第一部分

MiR-124-3p在非小细胞肺癌转移中的作用机制

摘要

背景：转移是影响非小细胞肺癌患者生存的一个重要因素。然而，目前对于NSCLC转移的分子调控机制了解还不够深入。因此，本研究在阐明miR-124-3p在NSCLC转移中的重要作用，从而为NSCLC患者提供了一种潜在的治疗干预方式。

方法：采用纳米粒子追踪分析法测定不同临床分期NSCLC患者外泌体的分泌量，使用荧光倒置显微镜测量细胞对外泌体的摄取量。通过荧光素酶报告实验验证了miR-124-3p与其上游及下游调控基因的结合位点。通过Transwell细胞迁移实验评估了NSCLC细胞的侵袭迁移能力。对NSCLC细胞进行转录组测序分析，以寻找miR-124-3p调控的潜在信号通路。通过蛋白免疫印迹实验探究NSCLC细胞系中PI3K、p-PI3K、AKT和p-AKT的蛋白表达水平。最后在NSCLC异种移植模型中验证了miR-124-3p抑制肿瘤转移的作用。

结果： 我们的研究发现，晚期肺癌患者（n=24）血清中的外泌体含量比早期肺癌患者（n=30）高，这提示外泌体的分泌量与NSCLC转移之间存在潜在的相关性。miR-124-3p能够显著抑制NSCLC细胞外泌体的分泌和摄取，同时NSCLC细胞迁移也受到明显抑制。LINC00511作为miR-124-3p的竞争性内源性RNA抑制miR-124-3p的表达以促进外泌体的分泌，同时，miR-124-3p还可以直接靶向Rab27a的3'-UTR，以抑制外泌体的分泌，从而抑制细胞迁移和侵袭。除此之外，miR-124-3p还抑制了细胞内PI3K/AKT信号的激活。最后，通过测量小鼠皮下肿瘤的重量和体积以及小鼠肺部转移证明miR-124-3p能够抑制体内NSCLC肿瘤的生长和转移。

结论：在NSCLC中，miR-124-3p通过细胞外的外泌体转运和细胞内对PI3K/AKT信号通路的调控抑制转移。这些发现为更好地理解NSCLC的转移机制提供了新的见解，并提出了一个潜在的NSCLC治疗方式。

第二部分

多组学探究HLTF缺失对肝细胞癌的影响

摘要

背景：HLTF是核小体重塑复合物 SWI/SNF家族的成员。有研究发现，在40%的结直肠癌患者中观察到HLTF高度甲基化现象，加速肠道肿瘤发生提示HLTF是一种肿瘤抑制因子。此外，在胃癌和宫颈癌患者中也存在HLTF基因高度甲基化的现象。然而两项关于HLTF的肝癌研究显示仅6.3%和9.8%患者显示启动子甲基化。因此，HLTF在HCC中的作用机制仍需要进一步研究。

方法：(1)构建HLTF稳定敲除的HCC细胞株；(2)应用RNA-seq检测并分析HCC细胞HLTF敲除前后的差异表达基因；(3)应用ATAC-seq检测HLTF敲除前后HCC细胞染色质可及性的改变；(4)采用RNA-seq和ATAC-seq多组学数据进行联合分析，寻找HLTF潜在的下游调控通路和关键基因。

结果：TCGA数据库分析表明HLTF在肝细胞癌中表达升高且其高表达与预后不良相关；RNA-seq测序结果显示与野生型细胞相比，HLTF敲除细胞中有563个基因的表达水平上调，656个基因的表达水平下调；根据ATAC-seq的结果分析，我们发现在HLTF缺失时，共有27818个区域的染色质可及性发生了显著变化。14225个区域的染色质可及性得到了增强，而另外13593个区域则出现了染色质可及性降低的情况。对染色质可及性改变的区域进行motif富集分析，数据显示在染色质可及性增强的区域，Atf3，Fra1和BATF等被富集，在染色质可及性减弱的区域，Fra1，Fra2和JunB等被富集；RNA-seq和ATAC-seq多组学数据联合分析表明，重叠基因主要富集在花生四烯酸代谢通路，Wnt信号通路，钙离子信号通路等和TGF-β信号通路等。HLTF的缺失使NXF3表达水平升高，NXF3高表达的患者预后较好。

结论：在HCC中，HLTF可能参与调控花生四烯酸代谢通路、Wnt信号通路和TGF-β信号通路等，NXF3可能是HLTF的潜在下游基因。

Abstract

Background: Metastasis is a significant factor that affects the survival of patients with non-small cell lung cancer (NSCLC). Nevertheless, the molecular regulatory mechanism underlying the metastasis is currently not fully understood. This study aims to identify the important role of miR-124-3p in metastasis of NSCLC, thereby providing a potential therapeutic intervention for the NSCLC patients.

