血浆lncRNAs作为胃癌诊断潜在生物标志的研究

发布时间：2018-04-12 06:09

本文选题：胃癌 + 血浆　；参考：《南京医科大学》2017年硕士论文

【摘要】：背景:我国一直是胃癌(gastriccancer,GC)高发国家,每年胃癌发病数和死亡数均占世界总数的50%左右,疾病负担严重,是肿瘤防治的重点。胃癌患者多没有特异性症状,因而诊断时大多数患者已处于进展期,进展期胃癌预后较差,术后五年生存率低于30%,而早期胃癌预后良好,术后五年生存率可以达到90%以上,因此寻找能够鉴别早期胃癌、且具有高敏感性和特异性的生物标志至关重要。长链非编码RNAs(long non-coding RNAs,lncRNAs)是指长度超过200个核苷酸且没有蛋白编码能力的RNA转录本。目前大量研究已经表明lncRNAs在肿瘤发生发展过程中发挥着重要作用。近年来,lncRNAs被发现可以释放到循环中且稳定存在,且循环lncRNAs的表达水平在肿瘤患者和健康对照体内存在显著差异,有可能作为肿瘤潜在生物标志而改善肿瘤的早期诊断。目前循环lncRNAs的研究尚处于起步阶段,循环lncRNA的来源及其功能还未系统阐明。方法:本研究采用Arraystar Human lncRNA/mRNA v3.0芯片技术,检测并分析5例胃癌病例及5例性别、年龄相匹配健康对照的全基因组血浆lncRNAs;以胃癌患者血浆中高表达最显著的前10条lncRNAs作为本研究的候选lncRNAs,运用实时定量 PCR(quantitative real-time polymerase chain reaction,qRT-PCR)技术进行两阶段血浆大样本(一阶段包括50例胃癌病例血浆和50例健康对照血浆,二阶段包括173例胃癌病例血浆和173例对照血浆)验证,通过受试者工作特征曲线(receive operating charactreristic curve,ROC)分析及计算曲线下面积(area under the curve,AUC),评价单个或一组lncRNAs对胃癌诊断的应用价值。同时,本研究利用实时定量PCR检测了血浆lncRNAs在经历反复冻融或室温放置一段时间后的稳定性,并通过不同的胃癌细胞系及胃癌病例术前术后的血浆样本探讨血浆lncRNAs可能的来源;此外,本研究利用细胞生物学的方法,初步分析了血浆lncRNAs对胃癌细胞恶性表型的影响。结果:芯片分析筛选出差异表达的lncRNAs共3,878条,其中在胃癌患者血浆中表达上调的有603条,表达下调的有3,275条。两阶段血浆样本验证发现RP11-47304.5、GUSBP11 和 CTD-2303H24.2 这 3 条 lncRNAs 在胃癌病例血浆中显著高表达;lncRNAs稳定性检测发现,在血浆样本经历反复冻融或室温放置一段时间后,这3条lncRNAs的表达量均无显著差异;ROC曲线分析发现,这3条lncRNAs单个或者组合对胃癌诊断均有一定的价值,其中CTD-2303H24.2单独诊断的价值最大,而这3条lncRNAs的联合诊断价值要高于单个lncRNA的诊断价值,AUC为0.818,灵敏度为77.5%,特异度为73.9%;lncRNAs来源分析发现,不同的胃癌细胞培养液中均可检测到这3条lncRNAs,且胃癌患者术后血浆中lncRNAs的表达水平显著低于手术前的表达量;细胞功能实验发现,过表达RP11-47304.5可促进胃癌细胞的增殖和侵袭能力,干扰RP11-47304.5表达可抑制胃癌细胞的增殖和侵袭能力。结论:本研究发现了三条新的胃癌相关的血浆lncRNAs RP11-47304.5、GUSBP11和CTD-2303H24.2,这3条lncRNAs在血浆中稳定存在且在胃癌患者血浆中显著高表达,可以较好地区分健康对照和胃癌患者,可作为潜在的胃癌早期诊断的循环生物标志。进一步的功能学研究发现,RP11-47304.5与胃癌细胞的增殖和侵袭作用相关。
[Abstract]:Background: our country has been in gastric cancer (gastriccancer, GC) in the country every year, gastric cancer incidence and mortality in the world accounted for about 50% of the total burden of disease, seriously, is the focus of tumor prevention and treatment in patients with gastric cancer. No specific symptoms, the diagnosis most patients have been in advanced stage, the prognosis of advanced gastric cancer is poor. The postoperative five years survival rate is less than 30%, while the good prognosis of early gastric cancer, postoperative five year survival rate can reach more than 90%, so look for to be able to identify early gastric cancer, and Gao Min has the sensitivity and specificity of biomarkers is critical. Long chain non encoding RNAs (long non-coding RNAs, lncRNAs) refers to the length of more than RNA transcription 200 nucleotides and no protein encoding ability. At present, many studies have shown that lncRNAs play an important role in tumor development. In recent years, lncRNAs was found to be released into the circulation and Stable, and the expression level of circulating lncRNAs in cancer patients and healthy controls are significantly different in vivo, may be used as a potential biomarker of tumor and improve the early diagnosis of tumors. The study of circulating lncRNAs is still in the initial stage, and the power source of circulating lncRNA can not clarify the system. Methods: This study used Arraystar Human lncRNA/mRNA V3.0 chip technology, detection and analysis of 5 cases of gastric cancer and 5 cases of sex and age matched