【作者】 赵岩；

【导师】 孙跃明；

【作者基本信息】 南京医科大学 ， 普外科， 2014， 硕士

【摘要】 [背景]在全球,胃癌的死亡率位居第二位,据统计,每年约有750,000例新发病例且五年生存率不足25%,早期发现及诊断是提高胃癌患者生存率的重要手段,近年来,随着胃癌发生与发展的分子机制及治疗措施的进展,研究者们越来越意识到寻找出新的胃癌治疗靶点及早期诊断标志物是控制其发生发展不可避免的事情。长链非编码RNA (long non-coding RNA, lncRNA)是一类新发现的非编码RNA,其中隶属于lncRNA的HULC, H19等已被研究者们证实在人类肝癌,结直肠癌的发生发展中起到重要的作用,但其在胃癌发生发展过程中的相关作用迄今不清。本文拟通过检测长链非编码RNA HULC在胃癌组织及细胞的表达,研究在胃癌细胞中不同表达水平的HULC对胃癌细胞生物学特性的影响,最后,初步的揭示了长链非编码RNAHULC在胃癌中的相关分子机制。[目的]明确HULC在58例胃癌组织标本及细胞株的表达含量,通过体外实验重点探讨其对胃癌细胞株增殖,迁移,侵袭,凋亡的影响及相关的分子机制,从而更好的揭示新的胃癌标志物和预防治疗胃癌的分子机理。[方法]1.收集58例胃癌手术标本(癌组织及对应的癌旁组织),荧光实时定量PCR检测标本中HULC的表达量,并进一步的分析其与临床病理资料的关系；荧光实时定量PCR分别检测出胃癌细胞株MKN28、MKN45、SGC-7901、BGC-823、AGS及正常胃粘膜上皮细胞GES-1的表达量。2.利用慢病毒转染技术,建立HULC表达量不同的胃癌稳定转染细胞株,运用CCK-8, Transwell跨膜实验,细胞划痕实验,流式细胞技术检测细胞生物学特性的差异。3.倒置显微镜下观察HULC表达量不同的稳转细胞株形态学的变化；运用荧光实时定量PCR,Westernblot技术分别在RNA和蛋白水平上检测处理组以及对照组细胞株中EMT(上皮间质转化)标志物E-Cadherin, Vimentin的表达量。4.利用自噬抑制剂3-MA作用于上述稳转细胞株,通过流式细胞仪检测各组凋亡率的改变,Western-blot技术检测自噬标志物LC3-Ⅱ/LC3-Ⅰ比值的变化。[结果]1. lncRNA HULC在癌组织的表达量高于所对应的癌旁组织,两者差异具有统计学意义,通过临床病理资料分析,HULC表达量与区域淋巴结的转移,远处转移,TNM分期相关,ROC曲线分析示HULC有可能成为预测胃癌的标志物；胃癌细胞株BGC-823、SGC-7901、AGS中HULC表达量显著高于胃正常粘膜上皮细胞GES-1,其中,在SGC-7901细胞株的表达量最高,具有统计学差异,而在MKN28、MKN45中的表达量与GES-1相比,无统计学意义。2.稳定转染胃癌细胞株SGC-7901,成功构建HULC高表达和低表达细胞株,CCK-8增殖实验结果显示HULC可促进胃癌细胞株增殖。3.细胞划痕实验,Transwell跨膜实验示HULC可促进胃癌细胞的转移、侵袭；倒置电子显微镜观察下调HULC表达的稳转细胞株,细胞形态变成圆形,伪足变少,有逆转EMT的趋势；Western blot、荧光实时定量PCR结果示：下调HULC表达后,在蛋白和RNA水平上,E-Cadherin表达量增加,Vimentin表达量减少。4.加入自噬抑制剂3-MA后,通过流式细胞仪,Western blot技术观察显示可以得出通过上调HULC的表达可以促进细胞发生自我吞噬作用从而减少凋亡。[结论]长链非编码RNA HULC可作为促癌基因影响胃癌细胞增殖、凋亡、迁移、侵袭等生物学特性,其表达含量的变化与胃癌的发生发展密切相关,其潜在作用机制可能是EMT和自噬作用。更多还原

