片中的表达水平,结合之前KDM6A的免疫组化染色结果,分析二者在组织中表达是否具有一致性。(5)为了探究KDM6A是否通过该靶基因影响膀胱癌细胞表型,在KDM6A过表达细胞中敲低靶基因的表达水平,观察对细胞表型是否具有拯救作用。(6)为了探究KDM6A调控靶基因表达的是否依赖其去甲基化酶活性,在膀胱癌细胞中加入去甲基化酶抑制剂或构建去甲基化酶结构域突变失活载体,观察对靶基因表达水平的影响。5.探究KDM6A上游调控机制(1)通过PCR获得KDM6A转录起始位点上游2000bp的启动子片段,将其构建于pGL3-Basic载体中。通过启动子截短实验和双荧光素酶实验,确定KDM6A核心启动区域。(2)利用生物信息学网站预测与核心启动区域结合的转录因子,构建转录因子过表达载体,结合双荧光素酶实验、启动子结合位点突变实验及染色质免疫共沉淀(chromatin immunoprecipitation,CHIP)实验筛选对 KDM6A 核心启动区域具有调控作用的转录因子。(3)在膀胱癌细胞中利用siRNA敲低转录因子的表达水平,检测KDM6A及下游靶基因的RNA和蛋白水平变化。(4)在膀胱癌细胞中敲低转录因子的表达水平,检测细胞表型的改变。结果1.KDM6A在膀胱癌细胞和组织中的表达水平及其临床价值(1)Western blot结果显示,相较于永生化膀胱上皮细胞系(SV-HUC-1),KDM6A在膀胱癌细胞系RT4、T24、5637中表达水平较低,其中5637细胞中表达水平最低。(2)膀胱癌组织芯片免疫组化结果显示,KDM6A高表达的肿瘤患者预后较好(P=0.008),但其表达水平与肿瘤大小(P=0.580)、淋巴结转移(P=0.612)、肌层浸润(P=0.362)、TNM分期(P=0.705)无显著关系,并且KDM6A在膀胱癌和癌旁组织中的表达水平无差异(P=0.874)。2.KDM6A对于膀胱癌细胞增殖能力的影响具有细胞特异性(1)Western blot和RT-qPCR结果显示,与感染pLVX-IRES-Puro病毒的对照组细胞相比,感染pLVX-IRES-KDM6A病毒的细胞中KDM6A表达水平明显上调;与感染pLKO.l-Puro病毒的细胞相比,感染pLKO.1-shKDM6A病毒的细胞中KDM6A表达水平明显降低。(2)MTT和克隆形成实验结果表明,在RT4细胞中,过表达KDM6A显著降低其增殖活性和克隆形成能力;而在T24和5637细胞中,过表达或敲除KDM6A对于细胞增殖活性和克隆形成能力无显著影响。3.KDM6A抑制膀胱癌细胞的迁移浸润(1)划痕愈合实验和Transwell实验结果显示,过表达KDM6A在体外明显抑制膀胱癌细胞的迁移和浸润能力,而敲除KDM6A则促进膀胱癌细胞的迁移浸润。(2)裸鼠肺成瘤实验结果显示,KDM6A过表达组裸鼠肺成瘤的数目显著低于对照组,而KDM6A敲除组裸鼠肺成瘤数目明显多于对照组,表明KDM6A在体内抑制膀肮癌细胞的浸润和转移。4.ARHGDIB为KDM6A的下游靶基因之一(1)转录组测序数据显示,KDM6A过表达组有1055个基因上调,834个基因下调;而KDM6A敲除组有1285个基因上调,715个基因下调。经过RT-qPCR验证,多数基因(33/42)的表达结果与测序结果相符。(2)根据测序结果,ARHGDIB在KDM6A过表达组上调38.3倍,在KDM6A敲除组下调14.7倍,变化显著。Western blot和RT-qPCR结果显示,与对照组细胞相比,KDM6A过表达的膀胱癌细胞中ARHGDIB表达水平明显上升,KDM6A敲除的细胞中ARHGDIB水平显著下降。组织芯片的免疫组化结果显示,ARHGDIB和KDM6A表达水平具有明显一致性。5.KDM6A通过下游ARHGDIB抑制膀胱癌的浸润转移(1)划痕愈合实验和Transwell实验结果显示,体外敲低ARHGDIB后膀胱癌细胞的迁移浸润能力明显上升。在KDM6A过表达细胞中,敲除ARHGDIB后膀胱癌细胞的迁移浸润能力得到恢复。(2)组织芯片的免疫组化结果显示,与KDM6A结果相同,ARHGDIB高表达的肿瘤患者预后较好(P=0.008)。6.KDM6A依赖去甲基化酶功能域调控ARHGDIB的表达(1)体外实验结果表明,KDM6A过表达的膀胱癌细胞中H3K27me3蛋白水平明显下降,而KDM6A敲除的细胞中H3K27me3水平上调。(2)KDM6A去甲基化酶功能域发生失活突变后,其对于ARHGDIB表达的促进作用基本消失。而且,使用去甲基化酶抑制剂GSKJ4处理后ARHGDIB表达水平降低,而敲除EZH2后ARHGDIB水平升高。(2)在裸鼠肺成瘤实验中,尾静脉注射KDM6A敲除组和对照组肿瘤细胞后,给予腹腔注射EZH2抑制剂GSK126可以明显降低KDM6A敲除组裸鼠肺成瘤的数目。(3)CHIP结果显示,KDM6A和H3K27me3在ARHGDIB的启动子区域有三个共同的结合区域。与对照组相比,在KDM6A过表达细胞中,该三个位点上KDM6A蛋白结合增多,H3K27me3结合减少。7.FOXA1在转录水平促进KDM6A的表达(1)荧光素酶实验结果显示,KDM6A转录起始位点上游-127bp至-71bp为KDM6A的核心启动子区域。JASPAR网站预测FOXA1、USF1、USF2和TFE3四个转录因子可以和该区域结合。将该四个转录因子分别过表达后,双荧光素酶实验结果表明只有FOXA1可增加KDM6A核心启动子区域的转录活性。将预测的FOXA1结合位点突变之后,过表达FOXA1对启动子活性的促进作用消失。CHIP结果表明,FOXA1直接结合在KDM6A的核心启动子区域。(2)体外实验结果表明,敲低FOXA1水平后KDM6A和ARHGDIB的RNA和蛋白水平均明显降低。8.FOXA1通过正向调控KDM6A和ARHGDIB的表达抑制膀胱癌细胞的迁移浸润(1)在膀胱癌细胞中敲低FOXA1表达水平后,膀胱癌细胞的迁移浸润能力明显提高。(2)在体外实验中,过表达KDM6A或ARHGDIB可显著抑制敲低FOXA1造成的细胞运动能力增强。结论本课题研究表明,KDM6A可通过其去甲基化酶功能正向调控ARHGDIB的表达水平,从而抑制膀胱癌的迁移和浸润。转录因子FOXA1可直接结合在KDM6A的启动子区域,促进KDM6A的转录表达。因此,FOXA1-KDM6A-ARHGDIB轴对于抑制膀胱癌细胞的浸润转移发挥重要作用。第二部分术前血小板分布宽度(PDW)对于膀胱肿瘤诊断价值的探究研究背景在全球范围内,膀胱癌发病率位列所有肿瘤第十位,死亡率位列第十四位。大多数膀胱癌为尿路上皮型,可分为非肌层浸润性(NMIBC;Ta,T1)和肌层浸润性(MIBC;T2-T4),占比分别为75%和25%左右。NMIBC手术治疗后复发率高达60%-70%,并且大约20%NMIBC会进展为MIBC。经腹超声、膀胱镜和细胞学检查是诊断膀胱肿瘤的常规检查方法。由于复发率高,膀胱癌病人术后需要常规进行膀胱镜复查,复发后需要二次治疗,给病人造成较大经济负担。膀肮内翻性乳头状瘤属于良性肿瘤,约占膀胱肿瘤的1%-2%。因此,我们拟探寻鉴别膀胱肿瘤良恶性以及区分恶性程度的无创性检查方法。据文献报道,血小板在肿瘤发展和转移过程中发挥重要作用。血小板参数主要包括血小板计数(platelets,PLT)、平均血小板体积(mean platelet volume,MPV)、血小板分布宽度(platelet distribution width,PDW)、血小板压积(plateletcrit,PCT)等。MPV反映单个血小板的平均体积,PDW反映血小板容积的变异程度,PCT则反映单位体积的血液中血小板所占的体积比。研究表明,在多种肿瘤中存在血小板相关参数异常,如胃癌、肺癌、结肠癌、乳腺癌等。