healthy controls of the whole genome of plasma lncRNAs in 10 lncRNAs; the most significant expression in the plasma of gastric cancer patients as candidates for lncRNAs in this study, using real-time quantitative PCR (quantitative real-time polymerase chain reaction, qRT-PCR) two phase a large sample of plasma technology (including 50 cases of stage gastric cancer cases and 50 healthy controls plasma plasma, two stage gastric cancer cases including 173 cases of blood Pulp and 173 cases of control plasma) verified by receiver operating characteristic curve (receive operating charactreristic curve, ROC) area under the curve analysis and calculation (area under the curve, AUC), evaluation of a single or a group of lncRNAs on the application value of the diagnosis of gastric cancer. At the same time, this study using real-time quantitative PCR detection of plasma lncRNAs after repeated freeze-thaw stability at room temperature or after a period of time, and through the plasma samples of different gastric cancer cell lines and patients before and after surgery to investigate the source of plasma lncRNAs possible; in addition, this research using the methods of cell biology, preliminary analysis of the effects of plasma lncRNAs on the malignant phenotype of gastric cancer cells. Results: chip analysis showed that 3878 lncRNAs were differentially expressed, the expression in the plasma of patients with gastric cancer in 603, down regulated 3275 stage. The two plasma samples proved to R P11-47304.5, GUSBP11 and CTD-2303H24.2 lncRNAs in 3 gastric cancer cases was significantly higher in plasma expression; lncRNAs stability testing, experience in plasma samples or repeated freezing and thawing at room temperature for a period of time, there were no significant differences in the expression of the 3 lncRNAs; ROC curve analysis shows that the 3 lncRNAs single or combination of have certain value in diagnosis of gastric cancer, the diagnostic value of CTD-2303H24.2 alone, and the diagnostic value of combined diagnostic value of the 3 lncRNAs was higher than that of single lncRNA, AUC was 0.818, the sensitivity was 77.5%, specificity was 73.9%; the lncRNAs source analysis found that the liquid can be detected in the 3 lncRNAs different gastric cancer cell culture that expression level of lncRNAs in plasma and gastric cancer patients was significantly lower than the expression level before surgery; found cell function experiments, overexpression of RP11-47304.5 can promote the proliferation of gastric cancer cells and The invasion ability, interfering the expression of RP11-47304.5 proliferation and invasion inhibition of gastric cancer cells. Conclusion: This study found three new gastric cancer related to plasma lncRNAs RP11-47304.5, GUSBP11 and CTD-2303H24.2, the 3 lncRNAs is stable in plasma in patients with gastric cancer and blood plasma tissues, we can distinguish the healthy control and gastric cancer, early diagnosis of gastric cancer can be used as a circulating biological potential marker. Further functional studies found that RP11-47304.5 cells related to gastric cancer cell proliferation and invasion.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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