【Abstract】 [Background] Gastric cancer (GC) is the second leading cause of cancer-related mortality globally. There are～750,000new cases diagnosed annually around the world and5-year overall survival rates are <25%.Early diagnosis and treatment is an important means to improve survival.With the progress in understanding the pathophysiological mechanisms and treatment for GC in recent years, a deeper understanding of the molecular and genetic networks that control the initiation and progression of GC is imperative.Long non-coding RNAs (lncRNAs) are a class of newfound non-coding RNAs,such as HULC, H19have been suggested to have a functional role in tumorigenesis and tumor progression.However, investigations of HULC in GC are scarce. This study tries to detect the expression level of HULC in GC and the effects of HULC on gastric cancer biological function, to reveal the function and molecular mechanisms of HULC in GC preliminary.[Objective] In the present study, we examined the role of HULC in GC and the potential mechanisms involved by a retrospective analysis of58GC patients, and by carrying outin vitro experiments to clarify the contribution of HULC to various aspects of the malignant phenotype of human GC,which provides us with a new biomarker in GC and perhaps a potential target for GC prevention, diagnosis and therapeutic treatment.[Methods]1. Fifty eight surgical samples of GC were collected, the expression levels of HULC were detected by qRT-PCR, the relationship between the expression of HULC and the clinicopathological factors of GC was analyzed; the expression of HULC levels in GC cell lineMKN28,MKN45,SGC7901,BGC823,AGS and human gastric epithelial mucosa cell line GES-1were detected by qRT-PCR.2. By lentivirus construction, stably transfected gastric cancer cell lines of HULC-downregulated and HULC-upregulated were established respectively, the effect of HULC on cell proliferation,apoptosis and invasion in vitro were detected by CCK-8assays, transwell assays,wound healing assays and flow cytometry.3. Changes in cell morphology were observed under inverted microscope before and after the HULC expression was changed; the changes on the protein level and mRNA level of EMT associated gene (E-Cadherin and Vimentin) in HULC-downregulated were detected by qRT-PCR and Western blot, respectively.4. Cells were treated with3-MA (an inhibitor of autophagic sequestration blocker),we also determined a change in the ratio of LC3-Ⅱ/LC3-Ⅰ using wstem blot, and apoptosis was detected using flow cytometry.[Results]1. lncRNA HULC is upregulated in GC tissues in comparison with the adjacent normal gastric tissues. Of note, its level was significantly associated with lymph node metastasis, distant metastasis. In addition, a receiver operating characteristic (ROC) curve was constructed to evaluate the diagnostic values and HULC could be used as a marker of GC. Compared with human gastric epithelial mucosa cell line GES-1, expression of HULC in BGC-823,SGC-7901and AGS cells increased significantly,while no significant difference were found in MKN28or MKN45cells.2. Stably transfected cell lines of HULC-upregulated and HULC-downregulated were established successfully, CCK-8assays showed that abnormally expressed HULC promotes the proliferation of GC cells.3. Wound healing assays and transwell assays confirmed that HULC positively regulates GC cell migration and invasion and deletion of HULC reverses EMT, indicating that HULC could act as a possible regulator of EMT. Under an inverted microscope, silencing of HULC in SGC-7901cells induced round spheroids with no or few protrusions. Western blot and qRT-PCR results showed that the downregulation of HULC levels in SGC7901cells was associated with upregulated E-cadherin and downregulated Vimentin both on the protein and mRNA levels.4. After using autophagy inhibitor3-MA, the flow cytometry assays and western blot showed upregulated HULC inhibits cell apoptosis by activating autophagy.[Conclusion] HULC,a cancer-related lncRNA,contributed to the malignant phenotype of GC cells through its regulation of diverse cellular processes, including proliferation, apoptosis, migration and invasion and its potential effect on autophagy and EMT.更多还原