【作者】 李平；

【导师】 田野；

【作者基本信息】 苏州大学 ， 肿瘤学（专业学位）， 2019， 博士

【摘要】 研究背景和目的目前宫颈癌的发病率及死亡率居我国妇科恶性肿瘤第一位。包括组蛋白乙酰化在内的基因表观遗传修饰异常是宫颈癌发生发展的重要因素。参与组蛋白乙酰化的赖氨酸乙酰转移酶(lysine acetyltransferases,KATs)对染色体的结构和功能至关重要。在编码KATs的基因中,KAT6B是一个非常重要的肿瘤驱动基因,其扩增在多种肿瘤中发现,比如卵巢癌、乳腺癌、肺癌、肠癌等。也有研究发现KAT6B具有抑癌基因的作用。目前,KAT6B的有关研究较少,其对宫颈癌恶性生物学行为的作用及分子机制尚未明确。本项目拟运用分子、细胞生物学等方法,研究KAT6B在宫颈癌发展中的作用及相关信号机制。进一步研究KAT6B在宫颈癌患者中表达及对临床预后的影响。探讨宫颈癌治疗新的潜在靶点(KAT6B-AMPKα)。研究方法及材料首先,收集江苏大学附属昆山医院手术切除的未做任何放疗、化疗的新鲜原发宫颈癌标本6例,运用PCR和/或western blot方法检测宫颈癌组织中KAT6B、AMPKα表达。随后,人宫颈癌Hela、C33a细胞株(购自上海生命科学研究院),分别转染si RNA、CRISPR/Cas9-KO及过表达(over expression)KAT6B病毒,建立不同KAT6B表达、稳转的宫颈癌细胞株。显微镜下每日记录固定视野细胞数量、克隆形成实验检测各组细胞生长;EdU实验检测细胞增殖;细胞周期由流式细胞仪检测;细胞迁移由划痕实验、transwell实验检测;细胞侵袭能力由Matrigel transwell实验检测。Western blot检测KAT6B、AMPKα、磷酸化AMPKα、磷酸化AKT及m TOR、S6和磷酸化S6蛋白表达,并检测相关生长因子受体EGFR、PDGFRα、PDGFRβ等蛋白的表达变化。最后,收集江苏大学附属昆山医院活检及手术切除的未做任何放疗、化疗的宫颈癌标本(新鲜及石蜡包埋)119例,取手术周边正常组织(35例)作为对照。宫颈癌及正常宫颈组织中KAT6B表达由免疫组化方法检测,并分析其与宫颈癌各临床指标的相关性,包括病理类型、分期、分级、预后。进一步研究KAT6B在宫颈癌发展中的作用。实验操作中细胞种板的数量、试剂的浓度及具体实验方法基于发表文献和预实验的结果。统计学处理在细胞学实验中,至少设置3个副孔,每个实验重复≥3次。分析统计及作图使用SPSS 22.0及Graph Pad prism 7.0软件,并运用one-way ANOVA,Scheffe和Tukey检验、Chi-square检验、Kaplan-Meier法、COX回归分析等方法分析实验数据。有统计学意义的判定标准:P<0.05。研究结果1.宫颈癌组织KAT6B相比癌周围正常组织表达增加。收集江苏大学附属昆山医院手术切除的未做任何放疗、化疗的新鲜原发宫颈癌标本6例,PCR检测组织中KAT6B mRNA,结果发现宫颈癌组织中KAT6B mRNA水平较周围正常组织明显增加。宫颈癌组织中KAT6B、EGFR、PDGFRα蛋白表达明显增加。AMPKα则较正常组织相比减少,但无统计学意义。2.KAT6B促进宫颈癌细胞生长、增殖、迁移、侵袭。建立不同KAT6B表达、稳转的宫颈癌细胞株。之后对各组细胞连续6天计数,记录其生长情况。下调KAT6B减慢细胞增殖速度,细胞增殖明显抑制是从第四天开始。细胞克隆实验、EdU实验也提示下调KAT6B细胞增殖速度下降。而KAT6B上调,促进宫颈癌细胞生长、增殖。划痕实验、transwell、Matrigel transwell实验提示抑制KAT6B后,细胞迁移、侵袭能力明显抑制,而KAT6B过表达组均出现促进迁移、侵袭的结果。3.KAT6B促进细胞分裂增殖。敲低或敲除KAT6B后,细胞处于G0/G1期数量增多、处于S期明显减少。而KAT6B过表达的宫颈癌细胞株则出现相反的结果,G0/G1期细胞减少,处于DNA复制的S期细胞明显增多。4.KAT6B抑制AMPKα信号通路。western blot结果表明,KAT6B下调的宫颈癌细胞KAT6B表达减少,磷酸化的AMPKα表达增加,并且磷酸化的m TOR、AKT、S6表达也减少。EGFR、PDGFRα、PDGFRβ这些酪氨酸激酶受体也发现表达减少。而KAT6B过表达的细胞中则出现相反的结果。5.KAT6B高表达是宫颈癌的不良预后因素。在两种组织细胞浆、细胞核中均检出KAT6B表达。其中宫颈癌KAT6B高表达为30.43%(胞浆)、33.91%(胞核),差异无统计学意义。正常宫颈KAT6B高表达占2.94%(胞浆)、23.53%(胞核),差异有统计学意义。运用Kaplan-Meier方法分析发现KAT6B高表达(胞浆/胞核)宫颈癌患者总生存期(Overall Survival,OS)、无病生存期(Progression Free Survival,PFS)明显缩短。单因素及COX回归方法分析KAT6B表达与临床指标的相关性,提示FIGO分期、高KAT6B表达是OS的独立预后因素(FIGO分期:HR 3.531,95%CI1.763-7.071,p<0.05;胞浆KAT6B:HR 2.949,95%CI 1.443-6.025,p<0.05;胞核KAT6B:HR 2.529,95%CI 1.248-5.128,p<0.05),亦是PFS的独立预后因素(FIGO分期:HR 3.531,95%CI 1.763-7.071,p<0.05;胞浆KAT6B:HR 2.968,95%CI 1.541-5.713,p<0.05;胞核KAT6B:HR 2.529,95%CI 1.321-4.816,p<0.05)。结论1.宫颈癌组织中KAT6B表达增加,EGFR、PDGFRα蛋白也相应的表达增加,KAT6B可能是促进宫颈癌增殖的因素之一。AMPKα则较正常组织相比减少(p>0.05)。2.下调KAT6B后宫颈癌细胞增殖、迁移、侵袭能力均明显下降,而KAT6B过表达的宫颈癌细胞则表现出相反的结果。KAT6B在宫颈癌增殖、迁移、侵袭中具有重要作用。3.宫颈癌KAT6B抑制AMPKα激活,伴随多种酪氨酸激酶受体表达增加,并激活AKT-m TOR信号通路,这可能是KAT6B促进宫颈癌增殖的信号机制。4.宫颈癌KAT6B表达增加,且胞浆与胞核表达无明显差异。宫颈癌KAT6B表达与年龄、FIGO分期、淋巴结相关,不同病理类型、肿瘤分级的宫颈癌中表达无明显差异。KAT6B高表达患者预后较差,有更短的OS及PFS。COX分析发现FIGO分期、KAT6B表达为OS、PFS的独立预后因素。更多还原

【Abstract】 Background and ObjectivesCervical cancer ranks first in the incidence and death of gynecological tumors in China.More and more studies have found that abnormal gene epigenetic modification plays a vital role in cervical cancer development.Lysine acetyltransferases(KATs)histone acetylation is essential in the structure and function of chromosomes.In the gene encoding KATs,KAT6B is a very important tumor driver gene,whose amplification has been found in many kinds of tumors,such as ovarian cancer,breast cancer,lung cancer,and bowel cancer,etc.KAT6B is also identified as tumor suppressor genes.At present,there are few relevant studies on KAT6B.The malignant biological behavior of KAT6B and its molecular