然而,目前尚无关于血小板参数与膀肮肿瘤鉴别诊断及预后的具体研究。方法研究对象和内容此回顾性研究收纳了 2016年1月至2018年6月在山东大学齐鲁医院泌尿外科接受治疗的210例膀胱癌患者和76例膀胱乳头状瘤患者,并纳入了 132例健康对照。研究内容包括性别、年龄、吸烟史、白细胞(WBC)计数、血小板相关参数、肿瘤相关参数等。统计学方法分析不同人群之间血小板参数等变量的差异使用Kruskal-Wallis检验和Nemenyi检验。连续变量资料之间的比较使用Student’s t检验或Mann-Whitney U检验。分类变量资料之间的比较使用卡方分析。受试者工作特征曲线(receiver-operating characteristic curve,ROC)和曲线下面积(areaunder the curve,AUC)用于评估PDW的诊断价值。Youden参数用于确定PDW的cut-off值。结果(1)血小板相关参数在各组之间的差异与对照组相比,膀胱癌和乳头状瘤患者的MPV和PCT水平显著升高,PDW水平显著降低。与膀胱乳头状瘤患者相比,膀胱癌患者的PDW水平进一步降低。PLT水平在三组之间无显著差异,MPV和PCT在膀胱癌和乳头状瘤患者之间亦无差异。(2)PDW对于膀胱肿瘤的鉴别诊断价值ROC分析结果表明,PDW对于鉴别膀胱癌和乳头状瘤的AUC为0.583(95%CI:0.507,0.659;P=0.033),鉴别NMIBC和MIBC的AUC为0.612(95%CI:0.534,0.690;P=0.006),鉴别高低级别膀胱癌的AUC为0.662(95%CI:0.588,0.736;P<0.001),cut-off值分别为 11.25(灵敏度71.1%,特异度 42.4%),11.95(灵敏度51.6%,特异度65.5%),11.95(灵敏度59.6%,特异度66.1%)。(3)PDW和膀胱癌病人临床病理学参数的关系PDW降低与血小板计数升高(>223×109/L,P<0.001)、较大肿瘤体积(>3cm,P=0.029)、肿瘤高级别(P<0.001)、TNM高分期(P=0.018)、淋巴结转移阳性(P=0.048)、MIBC(P=0.003)、G分期高(P=0.022)相关。在MIBC人群中,PDW水平与淋巴结转移无显著相关性(P=0.125)。另外,PDW与膀胱癌患者的年龄、性别、吸烟史、白细胞数目、肿瘤多发性无显著关系。由于ROC曲线分析表明MIBC和NMIBC、低级别和高级别两组间的PDW cut-off值均为 11.95,我们利用此cut-off值将PDW分为PDW≤11.95(116/210,55.2%)和PDDW>11.95(94/210,44.8%)两组。肿瘤大小(P=0.042)、高低级别(P<0.001)、TNM分期(P=0.005)、T分期(P=0.004)、淋巴结转移(P=0.030)、肌层浸润(P=0.015)、G分期(P=0.004)在两组之间具有显著差异,而年龄、性别、吸烟史在两组间无差异。此外,PDW≤11.95与低MPV水平(P<0.001)、高血小板计数(P=0.002)显著相关。结论PDW可作为鉴别诊断膀胱癌和膀胱乳头状瘤的参考指标,并对膀胱癌恶性程度的判断具有重要价值。更多还原

【Abstract】 Part Ⅰ The FOXA1-KDM6A-ARHGDIB axis suppresses cell migration and invasion in human bladder cancerBackgroundBladder cancer(BCa)is the tenth most common malignancy worldwide,with approximately 549,000 new cases diagnosed and 200,000 deaths in 2018.Of all BCa cases,nearly 75%of patients are non-muscle-invasive type(NMIBC;stage Ta or T1)and have a favorable prognosis,with a 5-year overall survival rate reaching 90%.The remaining 25%are muscle-invasive(MIBC:stages T2 to T4)and have a 5-year survival rate decreasing to 60%,even worse for patients with positive lymph nodes or distant metastasis.The main cause for stage-dependent survival reduction is the presence of micrometastases when cystectomy is performed.Therefore,a new diagnostic and therapeutic target for early metastatic BCa is urgently needed.Epigenetics,participating in the regulation of gene expression via modification of DNA or chromatin proteins without DNA sequence alteration,is tightly involved in tumorigenesis,metastasis and recurrence of BCa.Notably,whole-exome sequencing of the genomic DNA identified genetic aberrations of the chromatin remodeling genes were highly prevalent in BCa,suggesting that epigenetic dysregulation is an important feature of BCa.Among all chromatin remodeling genes,KDM6A is the second most common mutant cancer-related gene in BCa.KDM6A,also known as UTX,containing a JmjC domain,is a histone demethylase which specifically demethylates di-and trimethylated histone 3 lysine 27(H3K27me2/3)and antagonizes EZH2-mediated transcriptional repression.Besides,KDM6A could promote H3K27 acetylation in a demethylase-independent pattern by recruiting the histone acetyltransferase p300.It is well established that KDM6A is involved in various biological processes and human diseases.Loss-of-function mutations in KDM6A were associated with a specific hereditary disease-Kabuki syndrome.Remarkably,it was