【作者】 张玲；

【导师】 孙耕耘；

【作者基本信息】 安徽医科大学 ， 内科学（呼吸系病）， 2019， 博士

【摘要】 背景肺癌是世界上最常见和最致命的恶性肿瘤之一,非小细胞肺癌(NSCLC)是最主要的组织病理学类型。研究发现,GATA2对RTK/RAS信号通路中KRAS或其他致癌基因突变的NSCLC细胞存活和生长至关重要;GATA2缺失降低了KRAS突变的NSCLC细胞生存能力,并显著抑制NSCLC发展。此外,GATA2还能通过调节蛋白酶体复合物、IL-1/NF-κB和Rho信号通路维持NSCLC细胞的存活。已知GATA2是GATA家族的一员,其作为转录因子能调控众多发育过程,并且GATA转录因子之间的相互作用在调控GATA家族基因的表达方面发挥重要作用。GATA2能与自身启动子区域结合并激活转录。然而,GATA1又与GATA2竞争性结合该启动子区域,通过破坏GATA2基因自身的转录来抑制GATA2的表达。因此,GATA1在GATA2的转录过程中发挥抑制作用。c-Myc在多种类型的癌细胞中都有组成性表达,它与启动子区域的E-boxes序列结合后,能激活众多参与细胞异常增殖的促癌基因转录,从而导致恶性肿瘤的形成。c-Myc基因的上调在胃癌、宫颈癌、结肠癌和NSCLC等恶性肿瘤中均有被发现。因此,c-Myc被认为是治疗癌症的一个潜在目标。近年人们发现长非编码RNA(lncRNA)在NSCLC等多种肿瘤的发生机制中发挥着重要的调控作用。lncRNA可以靶向转录激活因子或抑制因子调控下游基因的表达。目前研究报道了一种lncRNA的作用模式,即基因附近转录出来的lncRNA能通过招募转录因子或染色质修饰因子调控邻近基因的表达。例如,CCND1基因附近转录的lncRNA CCND1,它能结合TLS蛋白并使其转变为活性构象,从而结合到CCND1启动子上的CBP/p300复合物,最终抑制CCND1基因的转录。目的探讨lncRNA GATA2-AS1调控NSCLC生长及其受到c-Myc靶向调节的具体分子机制,为开拓NSCLC新的治疗靶点提供实验依据。材料与方法1.细胞培养和转染:在添加10%胎牛血清、1mM丙酮酸钠和100 mg/ml链霉素的DMEM培养基中培养A549细胞(KRAS突变)和HEK293T细胞。2.RNA干扰:HEK293T细胞转染对照组或实验组siRNA后,在Opti-MEM培养基培养12小时后换用10%胎牛血清的DMEM培养基,继续培养48小时。收集细胞,采用免疫印迹法和荧光定量PCR方法对敲除效率进行评估。3.实时荧光定量PCR(qRT-PCR):用Trizol提取总RNA,然后逆转录成cDNA;采用SYBR和ROX对照染料进行实时荧光定量PCR分析。4.免疫印迹(WB):收集细胞,加入缓冲液后95℃煮蛋白10~15分钟,然后将蛋白按照大小分离。5.染色质免疫共沉淀(ChIP):室温下将A549细胞用1%甲醛交联10分钟,甘氨酸终止反应。处理后的细胞用裂解缓冲液重悬。用GFP抗体或对照IgG偶联的蛋白与细胞裂解液孵育。洗脱后的产物经PCR扩增。6.RNA免疫共沉淀(RIP):1~3×10~7个细胞用低渗缓冲液裂解和离心。细胞裂解液经蛋白A/G beads预处理后,在4℃条件下与偶联相应抗体的A/G beads孵育。经多次洗涤后,用洗脱缓冲液洗脱beads上结合的免疫复合物,然后用蛋白酶K分离提取出蛋白结合的RNA。纯化的RNA可用于RT-PCR分析。7.克隆形成实验:将转染了对照组、GATA2或GATA2-AS1 siRNA的A549细胞于6孔板中进行传代。一周后,用预冷的10%甲醇固定细胞5分钟,室温下用结晶紫染色30分钟。经过多次冲洗,拍照。8.细胞周期实验:收集细胞后洗涤,在4℃条件下用70%乙醇固定过夜;然后离心5分钟,PBS洗涤2次,室温下加入PBS孵育30分钟;最后用流式细胞仪检测细胞周期的分布。结果1.GATA2-AS1抑制NSCLC细胞的增殖lncRNA GATA2-AS1是位于人类基因组第3号染色体GATA2基因反义链上的非编码性抑癌基因。首先在A549细胞中通过siRNA敲低GATA2-AS1,发现GATA2转录的mRNA水平上调;在GATA2-AS1缺失的细胞中,GATA2蛋白水平也升高。然后通过对蛋白酶体复合物、IL-1/NF-κB和Rho信号这三种途径中GATA2靶基因表达情况进行检测,发现在GATA2-AS1敲低细胞中均表现出较高的表达水平。随后在A549细胞中利用siRNA敲低GATA2-AS1,发现一旦缺少GATA2-AS1就会加快A549细胞的增殖,而同时敲低GATA2-AS1和GATA2,GATA2-AS1就无法加快细胞的增殖。以上表明GATA2-AS1通过抑制GATA2的表达从而阻止NSCLC细胞的生长。2.GATA2-AS1结合GATA1通过在A549细胞中进行核质分离实验分析GATA2-AS1的亚细胞定位,发现它主要位于细胞核。随后进行RIP证明异位表达的GATA1能够与GATA2-AS1相互结合;反之内源性GATA2-AS1能够与GATA1共沉淀,说明GATA2-AS1与GATA1蛋白在细胞核内存在相互作用。然后采用两阶段RIP分析GATA家族共调节因子FOG1是否包含在GATA2-AS1和GATA1复合物中。第一阶段使用Flag抗体去沉淀Flag-GATA1,它可以捕获高水平的FOG1和GATA2-AS1;第二阶段使用FOG1抗体去沉淀FOG1,发现能够共沉淀Flag-GATA1和GATA2-AS1。结果表明,GATA1、FOG1和GATA2-AS1在GATA2启动子处形成一个三聚体,然后调控GATA2转录。3.GATA2-AS1增强GATA1的抑制功能进一步利用GATA1抗体进行ChIP实验,检测到在GATA2启动子上游约2.8kb处有一段GATA结合序列,该序列与GATA1的结合率会随着GATA2-AS1的减少而降低。这表明GATA2-AS1增加了GATA1与GATA2启动子的结合,从而增强GATA1对GATA2表达的抑制作用。随后在过表达GATA2-AS1的同时敲低GATA1,发现GATA2-AS1对GATA2表达的抑制作用可以通过GATA1沉默得到恢复,进一步证实GATA2-AS1通过GATA1调控GATA2。4.GATA2-AS1通过GATA2抑制NSCLC细胞的增殖首先检测到GATA2-AS1敲低的A549细胞中GATA2靶基因的表达上调,而在敲低GATA2-AS1时再去敲低GATA2,发现对GATA2靶基因的作用消失,表明GATA2介导了GATA2-AS1对NSCLC细胞生长的影响。