ABSTRACT

Purpose: MicroRNAs (miRNAs or miRs) play an important role in the initiation and development of chondrosarcoma (CS). However, the role of miR-21-5p in CS progression and its underlying molecular mechanisms remains unclear.

Materials and Methods: miR-21-5p expression was measured by qRT-PCR. Cell proliferation, migration, and invasion were detected by CCK-8 and Transwell assay. Dual-luciferase reporter assay was used to validate the target of miR-21-5p. Western blot was applied to explore the expressions of CCR7 and EMT biomarkers. Then, the xenograft model was established to confirm the effects of miR-21-5p.

Results: miR-21-5p was significantly downregulated in CS tissues and cells and negatively correlated with grade, recurrence, and 5-year overall survival. In vitro, miR-21-5p caused G0/G1 cell cycle arrest and induced apoptosis by decreasing cyclin D1 expression and Bcl-2/Bax ratio. miR-21-5p suppressed cell migration and invasion of CS cells by inhibiting epithelial–mesenchymal transition (EMT). In vivo, miR-21-5p inhibited xenograft tumor formation of SW1353 cells. Mechanistically, miR-21-5p targeted the 3’-UTR of C-C chemokine receptor 7 (CCR7) mRNA to inhibit its expression. Overexpression of CCR7 reversed the inhibitory effects of miR-21-5p on CS cell behaviors. However, the silencing of CCR7 enhanced the inhibitory effects of miR-21-5p on CS cells. Besides, the overexpression of miR-21-5p or silencing of CCR7 obviously reduced the expression levels of p-STAT3, p-p56, and p-IκBα.

Conclusion: miR-21-5p targeted CCR7 expression to inhibit the STAT3 and NF-κB signaling, thereby suppressing cell proliferation, migration, invasion, and EMT in CS cells, which might be a novel mechanistic study for CS therapy.

Abbreviations: 3’-UTR: 3’-untranslated region; CCR7: C-C chemokine receptor type 7; CS: chondrosarcoma; DMEM: dulbecco’s modified eagle’s medium; EMT: epithelial–mesenchymal transition; HEK-293T: human embryonic kidney-293T; miR-21-5p: microRNA-21-5p; miR-NC: negative control miRNA; SD: standard deviation; si-CCR7: CCR7 siRNAs

摘要

目的：MicroRNAs（miRNAs或miRs）在软骨肉瘤（CS）的发生和发展中发挥着重要作用。然而，miR-21-5p 在 CS 进展中的作用及其潜在的分子机制仍不清楚。

材料和方法：通过qRT-PCR测量miR-21-5p的表达。CCK-8和Transwell法检测细胞增殖、迁移和侵袭。双荧光素酶报告基因检测用于验证 miR-21-5p 的靶标。应用Western blot检测CCR7和EMT生物标志物的表达。然后，建立异种移植模型以确认miR-21-5p的作用。

结果： miR-21-5p 在 CS 组织和细胞中显着下调，并且与分级、复发和 5 年总生存率呈负相关。在体外，miR-21-5p 通过降低细胞周期蛋白 D1 表达和 Bcl-2/Bax 比率引起 G0/G1 细胞周期停滞并诱导细胞凋亡。miR-21-5p 通过抑制上皮-间质转化 (EMT) 来抑制 CS 细胞的细胞迁移和侵袭。体内, miR-21-5p 抑制 SW1353 细胞的异种移植肿瘤形成。机制上，miR-21-5p 靶向 CC 趋化因子受体 7 (CCR7) mRNA 的 3'-UTR 以抑制其表达。CCR7的过表达逆转了miR-21-5p对CS细胞行为的抑制作用。然而，CCR7的沉默增强了miR-21-5p对CS细胞的抑制作用。此外，miR-21-5p的过表达或CCR7的沉默明显降低了p-STAT3、p-p56和p-IκBα的表达水平。

结论: miR-21-5p 靶向 CCR7 表达抑制 STAT3 和 NF-κB 信号通路，从而抑制 CS 细胞的增殖、迁移、侵袭和 EMT，这可能是 CS 治疗的新机制研究。

缩写：3'-UTR：3'-非翻译区；CCR7：CC趋化因子受体7型；CS：软骨肉瘤；DMEM：杜尔贝克改良鹰培养基；EMT：上皮-间质转化；HEK-293T：人胚肾-293T；miR-21-5p：microRNA-21-5p；miR-NC：阴性对照 miRNA；SD：标准差；si-CCR7：CCR7 siRNA