reported that 6 Kabuki patients developed different types of cancer,indicating cancer susceptibility for Kabuki patients.Besides,as a tumor suppressor in most malignancies,inactivating mutations in KDM6A were widely observed in human cancers.In colon cancer,KDM6A inhibits cell migration and invasion by increasing E-cadherin expression via coordinate regulation of H3K27 demethylation and acetylation.In clear cell renal cell carcinoma,low expression of KDM6A was related with reduced overall survival and disease free survival.In T-cell acute lymphoblastic leukemia(T-ALL),KDM6A escapes X chromosome inactivation in female T-ALL lymphoblasts and KDM6A mutations only occurs in male patients,who exhibits sensitivity to EZH2 inhibitor.Interestingly,it is controversial about the role of KDM6A in breast cancer.Notably,KDM6A have the highest mutation rate up to nearly 30%in BCa and KDM6A inactivation enhances PRC2-mediated transcriptional repression and can be targeted by EZH2 inhibitor in BCa.However,the effect of KDM6A on migration and metastasis of BCa and the underlying mechanism are not fully clear.Here we demonstrate that KDM6A suppresses the migration and invasion of BCa cells through transcription activation of ARHGDIB in a demethylase-dependent manner.Additionally,we observe that FOXA1 transcriptionally upregulates KDM6A and acts synergistically as a tumor suppressor in BCa.MethodsRT-qPCR and western blot were performed to detect the expression of KDM6A in BCa cells.Immunohistochemistry(IHC)staining were used to investigate the expression of KDM6A in BCa tissues.Overexpression and knockdown assays were carried out to explore the effects of KDM6A on BCa cells.MTT and colony formation assays were performed to detect cell viability.Wound healing and transwell assays were used to investigate cell mobility.RNA-seq were carried out to explore the downstream mechanism of KDM6A.Dual-luciferase reporter assays and chromatin immunoprecipitation(CHIP)were performed to determine the transcription factor regulating KDM6A.Xenograft tumor models were used to explore the effect of KDM6A on BCa cell invasion and metastasis in vivo.ResultsExpression of KDM6A in BCa cell lines and tissuesThe protein level of KDM6A in SV-HUC-1 cells was much higher than those in BCa cells.Besides,the results of IHC on tissue microarray showed high levels of KDM6A is associated with favorable prognosis.However,no significant correlations were found between KDM6A expression level with tumor size,myometrial invasion,lymph node metastasis and TNM stages.KDM6A influences BCa proliferation in a cell type specific mannerStable KDM6A overexpressed RT4,T24 and 5637 cells were established.The results of MTT and colony formation assays showed that overexpression of KDM6A dramatically inhibited cell proliferation in RT4 cells.However,KDM6A overexpressed T24 and 5637 cells showed no significant difference in cell proliferation.Knockdown of KDM6A did not exhibit any effect on proliferation of T24 and 5637 cells,indicating that the role of KDM6A in cell proliferation varies in different BCa cells.KDM6A inhibits BCa migration and invasion in vitro and in vivoThe results of wound-healing assay and transwell assay showed that overexpression of KDM6A significantly suppressed cancer migration