【作者】 李云；

【导师】 伍权；

【作者基本信息】 安徽医科大学 ， 临床检验诊断学， 2021， 硕士

【摘要】 癌症是世界上造成人类死亡的最主要的原因之一,而肺癌是全世界发病率和死亡率最高的实体肿瘤之一。根据病理组织类型的不同,肺癌可以分为小细胞肺癌和非小细胞肺癌,其中非小细胞肺癌占肺癌的80%以上,肺腺癌是非小细胞肺癌最常见的分型。虽然用于肺癌治疗的方法在不断改善,但并没有从根本上改变肺癌高发病率和高死亡率的现状。由于肺癌易转移扩散和耐药等特征可能导致肿瘤反复发生的情况,因此,对肺癌的发生发展和转移扩散等分子机制仍需要进一步深入研究。miRNA是一类长度约为22个核苷酸,由内源基因编码的非编码单链RNA分子,已有大量文献报道其与肿瘤的发生发展等过程密切相关。本文通过对5对肺腺癌组织和肺组织正常对照的miRNA芯片数据分析,筛选出了576个在肺腺癌中差异表达的miRNA,通过进一步验证以及文献调研,我们选择了在肺腺癌中低表达的hsa-let-7e-5p进行研究。我们通过TCGA数据库进一步分析了hsa-let-7e-5p在肺腺癌组织中的表达情况,发现hsa-let-7e-5p在肺腺癌组织中低表达,并具有统计学意义。我们检测了肺腺癌细胞中hsa-let-7e-5p的内源性表达水平,发现与对照相比hsa-let-7e-5p在肺腺癌细胞中低表达。功能实验结果显示hsa-let-7e-5p能抑制肺腺癌细胞的迁移。我们运用三个miRNA靶基因预测数据库对hsa-let-7e-5p的潜在靶基因进行了预测,并取交集进行了验证,结果显示DTX2显著改变,通过蛋白免疫印迹分析和双荧光素酶报告实验进一步验证DTX2是hsa-let-7e-5p的靶基因。我们运用TCGA数据库分析了DTX2在肺腺癌组织中的表达水平,发现DTX2在肺腺癌中高表达,并具有统计学意义。我们同期通过细胞实验证实了DTX2在肺腺癌细胞中高表达,过表达DTX2能促进肺腺癌细胞的迁移。这些研究结果显示hsa-let-7e-5p在肺腺癌中可通过直接靶向调控DTX2的表达来抑制肺腺癌细胞的迁移,拓展了我们对肺腺癌发病机理的认识,也为后续深入研究hsa-let-7e-5p和DTX2在肺癌中的功能和机制研究奠定了基础。更多还原

【Abstract】 At present,cancer has become one of the main causes of human death in the world.Lung cancer is one of the malignant tumors with the highest incidence and mortality in the world,and the incidence is increasing year by year.According to different pathological types,lung cancer can be divided into small cell lung cancer and non-small cell lung cancer.Among them,non-small cell lung cancer accounts for more than 80%,and lung adenocarcinoma is the most common type of non-small cell lung cancer.Although the treatment of lung cancer is constantly improving,it has not fundamentally changed the high incidence and mortality of lung cancer.The characteristics of lung cancer such as easy metastasis and drug resistance are important reasons for tumor recurrence.Therefore,the mechanism of the occurrence,development,and metastasis of lung cancer still needs to be further studied.miRNA is a class of non-coding single-stranded RNA molecules with a length of about 22 nucleotides encoded by endogenous genes.There have been a large number of literature reports that it is closely related to tumor cell proliferation,differentiation,apoptosis,migration,invasion and cell cycles.In this paper,by analyzing the miRNA chip data of lung adenocarcinoma and normal controls,we screened 576 miRNAs that are differentially expressed in lung adenocarcinoma.After further verification by QRT-PCR and literature research,we selected the low-expressing hsa-let-7e-5p in lung adenocarcinoma for research.We further analyzed the expression of hsa-let-7e-5p in lung adenocarcinoma tissues in the TCGA database,and found that hsa-let-7e-5p is indeed at a low expression level in lung adenocarcinoma tissues,and has statistical significance.We tested the endogenous expression of hsa-let-7e-5p in lung adenocarcinoma cells and found that hsa-let-7e-5p is down-regulated in lung adenocarcinoma cells.We found that overexpression of hsa-let-7e-5p can inhibit the migration of lung adenocarcinoma cells by the wound healing assay.We predicted the possible target genes of hsa-let-7e-5p through three miRNA target gene prediction websites and verified the intersection of them.Combined with literature research,we further analyzed the possible target gene DTX2 through western blot and dual-luciferase reporter assays and verified that DTX2 is the target gene of hsa-let-7e-5p.We analyzed the expression level of DTX2 in lung adenocarcinoma tissues in the TCGA database,and found that DTX2 was expressed at a high level in lung adenocarcinoma,and it was statistically significant.We tested the endogenous expression level of DTX2 in lung adenocarcinoma cells and found that DTX2 is highly expressed in lung adenocarcinoma cells and can promote the migration of lung adenocarcinoma cells.Therefore,we found that hsa-let-7e-5p is down-regulated in lung adenocarcinoma,and it is possible to inhibit the migration of lung adenocarcinoma cells by directly targeting DTX2.This study may play very important role on subsequent research about the functions and mechanisms of hsa-let-7e-5p and DTX2 in lung cancer.更多还原