Hepatocellular carcinoma (HCC) is heterogeneous, rendering its current curative treatments ineffective. The emergence of single-cell genomics represents a powerful strategy in delineating the complex molecular landscapes of cancers. In this study, we demonstrated the feasibility and merit of using single-cell RNA sequencing to dissect the intra-tumoral heterogeneity and analyze the single-cell transcriptomic landscape to detect rare cell subpopulations of significance. Exploration of the inter-relationship among liver cancer stem cell markers showed two distinct major cell populations according to EPCAM expression, and the EPCAM+ cells had upregulated expression of multiple oncogenes. We also identified a CD24+/CD44+-enriched cell subpopulation within the EPCAM+ cells which had specific signature genes and might indicate a novel stemness-related cell subclone in HCC. Notably, knockdown of signature gene CTSE for CD24+/CD44+ cells significantly reduced self-renewal ability on HCC cells in vitro and the stemness-related role of CTSE was further confirmed by in vivo tumorigenicity assays in nude mice. In summary, single-cell genomics is a useful tool to delineate HCC intratumoral heterogeneity at better resolution. It can identify rare but important cell subpopulations, and may guide better precision medicine in the long run. 

目的 探讨肝细胞癌中miR-3682-3p的表达情况、临床意义和作用机制。方法 待实验所需肝癌细胞、正常肝细胞(L02)培养与转染后,采用实时定量PCR(qPCR)检测miR-3682-3p在肝癌组织、癌旁组织、L02细胞和肝癌细胞系中的表达情况,利用TCGA数据库分析正常肝组织、肝癌组织中miR-3682-3p的表达差异,分析miR-3682-3p的表达与肝癌患者临床病理特征、预后的关系;通过Transwell实验评价细胞侵袭、迁移能力;通过TargetScan数据库预测获得下游靶基因,采用qPCR、Western blotting法分析miR-3682-3p对生长抑制特异性基因8(GAS8)的表达的调控作用,并利用双荧光素酶报告基因实验加以验证。结果 与癌旁组织和正常肝细胞比较,miR-3682-3p在肝癌组织、细胞系中均高表达(P<0.05),并与门静脉侵犯、Edmondson分级、TNM分期有关;基于TCGA数据库数据的生存分析显示,miR-3682-3p高表达与不良预后相关;在MHCC97-H细胞中敲减miR-3682-3p显著抑制细胞的侵袭、迁移(P<0.05);通过生物信息学预测加以实验验证证实GAS8是miR-3682-3p的直接靶基因,并通过回复实验证实GAS8介导了miR-3682-3p对肝癌细胞侵袭及迁移能力的促进作用。结论 miR-3682-3p在肝癌中通过靶向抑制抑癌基因GAS8从而促进肝癌细胞侵袭、迁移。

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