mechanism is not yet clear in cervical cancer.This project intends to study the biological role of KAT6B and its related signal mechanism in the development of cervical cancer by molecular and cell biology methods.The relationship between KAT6B expression and the clinical prognosis was studied further.Try to explore new potential targets for cervical cancer treatment(KAT6B-AMPKα).Methods and materialsFirst of all,6 cases of fresh primary cervical cancer tissues without radiotherapy or chemotherapy before surgery were collected from Kunshan Hospital affiliated to Jiangsu University.KAT6B mRNA in the tissues was detected by PCR.The western blot method was used to detect the expression of KAT6B protein and AMPKα protein in cervical cancer tissues.Subsequently,the Cell Bank of Shanghai Biological Institution provided the Hela and C33 a cervical cancer cell lines.Two different lentiviral si RNA constructs targeting KAT6B,KAT6B over-expression construct,two CRISPR/Cas9 KAT6B knockout constructs,and nonsense scramble vectors were transfected into cervical cancer cells.Cell proliferation was performed by the daily recording of cell number,clonogenicity,and EdU assay.Cell cycle was detected by flow cytometry.Cell scratch assay and transwell assay was performed to study the migration.Matrigel transwell assay detected cell invasion.Western blot was performed to detect the expression of different proteins,including KAT6B,AMPKα,phosphorylated AMPKα,phosphorylated AKT,phosphorylated m TOR,S6,and phosphorylated S6.EGFR,PDGFRα,and PDGFRβ were also detected by Western blot assay.Finally,119 cervical cancer specimens(fresh and paraffin-embedded)after biopsy and surgery were collected from Kunshan Hospital affiliated to Jiangsu University.Surrounding normal tissues(35 cases)were taken as controls.The expression of KAT6B was detected by immunohistochemistry(IHC)staining.Analyzed the correlation between KAT6B,pathological type,stage,grade,prognosis,and other clinical characteristics.The protocols were approved by the Ethics Review Board in Kunshan Hospital affiliated to Jiangsu University.The cell number in the seed plate,the concentration of reagents,and specific experimental assays were based on literature and results from preliminary experiments.Statistical analysisThe in vitro experiments were replicated three times at least 3 auxiliary wells,One-way ANOVA,Scheffe and Tukey test,Chi-square test,Kaplan-Meier method,and COX regression analysis were performed by SPSS 22.0 and Graph Pad prism 7.0.p<0.05 was considered significant statistically.Results1.KAT6B expression in cervical cancer tissues increased compared with normal cervical tissues.KAT6B mRNA in cervical cancer tissues was significantly increased compared with surrounding normal tissues.Western blot method was performed to detect KAT6B protein and AMPKα protein in cervical cancer tissues and normal tissues.It showed that the KAT6B,EGFR,and PDGFRα protein increased significantly in cervical cancer tissues.AMPKα was reduced compared to normal tissues,but it was not significant statistically.