背景

可变剪接（Alternative splicing，AS）与可变多聚腺苷酸化（Alternative polyadenylation，APA）作为转录后调控的关键机制，具有生成多样化mRNA亚型的能力，从而增加蛋白质的多样性，并对mRNA的功能、定位、稳定性及翻译效率产生影响。这些机制的紊乱与包括癌症在内的多种疾病息息相关。恶性肿瘤的转移是癌症治疗面临的主要挑战之一，其根源是循环肿瘤细胞（Circulating tumor cell，CTC）。CTC可以通过循环系统在远处器官定植，引起转移性病变。虽然当前研究已经揭示了CTC在促进肿瘤转移及不良预后中的关键作用，但对CTC团簇这一具有更高转移潜力的特殊形式背后的转录后调控相关分子机制，特别是在AS和APA方面的了解还非常有限。近年来，基于RNA的治疗策略因其针对性强和开发过程相对简便而受到重视，为治疗癌症等难治疾病开辟了新途径。然而，对CTC团簇中AS和APA的了解不足阻碍了这些治疗策略在抑制癌症转移中的应用。

目的

本文的主要目标是深入揭示CTC团簇与单个CTC中AS和APA谱的特征及其动态紊乱，进一步探究这些关键的转录后调控机制发生变化的原因，并评估这些变化是否导致CTC团簇表现出更为恶性的表型。通过解答这些问题，本研究将能够提供关于单个CTC和CTC团簇的转录后调控谱的新见解，揭示CTC团簇转移能力增强的分子特征，并为设计针对CTC团簇的基于RNA的治疗提供潜在的靶点。这项研究有望为阻断癌症转移和改善患者预后提供新的治疗策略，具有重要的临床意义和科学创新性。

方法

本文基于乳腺癌CTC单细胞转录组测序数据，对单个CTC和CTC团簇之间的AS和APA谱进行了比较分析，并使用乳腺癌细胞系通过3′ RACE、重组质粒载体的构建、荧光素酶报告基因测定、基因过表达、流式细胞术检测及分析、Western Blot、RT-qPCR、伤口愈合、Transwell检测细胞迁移侵袭等实验技术逐步阐明CTC团簇相比于单个CTC更恶性表型的相关转录后调控机制。

结果

本文分别在单个CTC和CTC团簇中鉴定到994个和836个AS事件，其中约20%的AS调控事件在这两种细胞类型之间显示出差异。在这些差异剪接中，研究发现SRSF6处于调控核心地位，通过扰乱了广泛的AS谱进而促进CTC团簇恶性程度的增加。在APA方面，研究发现与单个CTC相比，CTC团簇中的3′ 非翻译区（Untranslated region ，UTR）延长的亚型比例普遍较高。这种改变主要由14个核心APA因子调控，特别是PPP1CA调控了数量最多的APA事件。改变后的APA谱有助于促进CTC团簇的细胞周期进展，并降低其对氧化应激的敏感性。进一步的研究表明，在CTC团簇中，H2AFY mRNA具有高比例的长3′ UTR亚型，通过增强mRNA的稳定性和翻译活性促进了细胞周期进展，增强了细胞增殖和迁移侵袭能力，减少了细胞凋亡，从而增强了CTC团簇的转移能力。

结论

本研究首次利用单细胞转录组测序数据揭示了单个CTC和CTC团簇中异常的AS和APA谱，发现了与CTC团簇转移能力增强的恶性表型相关的异常AS和APA事件及相关分子机制。这些研究成果加深了研究者对CTC转录后调控复杂性的认识，并为利用基于RNA的治疗靶向CTC团簇，从而抑制其活性的策略提供了潜在的靶点和坚实的理论支撑。

Background

Alternative splicing (AS) and alternative polyadenylation (APA) represent key mechanisms of post-transcriptional regulation, enabling the generation of diverse mRNA isoforms, thereby contributing to protein complexity and impacting mRNA function, localization, stability, and translation efficiency. Aberration of these mechanisms is closely associated with various diseases, including cancer. Metastasis of malignant tumors represents a significant challenge in cancer therapy, originating from circulating tumor cells (CTCs) which can disseminate through the circulatory system and seed in distant locations for subsequent progression. Although the crucial role of CTCs in promoting cancer metastasis and poor prognosis is acknowledged, our understanding of the post-transcriptional regulatory molecular mechanisms underlying CTC clusters, a specialized form with heightened metastatic potential, particularly in terms of AS and APA, remains limited. In recent years, RNA-based therapeutic strategies have attracted attention due to their high specificity and relatively straightforward development process, offering new avenues for treating challenging diseases such as cancer. However, the limited understanding of AS and APA in CTC clusters hinders the application of these therapeutic strategies in inhibiting cancer metastasis.

Objective

The primary objective of this study is to comprehensively elucidate the characteristics of AS and APA in CTC clusters and single CTCs, as well as their dynamic dysregulation. Further investigation is required to ascertain the underlying causes of these pivotal post-transcriptional regulatory mechanism alterations and to assess whether these changes lead to a more malignant phenotype in CTC clusters. By addressing these questions, we aim to provide new insights into the post-transcriptional regulation profiles of single CTCs and CTC clusters, revealing molecular features that promote metastasis and potentially identifying targets for RNA-based therapies targeting CTC clusters. This research has the potential to provide new therapeutic strategies for blocking cancer metastasis and improving patient prognosis, thus bearing significant clinical implications and scientific innovation.

Methods

This study, based on single-cell transcriptome sequencing data from CTCs in breast cancer, conducted a comparative analysis of AS and APA profiles between single CTCs and CTC clusters. We employed a range of experimental techniques, including 3′ RACE, construction of recombinant plasmid vectors, dual-luciferase reporter gene assays, gene overexpression, flow cytometry analysis, western blot, RT-qPCR, wound healing assays, and Transwell assays with breast cancer cell lines, in order to further elucidate the post-transcriptional regulatory mechanisms associated with the more malignant phenotype of CTC clusters compared to single CTCs.

Results

In this study, 994 and 836 AS events were identified in single CTCs and CTC clusters, respectively. Approximately 20% of AS events exhibited differential regulation between the two cell types. Among the differential splicing events we observed, SRSF6, occupying a central regulatory position, disrupted broad AS profiles and contributed to the increased malignancy of CTC clusters. With regard to APA, we observed a global lengthening of 3′ untranslated regions (UTRs) in CTC clusters in comparision to single CTCs. This alteration was primarily governed by 14 core APA factors, with PPP1CA controlling the largest number of APA events. The altered APA profiles facilitated the cell cycle progression of CTC clusters and indicated their reduced sensitivity to oxidative stress. Further investigation revealed that in CTC clusters, H2AFY mRNA exhibited a high proportion of long 3′ UTR isoforms, which promoted cell cycle progression, enhanced cell proliferation and migration, reduced apoptosis, and thus enhanced the metastatic potential of CTC clusters by enhancing mRNA stability and translation activity.

Conclusions

This study employed single-cell transcriptome sequencing data to elucidate aberrant AS and APA profiles in single CTCs and CTC clusters for the first time. We have identified aberrant AS and APA events and associated molecular mechanisms that are related to the enhanced metastatic phenotype of CTC clusters. These findings contribute to a deeper  understanding of the complexity of post-transcriptional regulation in CTCs and provide potential targets and robust theoretical support for strategies utilizing RNA-based therapies to target CTC clusters and inhibit their activity.