Table 1_Single-cell and spatial transcriptomics reveal correlation between RNA methylation-related miRNA risk model and immune infiltration in hepatocellular carcinoma.xlsx

IntroductionIncreasing evidence highlights the pivotal role of RNA methylation and miRNAs in hepatocellular carcinoma (HCC). However, the risk associated with RNA methylation-related miRNAs (RMRMs) in the HCC immune microenvironment remains largely unknown. Here, we predicted the correlation between RMRM risk and immune cell infiltration in HCC using machine learning.
MethodsMiRNA sequencing data was used to identify RMRMs. A risk score model of HCC was developed utilizing four RMRMs, including miR-551a, miR-4739, miR-326, and miR-210-3p.
ResultsPatients with high-risk scores exhibited poorer prognoses. Single-cell RNA sequencing (scRNA-seq) analysis revealed the high-risk group exhibited increased infiltration levels of several immune cell subtypes, including myeloid-derived suppressor cell (MDSC), macrophage, and T cells. The data integration of scRNA-seq and bulk RNA-seq showed the decreased TIDE score in the high-risk patients and the elevated levels of Macro-secreted phosphoprotein 1 (SPP1), MDSC-meiotic nuclear divisions 1 (MND1), γδ T cells, and Macro-complement C1q C chain (C1QC) predicted adverse prognosis. ScRNA-seq and spatial transcriptomics data integration unveiled the spatial distribution of RMRMs risk scores and their correlation with immune cell subtype localization. Risk model-based clustering of HCC samples revealed that cluster 2, characterized by a higher risk score, correlated with a poorer prognosis and reduced immune and stromal scores. In vitro, the overexpression of miR-4739 in Huh-7 cells significantly induced SPP1+ macrophages, and the culture medium derived from SPP1+ macrophages further promoted the proliferation and migration of Huh-7 cells. Furthermore, miR-4739 reduced m1A methylation by inhibiting tRNA methyltransferase 61A (TRMT61A) expression.
DiscussionOur study reveals that the RMRM risk model could effectively predict the prognosis of HCC, and SPP1+ macrophages regulated by miR-4739-RNA methylation promote the proliferation and migration of HCC cells. These results highlight the potential of RMRMs in predicting the prognosis of HCC.

越来越多的证据表明，RNA甲基化与微小RNA（microRNAs, miRNAs）在肝细胞癌（hepatocellular carcinoma, HCC）中发挥关键作用。然而，与RNA甲基化相关微小RNA（RNA methylation-related miRNAs, RMRMs）相关的风险在肝癌免疫微环境中的作用仍未得到充分阐明。本研究借助机器学习方法，预测了肝癌中RMRM风险与免疫细胞浸润的相关性。
本研究通过miRNA测序数据鉴定RMRMs，并基于4种RMRMs（包括miR-551a、miR-4739、miR-326及miR-210-3p）构建肝癌风险评分模型。
高风险评分患者的预后较差。单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）分析显示，高风险组的多种免疫细胞亚群浸润水平升高，包括髓系来源抑制性细胞（myeloid-derived suppressor cell, MDSC）、巨噬细胞及T细胞。对scRNA-seq与批量RNA测序（bulk RNA-seq）数据进行整合分析发现，高风险患者的TIDE评分（Tumor Immune Dysfunction and Exclusion score, TIDE score）降低，且巨噬细胞分泌的磷蛋白1（secreted phosphoprotein 1, SPP1）、MDSC相关减数分裂核分裂蛋白1（meiotic nuclear divisions 1, MND1）、γδ T细胞以及巨噬细胞补体C1q C链（complement C1q C chain, C1QC）的表达水平升高，上述指标均可预测不良预后。对scRNA-seq与空间转录组学数据的整合分析揭示了RMRM风险评分的空间分布特征，及其与免疫细胞亚群定位的相关性。基于风险模型对肝癌样本进行聚类分析发现，以高风险评分为特征的聚类2与不良预后、较低的免疫评分及基质评分相关。体外实验显示，在Huh-7细胞系中过表达miR-4739可显著诱导SPP1阳性巨噬细胞，而SPP1阳性巨噬细胞的培养上清可进一步促进Huh-7细胞的增殖与迁移。此外，miR-4739可通过抑制tRNA甲基转移酶61A（tRNA methyltransferase 61A, TRMT61A）的表达降低m1A甲基化水平。
本研究表明，RMRM风险模型可有效预测肝癌患者的预后，且受miR-4739-RNA甲基化调控的SPP1阳性巨噬细胞可促进肝癌细胞的增殖与迁移。上述结果凸显了RMRMs在预测肝癌预后方面的潜在应用价值。