血清HER2及长链非编码RNAp2819在胃癌肿瘤生物学中的作用研究

发布时间：2018-06-02 00:54

本文选题：胃癌 + HER2　；参考：《中国人民解放军医学院》2017年博士论文

【摘要】：背景胃癌在世界范围内一直是一个严重威胁人类健康的疾病,其发病率在所有恶性肿瘤中占第二位,死亡率占全部癌性致死性疾病的第二位。在消化道恶性肿瘤中占首位。虽然近年来内镜、手术、化疗、靶向等诊疗手段均有了一定的提高,但目前胃癌患者5年生存率仍低于30%。既往研究关注基因组学、表观遗传学、蛋白质组学等方面的变化。如原癌基因的激活或(和)抑癌基因的失活、蛋白相互作用,信号传导通路,调控网络等。有些研究有些成果已用于临床,如基因HER2靶向治疗在乳腺癌中的应用,为部分患者带来益处。HER2的检测在乳腺癌中已成常规,但在胃癌尚不是,多需专门提出,但准确测定并监测胃癌患者体内HER2状态,对于合理应用HER2靶向治疗及评估预后均至关重要。目前其检测手段是荧光原位杂交分析(FISH)和免疫组织化学,在临床应用中有一定的局限性,比如第一,两种方法有13%左右的不一致率,第二,免疫组化是半定量检测,主观性较强,而FISH检测耗时且昂贵。第三,血清中HER2的情况可以在曲妥珠单抗治疗过程中发生变化,而FISH及免疫组织化学都不能实时监测,这些均不完善,成为本课题第一部分产生的初衷。肿瘤分子生物学的研究,除了对只占全部基因组2%的编码基因的研究之外,还有很多未知领域,新一代基因测序技术及高通量筛选方法发展,使大量非编码RNA被发现,非编码RNA主要包括长链非编码RNA(long non-codingRNA,lncRNA)和微小RNA(microRNA,miRNA)。最初认为非编码RNA无功能,随着全部人类基因组测序的完成,人类的疾病并没有随之迎刃而解。所以人们开始关注数量众多却被忽视的非编码RNA的研究。近些年miRNA在肿瘤生物学的调控作用已有广泛研究。然而,lncRNA的数量众多,功能多样,可在表观遗传学、转录及转录后等水平参与基因表达的调控。相关研究尚较少,特别是在胃癌的研究上更是刚刚开始,本研究通过对胃癌及癌旁组织异常表达的lncRNA研究,有望发现新的与胃癌相关的lncRNA,作为胃癌诊断治疗新靶点,从而形成了论文的第二部分。第一部分胃癌患者血清HER2水平检测与组织HER2表达相关性及临床意义目的:探索血清(胞外域)HER2水平与组织内HER2水平的相关性,探索血清HER2水平对胃癌患者的作用。方法:收集解放军总医院2012年12月-2013年6月期间239例胃癌库中患者资料。应用化学荧光法检测血清HER2水平,应用荧光原位杂交分析(FISH)及免疫组织化学的方法,检测组织内HER2水平。画出受试者工作特征曲线(ROC),确定出预测胃癌患者生存的血清HER2水平的最佳截断值。结果:血清HER2水平与组织内HER2水平、肿瘤体积、肠型胃癌等均显著相关,血清HER2水平在组织内HER2阳性的患者与组织内HER2阴性患者间有显著的差异。得出曲线下最大面积为0.79(95%CI: 0.71-0.87, P 0.001)。敏感度及特异度分别为0.54 (95%CI:0.37-0.70)和 0.93 (95%CI:0.88-0.96)。此时的截断值为 24.75ng/mL。高的血清 HER2水平对胃癌患者总体生存率呈负相关关系。(hazard ratio: 1.93,95%CI: 1.32-4.38, P =0.006)。结论:血清HER2水平可以作为一个能替代组织内HER2表达情况的特异性较高的标志物,HER2水平24.75ng/mL提示预后不佳。第二部分过表达lncRNA-p2819对胃癌细胞BGC-823增殖、凋亡和侵袭的影响目的:为了明确胃癌及癌旁组织中有哪些差异表达的lncRNA,这些差异表达的lncRNA有无临床意义,干扰这些lncRNA对胃癌肿瘤生物学行为有何影响。方法:(1 )收集2014年6月-2015年10月期间解放军总医院胃癌患者标本90例,利用人类lncRNA芯片,取5对胃癌与癌旁组织标本筛查差异表达的lncRNA,按照变化倍数、可靠性p值、可能的靶基因信息分析,是否已有文献报道等筛选原则选定候选lncRNA,然后扩大样本量13例,对候选的4个lncRNA进行qRT-PCR验证,从中选出具有潜在临床意义的目标lncRNA。扩大样本量到72例,行qRT-PCR检测,并分析lncRNA表达量与临床参数如肿瘤部位、大小、淋巴结转移,分化程度等的相关性。(2)针对目标lncRNA做功能试验,通过基因重组,构建真核表达载体,将重组质粒转染BGC-823胃癌细胞系,通过MTT, AnnexinV/PI, Transwell检测过表达LncRNA对细胞的增殖,凋亡以及侵袭的影响。结果:(1)芯片筛查发现5445差异表达的lncRNA,其中差异倍数超过2倍,可靠性p值0.05的lncRNA为19个上调,56个下调,候选 4 个 lncRNAp2819 (下调)、P17382 (上调)、P24084 (上调)、P27873 (上调)扩大到13对独立胃癌及癌旁标本行qRT-PCR验证。验证结果均与芯片结果一致,4个候选lncRNA在癌与癌旁正常组织的表达差异均具有统计学意义(分别为P2819:2-△△ct, 0.012 VS0.401; P=0. 01; P17382: 2-△△Ct, 0.572 VS0.061; P=0.01; P24084:2-△△Ct, 0.243 VS0.081; P=0.03; P2819: 2-△△Ct,0.312 VS0.091; P=0.03;)。最终选出差异倍数最大的lncRNAp2819,对72例胃癌标本行qRT-PCR检测,发现其表达量与肿瘤大小,部位,TNM分期显著相关,与年龄、性病无显著性相关。(2)构建pcDNA3.1(+)-p2819质粒,成功通过验证,肿瘤细胞BGC-823转染后过表达lncRNAp2819,通过MTT检测发现p2819可明显抑制细胞增殖。通过流式检测p2819过表达可以明显促进肿瘤细胞凋亡。对照组细胞总凋亡0.9%;转染组总凋亡62.8%;(p0.01 )。Transwell实验检测p2819对细胞的侵袭的影响,见转染LncRNA-p2819组对细胞侵袭有一定抑制作用。结论:芯片筛查出多个胃癌相关lncRNA,与胃癌肿瘤特征具有相关性,其中LncRNA-p2819过表达后对BGC-823细胞产生抑制增殖,促进凋亡,抑制侵袭的作用,说明其具有抑癌基因的特性。其作用机理及潜在临床应用价值尚待进一步研究。
[Abstract]:Background gastric cancer has been a serious threat to human health worldwide. Its incidence is second of all malignant tumors, and the mortality rate accounts for second of all cancer fatal diseases. It is the first in the digestive tract malignant tumors. Although endoscopy, surgery, chemotherapy, and targeting treatment have been improved in recent years, But at present, the 5 year survival rate of gastric cancer patients is still lower than that of 30%. previously concerned with genomics, epigenetics, proteomics, such as the activation of proto oncogenes or (and and) inactivation of tumor suppressor genes, protein interaction, signal transduction pathway, regulatory network and so on. Some research results have been used in