高甲基化的PODN通过使TGF-β/Smad2/3信号通路失活来抑制骨肉瘤进展。

Hypermethylated PODN represses the progression of osteosarcoma by inactivating the TGF-β/Smad2/3 pathway.

作者信息

Han Xiuxin, Zhang Yan, Lu Feng, Feng Jinyan, Zhang Chao, Wang Guowen

机构信息

Department of Bone and Soft Tissue Tumors, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Tianjin 300060, China.

出版信息

Pathol Res Pract. 2022 Oct;238:154075. doi: 10.1016/j.prp.2022.154075. Epub 2022 Aug 18.

DOI:10.1016/j.prp.2022.154075

PMID:36037657

Abstract

BACKGROUND

PODN is reported to be an promising biomarker for prognosis of osteosarcoma (OS), while the specific function of PODN has not been explored in OS. This study is designed to explore the function and underlying mechanism of PODN in OS.

METHODS

The mRNA expression of PODN was determined using qRT-PCR. Protein levels of PODN, DNMT1, DNMT3A, DNMT3B, TGF-β1, Smad2/3 and p-Smad2/3 were detected using western blot. The methylation of PODN was determined with methylation-specific PCR. Moreover, CCK-8 assay and colony formation assay were used for assessing the proliferation of OS cells. Transwell assay was used to evaluate migration and invasion abilities of OS cells. Immunohistochemical staining was performed to determine the protein expression of Ki67 and PODN in tumor tissues. For constructing a xenograft tumor model, MG-63 cells were introduced into the right side of the mouse back via subcutaneous injection.

RESULTS

PODN was lowly expressed and was hypermethylated in OS tissues and cells. PODN overexpression prevented OS cells from proliferating, migrating and invading, and inhibited tumorigenesis in xenograft mice. After PODN overexpression, protein levels of TGF-β1 and p-Smad2/3 were decreased in OS cells. Meantime, the suppressive effects of PODN overexpression on proliferation, migration and invasion of OS cells as well as mouse tumorigenesis were partly counteracted by TGF-β1 overexpression.

CONCLUSIONS

PODN overexpression inactivated the TGF-β/Smad2/3 pathway to suppress OS development in vitro and in vivo.

摘要

背景

据报道，足突蛋白聚糖（PODN）是骨肉瘤（OS）预后的一个有前景的生物标志物，而PODN在OS中的具体功能尚未得到探索。本研究旨在探讨PODN在OS中的功能及潜在机制。

方法

采用qRT-PCR检测PODN的mRNA表达。使用蛋白质印迹法检测PODN、DNA甲基转移酶1（DNMT1）、DNA甲基转移酶3A（DNMT3A）、DNA甲基转移酶3B（DNMT3B）、转化生长因子-β1（TGF-β1）、Smad2/3和磷酸化Smad2/3（p-Smad2/3）的蛋白水平。用甲基化特异性PCR测定PODN的甲基化情况。此外，采用CCK-8法和集落形成试验评估OS细胞的增殖。采用Transwell试验评估OS细胞的迁移和侵袭能力。进行免疫组织化学染色以确定肿瘤组织中Ki67和PODN的蛋白表达。为构建异种移植肿瘤模型，将MG-63细胞通过皮下注射引入小鼠背部右侧。

结果

PODN在OS组织和细胞中低表达且高度甲基化。PODN过表达可阻止OS细胞增殖、迁移和侵袭，并抑制异种移植小鼠的肿瘤发生。PODN过表达后，OS细胞中TGF-β1和p-Smad2/3的蛋白水平降低。同时，TGF-β1过表达部分抵消了PODN过表达对OS细胞增殖、迁移和侵袭以及小鼠肿瘤发生的抑制作用。