异常表达的长链非编码 RNA H19 通过失衡成骨和破骨参与多发性骨髓瘤骨病。

Abnormal expression pattern of lncRNA H19 participates in multiple myeloma bone disease by unbalancing osteogenesis and osteolysis.

机构信息

Department of Hematology, The Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, PR China.

出版信息

Int Immunopharmacol. 2023 Jun;119:110058. doi: 10.1016/j.intimp.2023.110058. Epub 2023 Apr 13.

DOI:10.1016/j.intimp.2023.110058

PMID:37058751

Abstract

BACKGROUND

Accumulating genetic and epigenetic alterations in multiple myeloma (MM) have been demonstrated to be closely associated with osteolytic bone disease, generally characterized as increased osteoclast formation and decreased osteoblast activity. Previously, serum long non-coding RNA (lncRNA) H19 has been proved to be a biomarker for the diagnosis of MM. Whereas, its role in MM-associated bone homeostasis remains largely elusive.

METHODS

A cohort of 42 MM patients and 40 healthy volunteers were enrolled for evaluating differential expressions of H19 and its downstream effectors. The proliferative capacity of MM cells was monitored by CCK-8 assay. Alkaline phosphatase (ALP) staining and activity detection, either with Alizarin red staining (ARS) were employed to assess osteoblast formation. Osteoblast- or osteoclast-associated gene were detected using qRT-PCR and western blot analysis. Bioinformatics analysis, RNA pull-down, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP) were subjected to verify H19/miR-532-3p/E2F7/EZH2 axis, which was accounted for epigenetic suppression of PTEN. The functional role of H19 on MM development through unbalancing osteolysis and osteogenesis was also confirmed in the murine MM model.

RESULTS

Upregulation of serum H19 was observed in MM patients, suggesting its positive correlation with the poor prognosis of MM patients. Loss of H19 dramatically weakened cell proliferation of MM cells, promoted osteoblastic differentiation, and impaired osteoclast activity. While reinforced H19 exhibited the opposite effects. Akt/mTOR signaling plays an indispensable role in H19-mediated osteoblast formation and osteoclastgenesis. Mechanistically, H19 served as a sponge for miR-532-3p to upregulate E2F7, a transcriptional activator of EZH2, thereby accounting for modulating epigenetic suppression of PTEN. The in vivo experiments further validated that H19 exerted important impacts on tumor growth through breaking the balance between osteogenesis and osteolysis via Akt/mTOR signaling.

CONCLUSION

Collectively, increased enrichment of H19 in MM cells exhibits an essential role in MM development by disturbing bone homeostasis.

摘要

背景

多发性骨髓瘤（MM）中积累的遗传和表观遗传改变与溶骨性骨病密切相关，其特征通常为破骨细胞形成增加和成骨细胞活性降低。先前，血清长链非编码 RNA（lncRNA）H19 已被证明是 MM 诊断的生物标志物。然而，其在 MM 相关骨稳态中的作用在很大程度上仍不清楚。

方法

招募了 42 名 MM 患者和 40 名健康志愿者来评估 H19 及其下游效应物的差异表达。通过 CCK-8 测定法监测 MM 细胞的增殖能力。碱性磷酸酶（ALP）染色和活性检测（用茜素红染色（ARS））用于评估成骨细胞形成。使用 qRT-PCR 和 Western blot 分析检测成骨细胞或破骨细胞相关基因。生物信息学分析、RNA 下拉、RNA 免疫沉淀（RIP）和染色质免疫沉淀（ChIP）用于验证 H19/miR-532-3p/E2F7/EZH2 轴，该轴负责 PTEN 的表观遗传抑制。在 MM 小鼠模型中也证实了 H19 通过打破溶骨和成骨平衡对 MM 发展的功能作用。

结果

在 MM 患者中观察到血清 H19 的上调，表明其与 MM 患者的不良预后呈正相关。H19 的缺失显着削弱了 MM 细胞的增殖能力，促进了成骨细胞分化，并损害了破骨细胞的活性。而增强的 H19 则表现出相反的效果。Akt/mTOR 信号在 H19 介导的成骨细胞形成和破骨细胞生成中起着不可或缺的作用。机制上，H19 作为 miR-532-3p 的海绵，上调 E2F7，EZH2 的转录激活子，从而负责调节 PTEN 的表观遗传抑制。体内实验进一步验证了 H19 通过 Akt/mTOR 信号破坏成骨和溶骨之间的平衡对肿瘤生长产生重要影响。