然后通过克隆形成实验证明,A549细胞沉默GATA2-AS1的表型可以被GATA2 siRNA所恢复,通过细胞周期分析证实了GATA2敲低和GATA2-AS1及GATA2双敲低细胞之间的关系,说明GATA2-AS1通过GATA2调控NSCLC细胞生长的机制。5.c-Myc转录调控GATA2-AS1通过生物信息学分析,在GATA2-AS1启动子上游1964bp位点找到了c-Myc潜在的结合序列(CACGTG)。沉默c-Myc能诱导NSCLC细胞中GATA2-AS1表达上调,而在A549细胞中转染外源性c-Myc使其增加时,GATA2-AS1表达水平逐渐降低。随后使用c-Myc抗体进行ChIP实验,证实c-Myc与GATA2-AS1启动子上游1964bp位点结合。此外,通过siRNA沉默敲除MIZ-1的表达,发现缺失的MIZ-1能够恢复c-Myc过表达引起的GATA2-AS1下调,说明c-Myc通过与MIZ-1相互作用调控了对GATA2-AS1的转录抑制。6.GATA2-AS1在癌组织中表达较低对NSCLC标本中GATA2-AS1的表达情况进行检测发现,大多数癌组织表达的GATA2-AS1水平低于癌旁组织;同时这些癌组织也显示出相对较高的c-Myc和GATA2表达水平;但癌组织与癌旁组织组织的GATA1水平无明显变化。结果表明,GATA2-AS1通过抑制NSCLC细胞的增殖而发挥抑癌作用。结论1.lncRNA GATA2-AS1是一个非编码性抑癌基因,位于人类基因组第3号染色体GATA2基因的反义链上;其转录方向与GATA2相反,对NSCLC细胞的发生和发展具有抑制作用。2.lncRNA GATA2-AS1通过在GATA2启动子区域与GATA1蛋白相互结合,从而抑制GATA2基因的转录,阻止NSCLC细胞的增殖;此外,证实GATA2-AS1能通过调控蛋白酶体复合物、IL-1/NF-κB和Rho信号通路抑制NSCLC细胞的生长。3.lncRNA GATA2-AS1在NSCLC细胞中能被c-Myc转录抑制调控。4.本研究证明了c-Myc→GATA2-AS1→GATA1→GATA2信号通路在NSCLC发展中的作用,为NSCLC的治疗提供了潜在靶点。更多还原

【Abstract】 BackgroundLung cancer is one of the most common and lethal cancers in the world,and Non-small cell Lung Cancer(NSCLC)is the dominant histopathology.It is found that GATA2 is crucial for survival and growth of those NSCLC cells with mutations in KRAS and other oncogenes on the RTK/RAS pathway,and the deletion of GATA2 reduces survival of KRAS mutant NSCLC cells significantly inhibit the development of NSCLC.In addition,GATA2 can maintain the survival of NSCLC cells by regulating simultaneously proteasome complexes,IL-1/NF-κB and Rho signaling pathways.GATA2 is known to be a member of GATA family,which is a transcription factor that regulates many developmental processes,and the interaction of GATA transcription factors plays an important role in regulating gene expression in GATA family.GATA2binds at GATA2 gene promoter region and activates its own transcription.However,GATA1 can displace GATA2 from this position,and instigates GATA2 repression by means of disruption of its positive autoregulation.Therefore,GATA1-mediated displacement of GATA2 is tightly coupled to repression of GATA2 transcription.c-Myc is constitutively expressed in many types of cancer cells,which combined with the E-boxes sequence in the promoter region,and then activates the transcription of numerous oncogenes involved in abnormal cell proliferation,leading to the formation of malignant tumors.Constitutive up-regulation of c-Myc gene has been discovered in a lot of cancers,such as gastric,cervical,colon and non-small cell lung cancer.Therefore c-Myc has been identified as a potential target for cancer treatment.During recent years,long non-coding RNA(lncRNA)has been found to play an important regulatory role in a variety of tumorigenesis mechanisms including NSCLC.LncRNA can target transcriptional