Methods: Exosome secretion was determined by Nanoparticle Tracking Analysis (NTA) and the uptake was measured by fluorescence inverted microscope. The binding mechanism between miR-124-3p and its target genes was validated experimentally by Luciferase reporter. Cells migration was evaluated by Transwell assays. Transcriptome sequencing on NSCLC cells was carried out to verify the potential signaling pathway underlying miR-124-3p regulation. The protein expression levels of PI3K, p-PI3K, AKT and p-AKT in NSCLC cell lines were evaluated using Western blot analysis. The role of miR-124-3p to suppress the tumor metastasis was verified in NSCLC xenograft model.

Results:  Exosomes were more abundant in serum from patients with advanced lung cancer (n=24 patients) than in these from patients with early-stage lung cancer (n=30 patients), which suggested the potential correlation between amount of exosome secretion and the metastasis of NSCLC. Interestingly, the exosome release, uptake and the migration of NSCLC cells were significantly inhibited by miR-124-3p. LINC00511 suppressed the expression of miR-124-3p to facilitate exosome transport due to its role as the competitive endogenous RNA for miR-124-3p. The miR-124-3p could directly target the 3’-UTR of Rab27a in NSCLC cells to inhibit exosome secretion and thereby prevent cell migration and invasion. Aside from the inhibition of exosome transport, miR-124-3p inhibited the activation of PI3K/AKT signaling in the intracellular environment. Finally, by measuring subcutaneous tumor weight and volume and lung metastasis, we also demonstrated that miR-124-3p inhibited tumor growth in vivo.

Conclusion: In NSCLC, miR-124-3p significantly suppressed metastasis through extracellular exosome transport and intracellular PI3K/AKT signaling. These findings provide new insights toward a better understanding of the NSCLC metastasis and suggest a potential treatment method for NSCLC.

Abstract

Background: Helicase Like Transcription Factor (HLTF) is a member of the SWI/SNF family of nucleosome remodeling complexes. It has been found that HLTF is highly methylated in 40% of colorectal cancer patients and accelerated intestinal tumorigenesis suggesting that HLTF is a tumor suppressor. In addition, the HLTF gene was also highly methylated in patients with gastric and cervical cancers. However, two studies on HLTF in liver cancer showed that only 6.3% and 9.8% of patients showed promoter methylation. Therefore, the mechanism of HLTF in HCC still needs further investigation.

Methods: (1) HLTF was knockout through CRISPR/Cas9 in HCC cell line; (2) RNA-seq was applied to detect and analyze the differentially expressed genes of the wild type and HLTF knockout cells; (3) ATAC-seq was applied to detect the changes in chromatin accessibility of the wild type and HLTF knockout cells; (4) The multi-omics analysis of RNA-seq and ATAC-seq was used to find the key signaling pathways and downstream genes of HLTF.

Results: TCGA data base analysis showed that HLTF was highly expressed in hepatocellular carcinoma and was associated with poor prognosis; RNA-seq sequencing showed that 563 genes were up-regulated and 656 genes were down-regulated in HLTF knockout cells compared to wild-type cells; ATAC-seq analysis showed a total of 27,818 regions had significantly altered in chromatin accessibility with HLTF deletion, including 14,225 regions with enhanced chromatin accessibility and 13,593 regions with weakened chromatin accessibility; motif enrichment analysis showed that Atf3, Fra1 and BATF were enriched in regions with enhanced chromatin accessibility, Fra1, Fra2 and JunB were enriched in regions with weakened chromatin accessibility; the combination of RNA-seq and ATAC-seq analysis showed that the overlapping genes were mainly enriched in the Arachidonic acid metabolism pathway, Wnt signaling pathway, Calcium signaling pathway and the TGF-β signaling pathway; the deletion of HLTF increased the expression of NXF3 and highly-expressed NXF3 was associated with better prognosis in HCC patients.

Conclusions: Our study pointed out that HLTF may be involved in regulating Arachidonic acid metabolism pathway, Wnt signaling pathway, TGF-β signaling pathway, etc. NXF3 may be a potential downstream gene of HLTF, and our study provided directions and ideas to elucidate the mechanism of HLTF in hepatocellular carcinoma.