and invasion in both T24 and 5637 cells.Consistently,knockdown of KDM6A demonstrated facilitative effect on cell migration and invasion in both BCa cells.Moreover,KDM6A overexpressed T24 cells developed fewer lung metastases in nude mice than control cells,while KDM6A knockdown resulted in a significant increase in lung metastases.Identification of ARHGDIB as a target of KDM6AAs the results of RNA-seq shown,overexpression of KDM6A led to upregulated expression of 1055 genes and downregulation of 834 genes,while knockdown of KDM6A resulted in upregulation of 1285 genes and downregulation of 715 genes.To validate the data of the transcriptome profiling,we performed RT-qPCR on a separate set of RNA samples and confirmed the majority of target genes determined by RNA-Seq analysis.According to the results of RNA sequencing,ARHGDIB was one of the most significantly altered genes(38.3-fold increased expression in KDM6A group,14.7-fold decreased expression in shKDM6A group).We examine the expression of ARHGDIB in a separate set of samples by RT-qPCR and western blot,and the results showed that overexpression of KDM6A significantly increased both the mRNA and protein expression levels of ARHGDIB in T24 and 5637 cells,while knockdown of KDM6A decreased ARHGDIB levels.Moreover,the IHC results indicated the levels of KDM6A and ARHGDIB had high consistency.ARHGDIB was a downstream effector of KDM6A for suppressing BCa migration and invasionWe downregulated ARHGDIB expression in BCa cells using siRNA.Knockdown of ARHGDIB enhanced migration and invasion of T24 cells.Furthermore,the inhibition of KDM6A on BCa migration and invasion was counteracted by knockdown of ARHGDIB in T24 cells.Kaplan-Meier analysis showed high expression ARHGDIB were respectively associated with prolonged overall survival time,suggesting ARHGDIB is an independent prognostic factor in BCa/KDM6A transcriptionally promotes ARHGDIB expression in a demethylase-dependent mannerConsidering KDM6A as a gene expression activator demethylating H3k27me3,we first examine the H3K27me3 levels in KDM6A overexpression and knockdown BCa cells.The results showed KDM6A overexpression decreased prominently H3K27me3 levels in both T24 and 5637 cells,while KDM6A knockdown exhibited stimulative effect on H3K27me3 expression.Then,we introduced a catalytically dead KDM6A mutant(H1146A,E 1148A)into T24 cells.The results manifested that T24 cells overexpressing mutant KDM6A exhibited comparable ARHGDIB mRNA and protein levels compared with control cells,which were much lower than those of cells overexpressing wildtype KDM6A.Moreover,GSKJ4,a KDM6A demethylase inhibitor,could decrease ARHGDIB expression levels in wildtype T24 cells.Notably,EZH2 knockdown in T24 cells led to increased expression of ARHGDIB.Next,we sought to verify the ARHGDIB regulation mechanism in vivo.After tail vein injection with KDM6A knockdown T24 cells and control cells,the mice were administered EZH2 inhibitor-GSK126 or 20%SBE-β-CD by intraperitoneal injection.The results showed GSK126 counteracted the increased lung metastasis caused by KDM6A knockdown.To further elucidate the epigenetic regulatory mediated by KDM6A,we performed CHIP using wildtype T24 cells and found three common binding sites of KDM6A and H3K27me3 at