长链非编码RNA DLGAP1-AS2通过E2F1上调CD151表达促进直肠癌干细胞的放射抗性。

LncRNA DLGAP1-AS2 promotes the radioresistance of rectal cancer stem cells by upregulating CD151 expression via E2F1.

作者信息

Xiao Sheng-Ying, Yan Zhi-Guang, Zhu Xiao-Dong, Qiu Jun, Lu Yi-Chen, Zeng Fu-Ren

机构信息

Department of Oncology, Hunan Provincial People's Hospital (The First Affiliated Hospital of Hunan Normal University), No.89, Guhan Road, Changsha, Hunan Province 410024, P R China.

Department of Orthopedics, Ningxiang Hospital Affiliated to Hunan University of Chinese Medicine, Ningxiang, Hunan Province 410600, P R China.

出版信息

Transl Oncol. 2022 Apr;18:101304. doi: 10.1016/j.tranon.2021.101304. Epub 2022 Feb 7.

DOI:10.1016/j.tranon.2021.101304

PMID:35144091

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8844799/

Abstract

BACKGROUND

Radiotherapy resistance is one of the major causes of rectal cancer treatment failure. LncRNA DLGAP1-AS2 participates in the progression of several cancers. We explored the role and potential mechanism of DLGAP1-AS2 in the radioresistance of rectal cancer stem cells.

METHODS

HR8348-R cells, radioresistant cells from HR8348 after irradiation, were isolated into CD133 negative (CD133) and positive (CD133) cells. Cell proliferation, apoptosis, migration and tumorsphere formation were determined by CCK-8, flow cytometry, wound healing assay and tumorsphere formation assay, respectively. CD133, tumor stem cell drug resistance gene (MDR1 and BCRP1), DNA repair marker (γ-H2AX) and AKT/mTOR/cyclinD1 signaling were measured by Western blot. The relationship between DLGAP1-AS2 and E2F1 was verified using RIP. The interaction between E2F1 and CD151 promoter was confirmed using dual-luciferase reporter gene assay and ChIP. AKT inhibitor API-2 was employed for validating the effect of AKT/mTOR/cyclinD1 signaling in the radioresistance of rectal cancer cells.

RESULTS

The DLGAP1-AS2 level was increased in CD133 cells after irradiation. DLGAP1-AS2 knockdown inhibited the proliferation, migration and tumorsphere formation while stimulating apoptosis in CD133 cells. DLGAP1-AS2 inhibition downregulated the expression of CD133, MDR1, BCRP1 and γ-H2AX and suppressed AKT/mTOR/cyclinD1 activation. DLGAP1-AS2 upregulated the expression of CD151 by interacting with E2F1. API-2 neutralized the promotive effects of overexpressed CD151 on radioresistance.

CONCLUSION

DLGAP1-AS2 accelerates the radioresistance of rectal cancer cells through interactions with E2F1 to upregulate CD151 expression via the activation of the AKT/mTOR/cyclinD1 pathway.

摘要

背景

放疗抵抗是直肠癌治疗失败的主要原因之一。长链非编码RNA DLGAP1-AS2参与多种癌症的进展。我们探讨了DLGAP1-AS2在直肠癌干细胞放疗抵抗中的作用及潜在机制。

方法

将HR8348-R细胞（HR8348经照射后的放疗抵抗细胞）分离为CD133阴性（CD133⁻）和阳性（CD133⁺）细胞。分别通过CCK-8法、流式细胞术、伤口愈合试验和肿瘤球形成试验检测细胞增殖、凋亡、迁移和肿瘤球形成情况。通过蛋白质免疫印迹法检测CD133、肿瘤干细胞耐药基因（MDR1和BCRP1）、DNA修复标志物（γ-H2AX）以及AKT/mTOR/cyclinD1信号通路。使用RNA免疫沉淀法（RIP）验证DLGAP1-AS2与E2F1之间的关系。使用双荧光素酶报告基因试验和染色质免疫沉淀法（ChIP）证实E2F1与CD151启动子之间的相互作用。使用AKT抑制剂API-2验证AKT/mTOR/cyclinD1信号通路在直肠癌细胞放疗抵抗中的作用。

结果

照射后CD133⁺细胞中DLGAP1-AS2水平升高。敲低DLGAP1-AS2可抑制CD133⁺细胞的增殖、迁移和肿瘤球形成，同时促进其凋亡。抑制DLGAP1-AS2可下调CD133、MDR1、BCRP1和γ-H2AX的表达，并抑制AKT/mTOR/cyclinD1的激活。DLGAP1-AS2通过与E2F1相互作用上调CD151的表达。API-2可中和过表达的CD151对放疗抵抗的促进作用。