clinical, such as gene HER2 targeting treatment. The application of treatment in breast cancer, the detection of benefits for some patients with.HER2 has become routine in breast cancer, but it is not yet necessary for gastric cancer to be specially proposed, but it is very important to accurately determine and monitor the HER2 status in the patients with gastric cancer, which is very important for the rational use of HER2 targeting therapy and evaluation of prognosis. Analysis (FISH) and immunohistochemistry have some limitations in clinical application, such as the inconsistency rate of about 13% of the first, second methods, second, the immunohistochemistry is the semi quantitative detection, the subjectivity is strong, and the FISH detection is time-consuming and expensive. Third, the serum HER2 can be changed during the treatment of trastuzumab, and FISH The research on molecular biology of cancer, in addition to the study of the coding genes that only accounted for 2% of the whole genome, has a lot of unknown fields. The new generation of gene sequencing and high throughput screening methods have made a large number of non coding. The code RNA has been found that non coded RNA mainly includes long chain non coded RNA (long non-codingRNA, lncRNA) and tiny RNA (microRNA, miRNA). Initially, non coded RNA is unfunctional, and with the completion of all human genome sequencing, human diseases are not readily solved. MiRNA has been widely studied in recent years in the regulatory role of tumor biology. However, lncRNA has a large number of functions and can be involved in the regulation of gene expression at epigenetic, transcriptional and post transcriptional levels. The lncRNA study of abnormal expression of tissue is expected to discover new lncRNA related to gastric cancer, as a new target for diagnosis and treatment of gastric cancer, thus forming the second part of the paper. The correlation and clinical significance of serum HER2 level detection and tissue HER2 expression in the first part of gastric cancer patients: to explore the level of HER2 in serum (extracellular domain) and the level of HER2 in the tissues To explore the effect of serum HER2 level on patients with gastric cancer. Methods: to collect the data of 239 cases of gastric cancer patients during the period of June December 2012 in the General Hospital of the Liberation Army (General Hospital of the Liberation Army). The level of serum HER2 was detected by chemical fluorescence method, and the methods of fluorescence in situ hybridization (FISH) and immunohistochemical staining were used to detect the level of HER2 in the tissue. The work feature curve (ROC) was used to determine the best truncation value of the serum HER2 level for predicting the survival of gastric cancer patients. Results: serum HER2 level was significantly correlated with the level of HER2 in the tissue, tumor volume, intestinal type of gastric cancer, and the serum HER2 level was significantly different between the patients with HER2 positive in the tissues and the HER2 negative patients in the tissue. The maximum area under the line was 0.79 (95%CI: 0.71-0.87, P 0.001). The sensitivity and specificity were 0.54 (95%CI:0.37-0.70) and 0.93 (95%CI:0.88-0.96). The serum HER2 level with a truncated value of 24.75ng/mL. was negatively correlated with the overall survival rate of gastric cancer patients. (hazard ratio: 1.93,95%CI: 1.32-4.38, P). ER2 level can be used as a highly specific marker that can replace the expression of HER2 