【作者】 刘妍；

【导师】 李官成；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 第一章肿瘤标志物的生物信息学筛选及其表达谱初步分析目的:采用生物信息学方法筛选肿瘤差异表达基因,并对筛选得到的结果进行表达谱初步分析。方法:利用CGAP网站提供的cDNA xProfiler工具,以人类正常组织和肿瘤组织EST数据库为基础,筛选肿瘤差异表达基因,并对筛选得到的68个基因进行染色体定位。采用电子杂交以及RT-PCR的方法对筛选得到的结果进行表达谱初步分析。结果:通过生物信息学筛选得到在肿瘤组织中特异性表达的基因或EST片段2801个,其中已知基因68个,未知基因2733个。在这68个已知基因中,40个基因的功能已基本明确。在这40个基因中5个基因已经实验证实为肿瘤特异性基因,占12.5%。对筛选得到的68个基因进行染色体定位,发现在7号和10号染色体上筛选得到的肿瘤特异性基因较少,而在8号、21号以及X染色体中肿瘤特异性基因出现的频率明显多于其他染色体。进一步对筛选得到的2801个肿瘤特异性表达基因或EST片段进行电子杂交分析,9个基因结果较理想。RT-PCR检测这9个基因在不同细胞株中的表达,7个基因在被检测的11个细胞株中未得到目的片段,而GABRA3基因与HTA基因在肿瘤细胞中表达特异。结论:生物信息学筛选肿瘤差异表达基因是一种高效的方法。肿瘤特异性基因在某些染色体上相对“活跃”。第二章GABRA3基因的表达谱分析及功能研究目的:对GABRA3基因进行表达谱分析及功能初步研究。检测GABAA受体各亚单位在肿瘤组织中的表达。方法:采用RT-PCR以及免疫组化法分析GABRA3的表达谱。通过RNAi以及药物处理对GABRA3基因的功能进行初步研究。结果:在被检测的11株细胞株中,GABRA3基因在肝癌细胞株HepG2,乳腺癌细胞株MCF-7,MDA-MB-231和胶质瘤细胞株U251中表达,而在其他肿瘤细胞株以及正常细胞株中无表达。在被检测的20种正常组织中,该基因除在脑组织,子宫内膜,胎盘以及前列腺中有表达外,在其他正常组织中均未被检测到。在18例新鲜肺癌组织标本中GABRA3表达阳性率为66.67%,而对应的18例肺癌癌旁组织中均未检测到该基因。采用免疫组化的方法,分析GABRA3在60例肺癌及10例癌旁组织中的表达。10例癌旁组织均未染色,而60例肺癌组织标本中GABRA3蛋白表达的阳性率为41.67%,肺癌组织中细胞阳性染色主要见于胞质和胞膜。通过对GABRA3蛋白在肺癌组织中表达量与临床资料的相关性分析,发现GABRA3蛋白的表达量肺癌的病理分级密切相关(p＜0.05)。随着肿瘤恶性程度的增高,GABRA3蛋白表达的阳性率和表达量有增高的趋势。在8例肝癌组织标本中,共6例表达GABRA3基因,阳性率为65%。而在所有对应的癌旁组织中均未扩增得到该基因目的片断。在肺癌和肝癌组织中,还检测了GABAA受体其他亚单位的表达情况,除α3亚单位外还有GABAA受体α6、β3、γ2、δ、θ、ε和π亚单位的表达。在肝癌细胞株HepG2细胞中,检测到GABA生成过程中的关键酶GAD67表达。通过细胞增殖能力研究发现,GABA对肝癌细胞株HepG2具有促增殖的作用。当GABRA3基因被沉默后HepG2细胞的增殖能力明显降低。对GABRA3基因被沉默的HepG2细胞给予GABA药物处理,结果发现其增殖能力非但没有增强,反而有减弱的趋势。结论:GABRA3基因在肺癌和肝癌中表达上调,其表达量与肿瘤的分化程度密切相关。在肝癌中,肝癌细胞可经GAD67催化产生GABA;GABA发挥了促肿瘤细胞增殖的作用,其作用是通过上调表达的GABRA3介导的。除GABRA3外,肝癌和肺癌组织中还有GABAA受体其他亚单位的表达,当GABRA3被沉默时,GABA通过这些亚单位发挥了抑制肿瘤细胞增殖的作用。第三章HTA基因的表达谱分析及功能研究目的:对HTA基因进行生物信息学分析,并对该基因进行表达谱分析及功能初步研究。方法:采用RT-PCR法分析HTA基因的表达谱,通过RNAi对该基因的功能进行初步研究,采用各种在线工具软件对该基因进行生物信息学分析。结果:通过RT-PCR检测到HTA基因在人肝癌细胞株HepG2以及多种肿瘤组织中表达,在肝癌中表达阳性率较高,而在正常肝细胞株L-0以及20种正常组织标本均无表达。采用RNAi技术抑制HTA基因在肝癌HepG2细胞中的表达,通过细胞生长曲线、细胞倍增时间、软琼脂集落形成实验、细胞周期检测以及裸鼠成瘤实验研究发现,HTA基因被沉默后HepG2细胞的增殖能力降低。经生物信息学分析,HTA定位于人类染色体16q22.3,包含了3个外显子和2个内含子。其编码的蛋白质为一个分子量较小的碱性亲水性蛋白,定位于细胞核和细胞浆的可能性较大,可能含有7个磷酸化位点、6个糖基化位点以及4个抗原表位。HTA编码蛋白的二级结构中α-螺旋为33.70%,β-折叠为5.43%,属于混合蛋白。结论:HTA基因特异性表达于肿瘤组织,在肝癌中表达阳性率较高,可能是一种肿瘤特异性基因。该基因具有促进肝癌细胞增殖的作用。更多还原

【Abstract】 Part 1 In Silico Identification of Tumor Markers and the Expression Patterns of the Candidate GenesObjectives:To screen tumor markers through whole genome in silico.Methods:To identify the genes,which are differentially expressed in tumors compared with their corresponding normal tissues,a search was performed using the CGAP cDNA xProfiler based on EST data. E-Northern and RT-PCR was used to analyze the expression profiles of the identified tumor unique genes.Results:According to the cDNA xProfiler results,there were 2801 genes or ESTs unique in tumor tissues,68 of which were known and 2733 of which were unknown.The functions of 40 genes are clear.5 genes among the 40 genes were tumor-associated genes.On chromosome 7 and 10,few tumor markers were identified;while on chromosome 8,21 and X,much more markers were identified.Then,we performed E-Northern to analyze the expression profiles of the 2801 tumor unique genes.9 genes seemed like ideal.We then performed RT-PCR to verify the expression of these 9 genes in tumor and normal cell lines.7 of them could not be detected in 11 cell lines.GABRA3 and HTA were expressed specifically in cancer cells.Conclusions:In silico identification is an excellent and time-saving approache to identify genes that could be used as tumor diagnostic markers,prognostic indicators,and suitable targets for various forms of therapeutic intervention.Tumor associated genes were more "active" on some chromosomes.Part 2 Expression Profile and Function of GABRA3Objectives:To study the expression profile and function of GABRA3 gene and to detect the expression of GABAA receptor