activators or inhibitors to regulate the transcription and expression of downstream genes.One type of lncRNA accumulates in cis after transcription and regulate adjacent gene expression either by recruiting transcription factors or chromatin modifiers.For example,the lncRNA CCND1,which is transcribed near the chromosome of CCND1 gene,can bind to TLS proteins and transform TLS into the active conformation,which can bind to the CBP/p300 complex on the promoter of CCND1 and ultimately inhibit the transcription of CCND1 gene.ObjectiveTo explore the specific molecular mechanism of lncRNA GATA2-AS1 regulating the growth of NSCLC and its targeted regulation by c-myc,so as to provide experimental basis for exploring new therapeutic targets of NSCLC.Materials and methods1.Cell Culture and transfection:A549 cells(KRAS mutant)and HEK293T cells were cultured in DMEM supplemented with 10%Fetal Bovine Serum,1 mm Sodium pyruvate and 100 mg/ml streptomycin.2.RNA interference:After transfection of HEK293T cells into control group or experimental group,the cells were cultured in Opti-MEM for 12 hours and then in DMEM containing 10%Fetal Bovine Serum for 48 hours.The efficiency of knockdown was assessed by Western blotting and quantitative PCR(qPCR).3.Quantitative real-time PCR(qRT-PCR):Total RNA was extracted using Trizol and then reverse-transcribed into cDNA.qPCR was carried out and analyzed by using SYBR and ROX Reference Dye(ROX).4.Western blot(WB)analysis:Harvested cells,added SDS loading buffer solution,boiled protein at 95°C for 10~15 minutes,and separate protein on SDS-page.5.Chromatin immunoprecipitation(ChIP)assay:A549 cells were crosslinked with 1%formaldehyde at room temperature for 10 minutes.Glycine was added to stop the reaction.The treated cells were overhung with a lysis buffer.The protein coupled with anti-GFP or control IgG was incubated with cell lysates.The eluted product was amplified by PCR.6.RNA immunoprecipitation(RIP):1~3×10~7 cells were lysed and centrifuged with hypotonic buffer.The lysate was pretreated with protein A/G beads and incubated at 4°C for 3 hours with A/G beads coupled with the corresponding antibody.After extensive washing,the bead-bound immunocomplexes were eluted using elution buffer.To isolate protein bound RNA from the eluted immunocomplexes,samples were treated with protease K and RNAs were extracted.The purified RNAs were then subjected to RT-PCR analysis.7.Colony formation assay:A549cells transfected withscramble,GATA2 or GATA2-AS1 siRNAs were plated at a density of 500 cells per well on a six-well plate.After one week,cells were fixed with 10%cold methanol for 5 minutes and stained with crystal violet at room temperature for 30 minutes.After extensive wash,the colonies were photographed.8.Cell