(p>0.05).2.KAT6B promoted the growth,proliferation,migration,and invasion of cervical cancer.Cervical cancer cells expressing different levels of KAT6B were established.Counted cell number for 6 consecutive days.Cell proliferation rate was slowed down in KAT6B knock-down/knock-out cervical cancer cells.Cell proliferation was inhibited significantly on the 4th day.Clonogenicity and EdU assays also detected that cell proliferation was inhibited significantly after KAT6B down-regulated.The KAT6B overexpression promoted the growth and proliferation obviously in cervical cancer cells.After inhibiting KAT6B,cell migration and invasion were inhibited significantly compared with the control group by Scratch,Transwell,and Matrigel transwell assays.The KAT6B overexpression promoted cervical cancer migration and invasion.3.KAT6B promoted cell division and proliferationAfter knocking down or knocking out KAT6B,the number of cells in the G0/G1 phase increased and ones in the S phase decreased significantly.On the other hand,KAT6B overexpression cervical cancer cells showed the opposite results.Cells number decreased in G0/G1 phase but increased in the S phase significantly.4.KAT6B inhibited AMPKα signaling pathway.After knocking down or knocking out KAT6B,phosphorylated AMPKα increased,but phosphorylated m TOR,AKT,S6 decreased.Tyrosine kinase receptors EGFR,PDGFRα,PDGFRβ have also detected expression decreased.KAT6B inhibited the activation of AMPKα,reduced the expression of EGFR,PDGFRα,and PDGFRβ,and activated the AKTm TORC1 pathway.The opposite result appeared in KAT6B overexpressing cells.5.High expression of KAT6B is a poor prognostic factor of cervical cancer.KAT6B expression was detected in the cytoplasm and nucleus.The KAT6B high expression ratio in cervical cancer was 30.43%(cytoplasm)and 33.91%(nucleus).The difference was not significant statistically.The KAT6B high expression in the normal cervix accounted for 2.94%(cytoplasm)and 23.53%(nucleus).The difference is significant statistically.Kaplan-Meier analysis showed that the Overall Survival(OS)and ProgressionFree Survival(PFS)in high KAT6B expression(cytoplasm/nucleus)cervical cancer patients were shorter.COX regression method analyzed the correlation between KAT6B expression,tumor tissue type,grade,FIGO stage,and prognosis.It showed that FIGO stage,high KAT6B expression were independent prognostic factors in OS(FIGO stage: HR 3.531,95% CI 1.763-7.071,p<0.05;Cytoplasm: HR 2.949,95% CI 1.443-6.025,p<0.05;Nucleus: HR 2.529,95% CI 1.248-5.128,p<0.05)and PFS(FIGO stage: HR 3.531,95% CI 1.763-7.071,p<0.05;Cytoplasm: HR 2.968,95% CI 1.541-5.713,p<0.05;Nucleus: HR 2.529,95% CI 1.321-4.816,p<0.05).Conclusions1.The KAT6B expression was increased in cervical cancer tissues,and EGFR and PDGFRα protein was also increased.KAT6B may be one of the factors promoting the proliferation of cervical cancer.The expression of AMPKα was decreased compared with that one in surrounding normal tissues(p>0.05).2.After KAT6B knockdown/knockout,the proliferation,migration,and invasion abilities in cervical cancer cells were significantly decreased.It showed the opposite results for the cervical cancer cells with KAT6B overexpression.KAT6B played an important role in the proliferation,migration,and invasion of cervical cancer.3.KAT6B inhibited AMPKα activation,accompanied by increased expression of multiple tyrosine kinase receptors.KAT6B activated AKT-m TOR signaling pathway.This may be the signal mechanism that KAT6B promoted the proliferation in cervical cancer.4.The KAT6B expression was increased in cervical cancer tissues,and there was no significant difference between the expression in the cytoplasm and nucleus.The KAT6B expression in cervical cancer was correlated with age,FIGO stage,and lymph node status,but not with pathological type or tumor grade.Patients with KAT6B high expression had a poor prognosis with lower OS and PFS.FIGO stage and KAT6B were independent prognostic factors for OS and PFS.更多还原