Purpose

Our study aimed to study the role of lncRNA TP73-AS1/miR-539/MMP-8 axis in modulating M2 macrophage polarization in hepatocellular carcinoma (HCC).

Methods

The gene expression levels of TP73-AS1, miR-539 and MMP-8 were modified by transfection with the overexpression or knockdown vectors. The patient survival rate was analyzed using Kaplan-Meier method. The levels of TP73-AS1, miR-539, MMP-8 and M1/2 macrophage polarization markers were analyzed by qRT-PCR, western blot, and flow cytometry. The release of TGF-β1 in the supernatant was determined by ELISA assay. The interaction between TP73-AS1, miR-539 and MMP-8 was analyzed by bioinformatics analysis and dual-luciferase reporter assays. Mouse xenograft model was further established to examine the therapeutic effects of the TP73-AS1 knockdown and miR-539 overexpression in vivo.

Results

We found TP73-AS1 and MMP-8 upregulation, and miR-539 downregulation in HCC tissues and cell lines. Lower TP73-AS1 and MMP-8 expressions and higher miR-539 expression were associated with higher survival rate of patients. M2-macrophage markers CD206, Arg-1 and CD163 were significantly upregulated in the tumor tissues. TP73-AS1 negatively and directly regulated miR-539 and knockdown of TP73-AS1 inhibited MMP-8 expression and M2 macrophage polarization. Also, overexpression of miR-539 suppressed M2 macrophage polarization by negatively regulating MMP-8. Furthermore, knockdown of MMP-8 also restrained M2 macrophage polarization via inhibiting TGF-β1 signaling. We also found knockdown of TP73-AS1 or overexpression of miR-539 inhibited HCC tumor growth and M2 macrophage infiltration in vivo.

Conclusion

Our study demonstrated lncRNA TP73-AS1 negatively regulated miR-539 to promote MMP-8 expression, which activated TGF-β1 signaling to induce M2 macrophage polarization in HCC.

中文翻译：

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LncRNA TP73-AS1/miR-539/MMP-8 轴通过 TGF-β1 信号传导调节肝细胞癌中的 M2 巨噬细胞极化。

目的

我们的研究旨在研究 lncRNA TP73-AS1/miR-539/MMP-8 轴在调节肝细胞癌 (HCC) 中 M2 巨噬细胞极化中的作用。

方法

TP73-AS1、miR-539 和 MMP-8 的基因表达水平通过用过表达或敲低载体转染而改变。使用Kaplan-Meier方法分析患者存活率。通过 qRT-PCR、蛋白质印迹和流式细胞术分析 TP73-AS1、miR-539、MMP-8 和 M1/2 巨噬细胞极化标记的水平。通过ELISA测定测定上清液中TGF-β1的释放。通过生物信息学分析和双荧光素酶报告基因检测分析了 TP73-AS1、miR-539 和 MMP-8 之间的相互作用。进一步建立小鼠异种移植模型以检查体内 TP73-AS1 敲低和 miR-539 过表达的治疗效果。

结果

我们在 HCC 组织和细胞系中发现 TP73-AS1 和 MMP-8 上调，以及 miR-539 下调。较低的 TP73-AS1 和 MMP-8 表达以及较高的 miR-539 表达与较高的患者存活率相关。M2-巨噬细胞标志物CD206、Arg-1和CD163在肿瘤组织中显着上调。TP73-AS1 负向和直接调节 miR-539，敲除 TP73-AS1 可抑制 MMP-8 表达和 M2 巨噬细胞极化。此外，miR-539 的过表达通过负调节 MMP-8 来抑制 M2 巨噬细胞极化。此外，MMP-8 的敲低还通过抑制 TGF-β1 信号传导来抑制 M2 巨噬细胞极化。我们还发现 TP73-AS1 的敲低或 miR-539 的过表达抑制了体内 HCC 肿瘤生长和 M2 巨噬细胞浸润。

结论

我们的研究表明 lncRNA TP73-AS1 负调控 miR-539 以促进 MMP-8 表达，从而激活 TGF-β1 信号传导以诱导 HCC 中的 M2 巨噬细胞极化。