subunits in tumors.Methods:RT-PCR and immunohistochemistry were used to analyze the expression profile of GABRA3 gene.RNAi and GABA were used to study the function of GABRA3.Results:GABRA3 was highly expressed in hepatocellular carcinoma line HepG2,and weakly detected in glioma cell line U251 and breast cancer cell lines MCF-7 and MDA-MB-231.In normal tissues, GABRA3 was strongly expressed in brain and placenta,and weakly detected in endometrium and prostate,but undetectable in other tissues. More importantly,the expression of GABRA3 in cancers was rare or less abundant but with the exception of glioma,endometrial cancer,lung caner and liver cancer.By RT-PCR,GABRA3 was detected in 12 out of 18 lung cancer samples(66.67%),but not expressed in corresponding adjacent noncancerous lung tissues.By immunohistochemistry,GABRA3 was detected in 25 out of 60 lung cancer samples(41.67%),but not in 10 corresponding adjacent noncancerous lung tissues.The experimental results demonstrated cytoplasmic and membrane staining.The results further showed the correlation between GABRA3 protein expression and clinical-pathologic variables in lung cancers.GABRA3 protein expression was significantly higher in lower grade of lung cancer (p＜0.05).GABRA3 was also detected in 6 out of 8 liver cancer samples (65%),while not in corresponding adjacent noncancerous liver tissues. We also detected the other subunits of GABAA receptor in lung cancer and liver cancer by RT-PCR.In those samples GABAA-α6,β3,γ2,δ,θ,εandπsubunits were also expressed.GABRA3 may compose functional GABAA receptors with them.In liver cancer cell line HepG2,GAD67 were detected,suggesting that GABA could function in an autocrine/paracrine manner in HCC cells and promote cell growth.GABA promoted the proliferation of HepG2 cells.Knockdown of endogenous GABRA3 expression in HepG2 attenuated HCC cell growth.GABA enhanced the growth of GABRA3 expressing HepG2 cell.On the other hand,the proliferating ability of GABRA3-knockdown HepG2 was not enhanced but lowered by GABA.Conclusions:GABRA3 is overexpressed in lung cancer and hepatoma.GABRA3 protein expression was significantly higher in lower grade of cancer.HepG2 cells could produce GABA.GABA promoted the proliferation of HepG2 cells through GABRA3.In lung cancers and liver cancers,some other GABAA receptor subunits were also expressed. When GABRA3 was knocken down,GABA inhibited the proliferative ability of HepG2 cells through those subunits.Part 3 Expression Profile and Function of HTAObjectives:To study the expression profile and function of HTA.Methods:RT-PCR was used to analyze the expression profile of HTA gene and RNAi was used to study the function of it.Results:HTA was a tumor-specific gene,especially in hepatoma.It was detected in liver cancer cell line HepG2,some liver cancer tissue samples(75%) and some other cancer tissues.But in the normal tissues it was not detected.Knockdown of endogenous HTA gene was performed by small interfering RNA in malignant hepatocyte HepG2.Then we tested the cell proliferative ability of these cells in vitro and in vivo.The results showed that HTA could promote HCC cell growth.HTA is located on chromosome 16q22.3,including 3 exons and 2 introns.The encoded protein is an alkaline hydrophilic protein,whose molecular weight is 10KD,PI is 10.9.This protein may play an important role in signal conduction because it may contain 7 phosphorylation sites and 6 glycosylation sites.The secondary structure of the protein contains 33.7%α-helix and 5.43%β-sheet.HTA encoded protein may contains 4 antigen epitope,which are located at 4-14aa,37-43aa,54-60aa and 65-89aa.Conclusions:HTA was a tumor-specific gene,especially in hepatoma.It plays an important role in HCC cell viability and is a sustained event in the tumorigenic process.更多还原