cycle analysis:The cells were harvested,washed and fixed with 70%ethanol at4°C overnight.Set and centrifuged for 5 minutes.PBS was rinsed twice and incubated at room temperature for 30 minutes with PBS.Finally,the distribution of cell cycle was detected by Flow cytometry.Results1.GATA2-AS1 inhibits the proliferation of NSCLC cellsLncRNA GATA2-AS1 was a non-coding oncosuppressor gene located on the antisense chain of GATA2 gene on chromosome 3 of the human genome.Firstly,we knocked down GATA2-AS1 by siRNA in A549 cells and found mRNA level of GATA2 was up-regulated after GATA2-AS1 silencing,and the level of GATA2 protein was up-regulated in GATA2-AS1 deficient cells.Then the expression of GATA2 targets gene in GATA2-AS1 knock-down cells was examined through proteasome complex,IL-1/NF-κB and Rho signaling pathways.Subsequently,we used siRNA to knock down GATA2-AS1 in A549 cells,and found that the absence of GATA2-AS1 accelerated the proliferation of A549 cells,while the absence of GATA2-AS1 and GATA2 did not.These results demonstrate that GATA2-AS1 represses GATA2 expression and NSCLC cells growth.2.GATA2-AS1 binds to GATA1The subcellular localization of GATA2-AS1 in A549 cells was analyzed by means of nuclear-cytoplasmic separation.Then RIP assay demonstrated that ectopically expressed GATA1 was able to interact with GATA2-AS1,whereas endogenous GATA2-AS1 was capable of co-precipitate with GATA1,indicating that GATA2-AS1 and GATA1 protein physically interact with each other in the nucleus.Then two-step RIP assay was used to analyze whether GATA family coregulator FOG1 is included in the GATA2-AS1 and GATA1 complex.In the first phase IP we used anti-Flag IgG which captured high levels of FOG1 and GATA2-AS1 with Flag-GATA1,and in the second IP we used anti-FOG1IgG co-precipitated Flag-GATA1 and GATA2-AS1 in addition to FOG1.The results show that GATA1,FOG1 and GATA2-AS1 form a trimer at the GATA2 promoter,and then regulate its transcription.3.GATA2-AS1 enhances repressive function of GATA1Further ChIP assay using GATA1 antibody showed that there was a GATA binding sequence located at about 2.8 kb upstream of GATA2 gene,and a lower GATA1 occupancy on GATA2 promoter region was detected in GATA2-AS1 silencing cells.This suggests that GATA2-AS1 enhances GATA1 binding to GATA2 promoter and therefore enhances the repressive function of GATA1 on GATA2 expression.It was also found that suppressive effect of GATA2-AS1 on GATA2 expression was rescued by GATA1silencing,which further confirmed that GATA2-AS1 regulated GATA2 through GATA1.4.GATA2-AS1 inhibits the proliferation of NSCLC cells through GATA2Firstly,GATA2 targets expression in A549 cells with or without in GATA2-AS1 siRNA transfection were examined,and it was