Silencing LncRNA OIP5-AS1 increases radiosensitivity of non-small cell lung cancer A549 cell line by up-regulating miR-34c-5p expression

中华放射肿瘤学杂志 Pub Date : 2020-01-15 DOI:10.3760/CMA.J.ISSN.1004-4221.2020.01.013

Kai Mao, Xiaohua Ding, Liping Wu, Y. Mao, Liguo Zhang, Jun Li, Jiang Lu

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Abstract

Objective To investigate the effect of LncRNA OIP5-AS1 on radiosensitivity of non-small cell lung cancer (NSCLC) cells and its mechanism. Methods The radiation-resistant cell A549R was established by using A549 cells irradiated by X-ray 6Gy in 5 fractions. The expression levels of OIP5-AS1 and miR-34c-5p in A549 and A549R cells were detected by qRT-PCR. OIP5-AS1 inhibitor or miR-34c-5p mimetic was transfected into A549R cells, or OIP5-AS1 overexpression plasmid was transfected into A549 cells. Cell apoptosis was detected by flow cytometry. Cell radiosensitivity was analyzed by colony formation assay. The expression levels of p-Chk2 and p-ATM proteins were measured by Western blot. Dual luciferase assay was adopted to verify the relationship between OIP5-AS1 and miR-34c-5p. Results Compared with A549 cells, the expression of OIP5-AS1 was significantly up-regulated in A549R cells (1.97±0.11 vs.1.01±0.05, P<0.05), whereas the expression of miR-34c-5p was remarkably down-regulated (0.43±0.02 vs.1.02±0.06, P<0.05). The expression levels of p-Chk2 and p-ATM proteins in A549R cells in the silencing OIP5-AS1+ 6Gy group were significantly lower (0.43±0.03 vs.1.39±0.15, 0.51±0.0 5 vs.1.21± 0.11, both P<0.05), whereas the apoptotic rate was significantly higher than those in the silencing control+ 6Gy group [(13.29±1.25)% vs. (28.47±2.31)%, P<0.05)]. The expression levels of p-Chk2 and p-ATM proteins in A549 cells in overexpressing OIP5-AS1+ 6Gy group were significantly higher than those in overexpression control+ 6Gy group (1.23±0.13 vs.0.75±0.06, 1.08±0.11 vs.0.59±0.04, both P<0.05). Inhibiting miR-34c-5p expression reversed the effect of silencing OIP5-AS1 on survival fraction of A549R cells (SER=1.42). OIP5-AS1 negatively regulated the expression of miR-34c-5p. Conclusion Silencing OIP5-AS1 enhances the radiosensitivity of radiation-resistant A549 cells by up-regulating the expression of miR-34c-5p, providing a potential target for radiotherapy of NSCLC cells. Key words: A549 cell line; A549R cell line; OIP5-AS1 gene; miR-34c-5p gene; radiosensitivity

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沉默LncRNA OIP5-AS1通过上调miR-34c-5p表达增加非小细胞肺癌癌症A549细胞系的放射敏感性

目的探讨LncRNA OIP5-AS1对非小细胞肺癌（NSCLC）细胞放射敏感性的影响及其机制。方法用6 Gy射线照射A549细胞，分5个组分建立A549R细胞株。通过qRT-PCR检测OIP5-AS1和miR-34c-5p在A549和A549R细胞中的表达水平。将OIP5-AS1抑制剂或miR-34c-5p模拟物转染到A549R细胞中，或将OIP5-AS1过表达质粒转染到A549细胞中。流式细胞仪检测细胞凋亡。通过集落形成试验分析细胞的放射敏感性。通过蛋白质印迹测定p-Chk2和p-ATM蛋白的表达水平。采用双荧光素酶测定法验证OIP5-AS1与miR-34c-5p之间的关系。结果与A549细胞相比，A549R细胞中OIP5-AS1的表达显著上调（1.97±0.11 vs.1.01±0.05，P<0.05），而miR-34c-5p的表达显著下调（0.43±0.02 vs.1.02±0.06，P<0.05）。沉默OIP5-AS1+6Gy组A549R细胞中P-Chk2和P-ATM蛋白的表达水平显著降低（0.43士0.03 vs.1.39±0.15，0.51士0.05 vs.1.21±0.11，均P<0.05），细胞凋亡率显著高于沉默对照+6Gy组[（13.29±1.25）%vs.（28.47±2.31）%，P<0.05）。抑制miR-34c-5p的表达逆转了沉默OIP5-AS1对A549R细胞存活率的影响（SER=1.42）。结论沉默OIP5-AS1通过上调miR-34c-5p的表达，增强耐辐射A549细胞的放射敏感性，为NSCLC细胞的放射治疗提供了潜在的靶点。关键词：A549细胞系；A549R细胞系；OIP5-AS1基因；miR-34c-5p基因；放射敏感性

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来源期刊

中华放射肿瘤学杂志

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6375

期刊介绍： The Chinese Journal of Radiation Oncology is a national academic journal sponsored by the Chinese Medical Association. It was founded in 1992 and the title was written by Chen Minzhang, the former Minister of Health. Its predecessor was the Chinese Journal of Radiation Oncology, which was founded in 1987. The journal is an authoritative journal in the field of radiation oncology in my country. It focuses on clinical tumor radiotherapy, tumor radiation physics, tumor radiation biology, and thermal therapy. Its main readers are middle and senior clinical doctors and scientific researchers. It is now a monthly journal with a large 16-page format and 80 pages of text. For many years, it has adhered to the principle of combining theory with practice and combining improvement with popularization. It now has columns such as monographs, head and neck tumors (monographs), chest tumors (monographs), abdominal tumors (monographs), physics, technology, biology (monographs), reviews, and investigations and research.