Abstract

Background

Our previous studies have indicated that miR-198 reduces cellular methylguanine DNA methyltransferase (MGMT) levels to enhance temozolomide sensitivity. Transforming growth factor beta 1 (TGF-β1) switches off miR-198 expression by repressing K-homology splicing regulatory protein (KSRP) expression in epidermal keratinocytes. However, the underlying role of TGF-β1 in temozolomide resistance has remained unknown.

Methods

The distribution of KSRP was detected by western blotting and immunofluorescence. Microarray analysis was used to compare the levels of long noncoding RNAs (lncRNAs) between TGF-β1–treated and untreated cells. RNA immunoprecipitation was performed to verify the relationship between RNAs and KSRP. Flow cytometry and orthotopic and subcutaneous xenograft tumor models were used to determine the function of TGF-β1 in temozolomide resistance.

Results

Overexpression of TGF-β1 contributed to temozolomide resistance in MGMT promoter hypomethylated glioblastoma cells in vitro and in vivo. TGF-β1 treatment reduced cellular MGMT levels through suppressing the expression of miR-198. However, TGF-β1 upregulation did not affect KSRP expression in glioma cells. We identified and characterized 2 lncRNAs (H19 and HOXD-AS2) that were upregulated by TGF-β1 through Smad signaling. H19 and HOXD-AS2 exhibited competitive binding to KSRP and prevented KSRP from binding to primary miR-198, thus decreasing miR-198 expression. HOXD-AS2 or H19 upregulation strongly promoted temozolomide resistance and MGMT expression. Moreover, KSRP depletion abrogated the effects of TGF-β1 and lncRNAs on miR-198 and MGMT. Finally, we found that patients with low levels of TGF-β1 or lncRNA expression benefited from temozolomide therapy.

Conclusions

Our results reveal an underlying mechanism by which TGF-β1 confers temozolomide resistance. Furthermore, our findings suggest that a novel combination of temozolomide with a TGF-β inhibitor may serve as an effective therapy for glioblastomas.

中文翻译：

---

TGF-β1通过改变microRNA加工和升高MGMT调节胶质母细胞瘤中的替莫唑胺耐药性

摘要

背景

我们以前的研究表明，miR-198可以降低细胞甲基鸟嘌呤DNA甲基转移酶（MGMT）的水平，从而增强替莫唑胺的敏感性。转化生长因子β1（TGF-β1）通过抑制表皮角质形成细胞中的K同源性剪接调节蛋白（KSRP）表达来关闭miR-198表达。然而，尚不清楚TGF-β1在替莫唑胺耐药性中的潜在作用。

方法

通过蛋白质印迹和免疫荧光检测KSRP的分布。微阵列分析用于比较经TGF-β1处理和未处理的细胞之间的长非编码RNA（lncRNA）的水平。进行RNA免疫沉淀以验证RNA与KSRP之间的关系。用流式细胞术和原位及皮下异种移植肿瘤模型确定TGF-β1在替莫唑胺耐药性中的作用。

结果

在体外和体内，TGF-β1的过表达促进了MGMT启动子低甲基化的胶质母细胞瘤细胞中的替莫唑胺耐药性。TGF-β1处理通过抑制miR-198的表达降低了细胞的MGMT水平。然而，TGF-β1的上调并不影响神经胶质瘤细胞中的KSRP表达。我们鉴定并鉴定了2个lncRNA（H19和HOXD-AS2），它们通过Smad信号传导被TGF-β1上调。H19和HOXD-AS2表现出与KSRP的竞争性结合，阻止了KSRP与主要miR-198的结合，从而降低了miR-198的表达。HOXD-AS2或H19上调强烈促进替莫唑胺抗性和MGMT表达。此外，KSRP耗竭消除了TGF-β1和lncRNA对miR-198和MGMT的影响。最后，

结论

我们的结果揭示了TGF-β1赋予替莫唑胺抗性的潜在机制。此外，我们的发现表明，替莫唑胺与TGF-β抑制剂的新型组合可作为胶质母细胞瘤的有效疗法。