found those alterations in GATA2-AS1 silencing cells could be reversed by GATA2 siRNA treatment,suggesting GATA2 indeed mediated the effect of GATA2-AS1 on NSCLC cells growth.Then it was demonstrated that the phenotype of GATA2-AS1 silencing A549 cells could be rescued by GATA2 siRNA,which was further confirmed by cell cycle analysis between GATA2 knock-down,GATA2-AS1 and GATA2 double knock-down cells,illustrated that GATA2-AS1regulates the growth of lung cancer through GATA2.5.c-Myc transcriptionally regulates GATA2-AS1Through bioinformatic analyses,c-Myc response element sequence(CACGTG)was determined at the-1964 site of GATA2-AS1 promoter.c-Myc silencing could induce up-regulation of GATA2-AS1 level in NSCLC cells,and in contrast the level of GATA2-AS1reduced gradually when exogenous c-Myc was transfected into A549 cells.Then anti-c-Myc was used to carry out the ChIP assay,and confirmed c-Myc indeed bound to the region including the c-Myc response element at-1964 of the GATA2-AS1 promoter.Furthermore,MIZ-1was knocked down by siRNA silencing and it was found MIZ-1deficiency was able to rescue the GATA2-AS1 down-regulation induced by c-Myc over-expression,indicating c-Myc regulated transcriptional repression of GATA2-AS1 through interacting with MIZ-1.6.GATA2-AS1 expresses at a lower level in NSCLC tissuesTo further understand the function of GATA2-AS1 in the development of NSCLC,the expression of GATA2-AS1 was examined in clinical NSCLC samples.Analysis of surgically removed NSCLC samples showed that the expression level of GATA2-AS1 in most cancer tissues was lower than that in adjacent tissues.At the same time,these cancer tissures also showed relatively higher expression levels of c-Myc and GATA2.However,there was no obvious change in GATA1 level between cancer tissues and adjacent tissues.These results suggest that GATA2-AS1 could inhibit the proliferation of NSCLC cells.Conclusions1.LncRNA GATA2-AS1 was a non-coding cancer suppressor gene,which was located in the antisense chain of GATA2 gene on chromosome 3 of human genome.Its transcription direction was opposite to GATA2,which could inhibit the occurrence and development of NSCLC cells.2.LncRNA GATA2-AS1 binded to GATA1 protein in the promoter region of GATA2,thereby inhibiting the transcription of GATA2 and ultimately preventing the proliferation of NSCLC cells.In addition,GATA2-AS1 had been shown to inhibit the growth of NSCLC cells by regulating the proteasome complex,IL-1/NF-κB and Rho signaling pathways.3.LncRNA GATA2-AS1 could be regulated by transcription inhibition of c-Myc in NSCLC cells.4.This Study proved the role of c-Myc→GATA2-AS1→GATA1→GATA2 signaling pathway in the development of NSCLC and provide potential targets for the treatment of NSCLC.更多还原