ARHGDIB promoter.CHIP-qPCR assays showed KDM6A exhibited increased enrichment levels at ARHGDIB promoter region in KDM6A overexpressed T24 cells compared with control cells,while the enrichment levels of H3K27me3 decreased.Thus,these results indicated that KDM6A modulates ARHGDIB expression in a demethylase-dependent manner.FOXA1 transcriptionally enhances KDM6A expression in BCaThe dual-luciferase reporter assay showed that the KDM6A promoter region approximately between-127bp and-71 bp is critical for KDM6A transcription regulation.Next,we analyzed the core promoter region with JASPAR program(http://jaspar.genereg.net/).Four transcription factors(FOXAl,USF1,USF2,TFE3)were predicted to potentially bind with this region.To explore their effects in regulating KDM6A transcription,these four transcription factors were separately overexpressed in T24 cells.The luciferase reporter assay indicated the promoter activity from-127bp to+47bp(construct P5)increased prominently in FOXA1 overexpressed T24 cells.To verify the putative FOXA1 binding site,we introduced two point mutations into construct P5.The results showed that the elevated luciferase activity by FOXA1 overexpression was completely abolished in mutant P5 group.Furthermore,CHIP assay confirmed that FOXA1 could bind directly to the promoter of KDM6A.To explore the effect of FOXA1 on KDM6A expression,we knocked down FOXA1 with siRNAs in T24 cells.RT-qPCR and western blot assays demonstrated that FOXA1 knockdown significantly decreased KDM6A and ARHGDIB expression levels.Similar results were obtained in 5637 cellsFOXA1 suppresses migration and invasion of BCa cells through promotion of KDM6A and ARHGD1B expressionTo determine the effect of FOXA1 on BCa migration and invasion,we knocked down FOXA1 expression in wildtype T24 cells,wound healing and transwell assays were performed.The results showed that transient knockdown of FOXA1 enhanced cell migration and invasion in T24 cells.To explore whether KDM6A is essential for FOXA1 regulating cell mobility,we transfected FOXA1 siRNA into KDM6A overexpressed T24 cells and control cells.The increased migration and invasion ability caused by FOXA1 knockdown was nearly neutralized in KDM6A overexpressed cells.Moreover,overexpression of ARHGDIB could also abate the enhanced effect of FOXA1 knockdown on T24 migration.ConclusionsHere we demonstrated that KDM6A inhibited migration and invasion of BCa cells in vitro and in vivo,revealing that KDM6A is a key role in regulating BCa mobility.By RNA-sequencing we identified ARHGDIB as a novel downstream gene which is responsible for the anti-tumor effect of KDM6A in BCa.CHIP assays showed that KDM6A promoted ARHGDIB expression through demethylating H3K27me3 at its promoter region.Furthermore,We found FOXA1,acting as a transcription factor,upregulated KDM6A expression and acted synergistically as a tumor suppressor in BCa.These findings reveal that The FOXA1-KDM6A-ARHGDIB signaling axis plays important role in BCa metastasis,allowing for the identification of more aggressive BCa and providing a better definition of treatment strategies.Part II Evaluation of platelet distribution width(PDW)as a diagnostic and prognostic biomarker in bladder neoplasmBackgroundBladder cancer is the tenth most common cancer