【作者】 张鑫；

【导师】 王鹏；

【作者基本信息】 南方医科大学 ， 药学（专业学位）， 2025， 硕士

【摘要】 研究背景:肺腺癌作为肺癌的主要组织学亚型,全球发病率和死亡率居高不下,患者预后改善仍面临重大挑战,亟需探索新的治疗靶点。近年来,非编码RNA的调控机制成为肿瘤研究热点,其中微小核糖核酸(miRNA)通过转录后调控广泛参与肿瘤恶性表型(如增殖、迁移和侵袭)的调控。例如,miR-21-5p在肺腺癌中异常高表达且与不良预后显著相关,但其具体作用机制尚未完全阐明。值得注意的是,核激活miRNA(NamiRNA)的发现为miRNA功能研究提供了新视角:这类miRNA可通过结合靶基因增强子序列,直接激活基因转录,形成正向调控模式。然而,miR-21-5p是否通过NamiRNA机制调控下游靶基因还有待阐明。在潜在靶基因中,微管蛋白δ1(TUBD1)因其在肿瘤中的调控作用备受关注。作为微管蛋白家族成员,TUBD1参与细胞骨架构建和有丝分裂,并且其在乳腺癌中存在的基因扩增和过表达现象,与原发性非小细胞肺癌发生相关,提示其可能驱动肿瘤恶性进展。然而,TUBD1在肺腺癌中的表达模式及功能尚不明确。但生物信息学分析显示,肺腺癌组织中TUBD1表达水平显著升高。结合上述背景,我们提出miR-21-5p可能通过NamiRNA机制激活TUBD1转录,进而促进肺腺癌进展的假设。研究目的:探究miR-21-5p作为NamiRNA在肺腺癌中的作用及分子机制。研究方法:(1)通过生物信息学分析差异表达miRNA,筛选出在肺腺癌中上调基因miR-21-5p,并通过ENCORI数据库以及RT-qPCR进行验证。(2)在肺腺癌细胞系H1975及PC9转染miR-21-5p inhibitor,通过细胞功能实验探究敲低miR-21-5p后对肺腺癌体外增殖活力、迁移及侵袭能力的影响。(3)通过荧光原位杂交确定miR-21-5p亚细胞定位,UCSC数据库预测miR-21-5p是否位于增强子活性区域并通过Weestern blot验证miR-21-5p对增强子标志物H3K27ac的影响。(4)通过RT-qPCR及Western blot验证抑制miR-21-5p表达后能否降低邻近基因TUBD1的表达。(5)通过EnhancerAtlas 2.0数据库和RNA22工具验证NamiRNA-21-5p是否靶向TUBD1的增强子。研究结果:(1)生物信息学筛选出472个差异表达miRNA,其中miR-21-5p在肺腺癌高表达,并在肺腺癌细胞系中得到验证。(2)抑制miR-21-5p表达,肺腺癌细胞体外增殖活力、迁移及侵袭能力受到抑制。(3)亚细胞定位发现miR-21-5p于细胞核内分布,USCS验证其位于增强子活性区域以及Western blot证实抑制其表达后H3K27ac蛋白表达降低,支持miR-21-5p作为NamiRNA。(4)NamiRNA-21-5p敲低后,邻近基因TUBD1的表达及蛋白表达水平被抑制。(5)NamiRNA-21-5p 可能靶向 TUBD1的增强子HS02-04060和HS02-04057。研究结论:miR-21-5p在肺腺癌中高表达,并增强肺腺癌体外增殖、迁移及侵袭能力。miR-21-5p可能作为NamiRNA正向调节TUBD1表达,构建形成NamiRNA-21-5p-增强子-TUBD1调控网络,进而促进肺腺癌发生发展。更多还原

【Abstract】 Background:Lung adenocarcinoma(LUAD),as a major histologic subtype of lung cancer,has a high global morbidity and mortality rate,and the improvement of patients’ prognosis still faces major challenges,which urgently requires the exploration of new therapeutic targets.In recent years,the regulatory mechanism of non-coding RNAs has become a hotspot of tumor research,among which microRNAs(miRNAs)are widely involved in the regulation of malignant phenotypes of tumors(e.g.,proliferation,migration,and invasion)through post-transcriptional regulation.For example,miR-21-5p is aberrantly expressed in LUAD and significantly associated with poor prognosis,but its specific mechanism of action has not been fully elucidated.Notably,the discovery of nuclear activated miRNAs(NamiRNAs)has provided new perspectives on miRNA function:these miRNAs can directly activate gene transcription by binding to the enhancer sequences of target genes,resulting in a positive regulatory mode.However,it remains to be elucidated whether miR-21-5p regulates downstream target genes through the NamiRNA mechanism.Among the potential target genes,tubulin δ1(TUBD1)has attracted much attention for its regulatory role in tumors.As a member of the microtubulin family,TUBD1 is involved in cytoskeleton construction and mitosis,and its gene amplification and overexpression in breast cancer,which is associated with primary non-small cell lung carcinogenesis,suggests that it may drive malignant tumor progression.However,the expression pattern and function of TUBD1 in LUAD are not clear.However,bioinformatics analysis showed that the expression level of TUBD1 was significantly elevated in LUAD tissues.Combined with the above background,we propose the hypothesis that miR-21-5p may activate TUBD1 transcription through the NamiRNA mechanism and thus promote LUAD progression.Objective:To investigate the role and molecular mechanism of miR-21-5p as NamiRNA in LUAD.Methods:(1)Differentially expressed miRNAs were analyzed by bioinformatics to screen out miR-21-5p,a gene upregulated in LUAD,and verified by the ENCORI database as well as by RT-qPCR.(2)miR-21-5p inhibitor was transfected in LUAD cell lines H1975 and PC9,and the effects of knocking down miR-21-5p on the proliferative viability,migration and invasive ability of LUAD in vitro were investigated by cell function experiments.(3)Determine the subcellular localization of miR-21-5p by fluorescence in situ hybridization,predict whether miR-21-5p is located in the enhancer activity region using the UCSC database,and validate its effect on the enhancer marker H3K27ac by Western blot,supporting the role of miR-21-5p as the NamiRNA.(4)Assess whether inhibition of NamiRNA-21-5p expression can reduce the expression of the neighboring gene TUBD1 by RT-qPCR and Western blot.(5)Verification of whether NamiRNA-21-5p targets enhancers to mediate TUBD1 activation by EnhancerAtlas 2.0 and RNA22.Results:(1)Bioinformatics screening identified 472 differentially expressed miRNAs,among which miR-21-5p was highly expressed in LUAD and validated in LUAD cell lines.(2)Inhibition of miR-21-5p expression suppressed the proliferative vigor,migration,and invasive ability of LUAD cells in vitro.(3)Subcellular localization revealed that miR-21-5p is distributed within the nucleus.UCSC analysis confirmed its presence in enhancer activity regions,and Western blot further demonstrated that inhibition of miR-21-5p expression resulted in reduced H3K27ac protein levels.(4)After knocking down miR-21-5p,the expression of the neighboring gene TUBD1 and its protein expression level were suppressed.(5)NamiRNA-21-5p may target enhancers HS02-04060 and HS02-04057 of TUBD1.Conclusion:miR-21-5p is highly expressed in LUAD and enhances the proliferation,migration,and invasion ability of LUAD cells in vitro.miR-21-5p may act as a NamiRNA to positively regulate the expression of TUBD1,constructing a NamiRNA-21-5p-Enhancer-TUBD 1 regulatory network,which in turn promotes LUAD genesis and development.更多还原