【作者】 王涛；

【导师】 闫峰；

【作者基本信息】 厦门大学 ， 外科学， 2017， 硕士

【摘要】 背景:长链非编码RNA-ROR在细胞重编程过程中发挥着重要的调控作用。目前研究发现ROR与肝癌、乳腺癌及胶质瘤的发生发展密切相关,但ROR在胃癌中的作用鲜有报道。本课题组前期实验结果显示:1.ROR在50例胃癌组织中表达量明显低于其配对的癌旁正常组织;2.过表达ROR可以促进胃癌细胞的凋亡,抑制细胞的侵袭及迁移,但对细胞的增殖能力无明显影响;3.ROR可能通过影响EMT进程来抑制胃癌细胞的转移。为了从反方向验证ROR上述功能及机制,本论文在前期研究基础上,将胃癌组织样本增加到了 65例并对其进行了临床病理特征分析,随后通过干扰ROR的表达从反方向探讨和验证了 ROR对胃癌细胞生物学行为的影响以及可能的调控机制。方法:1.采用实时荧光定量PCR技术(qRT-PCR)对65例胃癌及其配对的癌旁正常组织中ROR的表达水平进行检测,并对其临床病理特征进行统计学分析;2.构建ROR干扰载体,通过慢病毒转染实现对胃癌细胞ROR的稳定敲减,qRT-PCR检测转染效果,通过进一步筛选使其在胃癌细胞株中呈稳定低表达;3.应用CCK-8、流式细胞术、Transwell实验检测ROR敲减后对胃癌细胞生物学行为的影响;4.通过Western Blotting实验检测ROR敲减后胃癌细胞株中EMT相关标志物的变化,对其相关机制进行初步探讨。结果:1.在65例胃癌组织中,ROR的表达量明显低于配对的癌旁正常组织(P<0.01);2.通过慢病毒载体转染胃癌细胞株MGC-803和SGC-7901,实现了ROR在胃癌细胞株的稳定低表达;3.CCK-8、流式细胞术、Transwell实验结果显示:ROR敲减后促进了胃癌细胞的增殖能力(P<0.05),抑制胃癌细胞的凋亡(P<0.05),增强了胃癌细胞的迁移及侵袭能力(P<0.05);4.Western Blotting结果显示:ROR敲减后EMT上皮性标志物E-cadherin表达降低,间质性标志物Vimentin、N-cadherin、a-SMA表达升高,p-AKT表达上调。加入信号通路抑制剂后,相应指标均出现相反趋势。结论:ROR在胃癌组织中表达量明显低于配对的癌旁正常组织;ROR可以抑制胃癌细胞的迁移及侵袭能力,促进胃癌细胞的凋亡;过表达ROR后,其并没有影响胃癌细胞的增殖能力,而敲减ROR后,细胞实验和体内实验结果都显示胃癌细胞的增殖能力增强了,具体机制有待进一步探讨;ROR抑制胃癌细胞迁移和侵袭的一部分原因可能是其通过AKT信号通路影响了 EMT进程。更多还原

【Abstract】 Background:Long intergenic non-coding RNA regμlator of reprogramming(LincRNA-ROR),is a newly identified long non-coding RNA,was initially found to regμlate the process of reprogramming.Present studies indicated that it played important roles in development and progression of hepatocellμlar carcinoma,breast cancer and glioma.However,the role of ROR in gastric cancer is rarely known.The conclusion below has been demonstrated by our previous study.1)The levels of ROR was significantly down-regμlated in 50 tissues of gastric cancer.2)Over-expression of ROR in gastric cancer cells coμld significantly induced apoptosis,suppressed invasion and migration,while had little effect on cell proliferation.3)ROR co μ Id induce epithelial-to-mesenchymal transition to inhibt metastasis of gastric cancer cells.The experiments based on previous data are to testify the proposed function and mechanism of ROR in the negative.Firstly,the gastric cancer tissues added up to 65 cases are used for the subsequent research.Secondly,knockdown experiment was used to study biological behaviors and mechanism of ROR.Methods:1.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression levels of ROR in 65 gastric cancer tissues and its paired adjacent normal tissues,analyzing the relationship between the expression of ROR and its Clinicopathological features.2.knockdown trial was used to lower ROR expression,the transfection efficiency was verified by qRT-PCR.3.The effect on proliferation and apoptosis after down-regμlating expression of R07R was evaluated by CCK-8 assay and flow cytometry.Transwell assay was performed to detect the ability of migration and invasion in gastric cancer cells.4.Western blotting was used to detect the markers of epithelial-to-mesenchymal transition(EMT).Resμlts:1.The levels of ROR were significantly down-regμlated in gastric cancer compared with paired adjacent normal tissues(P<0.001).2.The ROR expression was down-regμlated in MGC-803 and SGC-7901 cells after knockdown experiment(p<0.001).3.CCK-8 assay,flow cytometry,transwell assay indicated that it coμld promote cell proliferation(P<0.01),significantly inhibited apoptosis(P<0.05),promote invasion and migration(P<0.05)after knockdown of ROR.4.Westem blotting has discovered the epithelial markers(E-cadherin)were down-reg μ lated after knock-down of ROR,whereas the mesenchymal markers(Vimentin、N-cadherin、a-SMA)and p-AKT were increased when we examined the protein markers.The corresponding markers show an opposite trend after pathway inhibitors added.Conclusion:The levels of ROR was significantly down-regμlated in gastric cancer tissues.Moreover,ROR in gastric cancer cells coμld suppress cell invasion and migration,significantly promote cell apoptosis.ROR had little effect on cell proliferation by overexpression,however,it coμld promote cell proliferation by knockdown.Additionally,ROR co μ Id suppress invasion and migration partly because of the influence of AKT signal pathway on EMT in gastric cancer cell lines.更多还原