and fourteenth leading cause of cancer-related death worldwide.Most of the bladder cancer cases are urothelial type and pathologically divided into non-muscle-invasive bladder cancer(NMIBC;stage Ta or T1),and muscle-invasive bladder cancer(MIBC;stages T2 to T4),accounting for 75%and 25%of the newly diagnosed bladder cancer patients separately.Despite progression in surgery,there is still a 60%-70%recurrence rate in NMIBC patients and 20%of NMIBC will undergo progression to MIBC disease.Due to high recurrence rate,frequent cystoscopic surveillance and reoperation after recurrence is required,making it the most expensive malignancy to treat.Ultrasonography,cystoscopy,and cytology are routinely used diagnostic methods of bladder cancer currently,but they are limited due to complicated procedures,expensive for long-term and frequent surveillance.The inverted urothelial papilloma of bladder is generally categorized histologically and clinically as a benign tumor,accounting for 1-2%of all bladder neoplasms.Simple and non-invasive methods to distinguish urothelial papilloma from bladder cancer,and indicators of tumor malignancy of bladder cancer are still warranted.Platelets play a pivotal role in cancer progression and metastasis.The parameters of platelets are known as platelet indices,which mainly include platelet count(PLT),mean platelet volume(MPV),platelet distribution width(PDW),and plateletcrit(PCT)The MPV reflects the average platelet volume and is considered as a surrogate marker of platelet activation.PDW represents variation in platelet size,and PCT is an indicator of the platelet mass in a unit of volume,which is determined by platelet count and MPV.Several studies have identified aberrations in platelet indices in various cancer types including gastric cancer,lung cancer,colon cancer and breast cancer.Thus,platelet indices hold great importance as diagnostic values in cancer management.However,whether platelet indices were associated with bladder cancer and their diagnostic values in discriminating malignancy and tumor stages have not been put forwarded.MethodsPatients’ characteristicsA total of 210 bladder cancer patients were enrolled in this study.A total of 76 patients with urothelial papilloma,and 132 healthy sex-and age-matched healthy volunteers were enrolled as controls.All patients were diagnosed and received surgical intervention as primary treatment at the Department of Urology,Qilu Hospital,Shandong University,between January 2016 and June 2018.The clinicopathological data of all bladder cancer patients,including age,sex,smoking history,blood examination results,and tumor characteristics were obtained.Statistical analysisThe levels of platelet count,MPV,PDW and PCT of bladder cancer patients,urothelial papilloma patients and healthy controls were analyzed using Kruskal Wallis test with multiple comparisons as determined by Nemenyi test.The continuous variables were compared using Student’s t-test or Mann-Whitney U,and categorical variables were compared using the Chi-square test.Receiver-operating characteristic(ROC)curves were used to evaluate the diagnostic role of PDW.The diagnostic accuracy of the ROC curve was determined by the area under the curve(AUC).The Youden index was used to determine the optimal