in the tissue. HER2 level 24.75ng/mL suggests poor prognosis. Second the effect of lncRNA-p2819 on the proliferation, apoptosis and invasion of BGC-823 in gastric cancer cells is to determine what differentially expressed lncRNA in gastric and paracancerous tissues. The differential expression of lncRNA has no clinical significance, interfering with the effect of these lncRNA on the biological behavior of gastric cancer. Methods: (1) to collect 90 cases of gastric cancer specimens from the General Hospital of PLA during the period of June 2014 -2015, and use human lncRNA chip to obtain 5 differential expressions of lncRNA for gastric cancer and paracancerous tissue specimens, according to the multiplier number of changes, Depending on the p value, the possible target gene information analysis, whether the candidate lncRNA was selected for the selection principle of the literature, 13 samples were enlarged, and the candidate 4 lncRNA were verified by qRT-PCR, from which the potential clinical significance target lncRNA. was enlarged to 72 cases, the qRT-PCR detection was performed, and the lncRNA expression and clinical reference were analyzed. The correlation of tumor location, size, lymph node metastasis, differentiation degree and so on. (2) to do functional tests on target lncRNA, construct eukaryotic expression vector by gene recombination, transfect the recombinant plasmid to BGC-823 gastric cancer cell line, and detect the effect of LncRNA on cell proliferation, apoptosis and invasion through MTT, AnnexinV/PI and Transwell. Fruit: (1) chip screening found 5445 differentially expressed lncRNA, of which the difference multiplier was more than 2 times, the lncRNA of the reliability p value 0.05 was 19 up-regulated, 56 down-regulation, 4 lncRNAp2819 (down regulation), P17382 (up), P24084 (up), and P27873 (up) expanded to 13 pairs of independent gastric and paracancerous markers for qRT-PCR validation. The results were all with chips. The differences in the expression of the 4 candidate lncRNA were statistically significant (P2819:2- delta delta CT, 0.012 VS0.401, P=0. 01, P17382: 2- Delta Ct, 0.572 VS0.061, P=0.01, P24084:2- Delta Ct, 0.243, 0.312;). Large lncRNAp2819, qRT-PCR detection of 72 cases of gastric cancer specimens, found that the expression of tumor size, location, TNM staging is significantly related to age, no significant correlation with age, STD. (2) construction of pcDNA3.1 (+) -p2819 plasmid, the successful pass verification, tumor cells BGC-823 transfected lncRNAp2819, through MTT detection p2819 can obviously inhibit fine. Cell proliferation by flow cytometry could significantly promote apoptosis of tumor cells. The total apoptosis in the control group was 0.9%, the total apoptosis of the transfected group was 62.8%, and the effect of p2819 on the invasion of the cells in the transfected group was detected by.Transwell test, and the transfection of LncRNA-p2819 group had a certain inhibitory effect on the invasion of the cells. Conclusion: the chip was used to screen multiple gastric cancer related lnc. RNA is related to the tumor characteristics of gastric cancer, in which the overexpression of LncRNA-p2819 inhibits the proliferation of BGC-823 cells, promotes apoptosis, inhibits the effect of invasion, and indicates the characteristics of the tumor suppressor gene. Its mechanism and potential clinical value need to be further studied.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2

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