cut-off value of PDW.ResultsEvaluation of platelet indices in bladder neoplasm patientsThe levels of MPV and PCT in bladder cancer patients(MPV:P<0.001;PCT:P<0.001)and urothelial papilloma patients(MPV:P<0.001;PCT:P<0.01)were considerably higher than that in healthy controls,whereas the PDW in bladder cancer patients and urothelial papilloma patients was lower when compared to healthy controls(P<0.001).Moreover,the PDW levels were lower in bladder cancer patients when compared to urothelial papilloma patients(P<0.05).However,no significant differences regarding platelet count were observed among healthy controls,urothelial papilloma patients and bladder cancer patients enrolled in our study.PDW is a potential diagnostic indicator of tumor malignancy in bladder neoplasmPDW,as assessed using ROC analysis,assists in distinguishing bladder cancer from urothelial papilloma,MIBC from NMIBC,and high-grade bladder cancer from low-grade bladder cancer.The AUC regarding the prediction of bladder cancer,MIBC,and high-grade bladder cancer were 0.583(95%CI:0.507,0.659;P=0.033),0.612(95%CI:0.534,0.690;P=0.006),and 0.662(95%CI:0.588,0.736;P<0.001).The cutoff values of PDW for evaluating bladder cancer,MIBC,and high-grade bladder cancer were 11.25(sensitivity 71.1%,specificity 42.4%),11.95(sensitivity 51.6%,specificity 65.5%),and 11.95(sensitivity 59.6%,specificity 66.1%)respectively,according to the Youden index.The relationship between PDW and clinicopathological characteristics in bladder cancer patientsThe levels of PDW were significantly lower in patients with more advanced bladder cancer.Specifically,decreased PDW levels were observed in bladder cancer patients with higher platelet counts(>223×109/L,P<0.001),greater tumor size(>3cm,P=0.029),high tumor grade(P<0.001),more advanced TNM stages(P=0.018),MIBC(P=0.003),and higher G classification(P=0.022).Further grouping analysis according to TNM stages revealed that the PDW levels were associated with primary tumor stages(pT stages,P=0.011)and lymph node stages(pN stages,P=0.074).Furthermore,PDW level was lower in MIBC patients with lymph node metastasis compared with patients in NO stage,though the difference was not significant(pN stages in MI,P=0.125).No significant correlations were observed between PDW levels and age,gender,smoking history,WBC count,and multiplicity of tumor growth.According to the cutoff values of PDW in evaluating muscle invasiveness and tumor grade,bladder cancer patients were divided into two groups.Of the total 210 bladder cancer patients,116 patients(55.2%)had PDW<11.95,while 94 patients(44.8%)had PDW>11.95.Tumor size(P=0.042),tumor grade(P<0.001),TNM stages(P=0.005),pT stages(P<0.004),pN stages(P=0.044),muscle invasiveness(P=0.015),and G classification(P=0.004)showed significant differences between the two groups,whereas no significant differences in age,gender and smoking history were observed between these two groups of patients.Meanwhile,patients with PDW<11.95 showed considerably lower MPV levels(P<0.001),as well as higher platelet count(P=0.002),compared to patients with PDW>11.95.ConclusionsReduced preoperative PDW level is an indicator of malignancy and advanced bladder cancer stages,suggesting it as a potential biomarker in bladder cancer diagnosis.更多还原