Song Yu, Zuo Yun, Qian Xiao-Lan, Chen Zhi-Peng, Wang Shao-Kai, Song Lei, Peng Li-Ping
Department of Oncology, Zhangjiagang First People's Hospital, Zhangjiagang, China.
Department of Respiratory Medicine, The First Hospital of Jilin University, Changchun, China.
Cell Physiol Biochem. 2017;43(3):1258-1272. doi: 10.1159/000481839. Epub 2017 Oct 9.
This study aimed to explore the effects of microRNA-21-5p (miR-21-5p) on the radiation sensitivity of non-small cell lung cancer (NSCLC) and the involvement of human MutS homolog 2 (hMSH2) One hundred fourteen NSCLC patients at stage II or III who received surgery and postoperative radiotherapy were enrolled in this study.
The patients were assigned into radiation-sensitive and -insensitive groups. NSCLC A549 cells were transfected to generate control, Negative control (NC), miR-21-5p inhibitor, miR-21-5p mimic, small interfering hMSH2 (sihMSH2), miR-21-5p inhibitor + sihMSH2 and hMSH2 overexpression groups. Immunohistochemistry was performed to detect the hMSH2 expression in transfected and irradiated cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were performed to evaluate A549 miR-21-5p and hMSH2 expression in transfected and irradiated cells. A colony formation assay was adopted for cell survival analysis. The relationship between miR-21-5p and hMSH2 was verified by a luciferase reporter assay. Cell viability was measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and apoptosis was assessed by flow cytometry. NSCLC nude mouse models were established, and tumor volumes and tumor weights were recorded.
The radiation-sensitive group of patients exhibited lower miR-21-5p but higher hMSH2 expression than the insensitive group. For irradiated A549 cells, lower cell survival, higher apoptosis, increased miR-21-5p expression and decreased hMSH2 expression were observed at 6 and 8 Gy than at 0, 2 and 4 Gy; compared to 6 Gy, cell survival and hMSH2 expression were decreased and apoptosis and miR-21-5p expression were increased at 8 Gy. Additionally, miR-21-5p was found to target hMSH2. Compared with the control group, the cell survival rate was lower and the apoptosis rate higher in the miR-21-5p inhibitor group, whereas the opposite was observed for the miR-21-5p mimic and sihMSH2 groups. For the mouse model, decreased tumor volume and tumor weight and higher hMSH2 expression were found in the miR-21-5p inhibitor, radiation, hMSH2 overexpression, miR-21-5p inhibitor + radiation and hMSH2 overexpression + radiation groups compared with the control group. In addition, tumor volume and tumor weight were decreased and hMSH2 expression increased in the miR-21-5p inhibitor + radiation and hMSH2 overexpression + radiation groups compared with the radiation alone group.
These findings indicate that inhibition of miR-21 can promote the radiation sensitivity of NSCLC by targeting hMSH2.
本研究旨在探讨微小RNA-21-5p（miR-21-5p）对非小细胞肺癌（NSCLC）放射敏感性的影响以及人类MutS同源蛋白2（hMSH2）的参与情况。本研究纳入了114例接受手术及术后放疗的Ⅱ期或Ⅲ期NSCLC患者。
将患者分为放射敏感组和放射不敏感组。对NSCLC A549细胞进行转染，以产生对照组、阴性对照组（NC）、miR-21-5p抑制剂组、miR-21-5p模拟物组、小干扰hMSH2（sihMSH2）组、miR-21-5p抑制剂+sihMSH2组和hMSH2过表达组。采用免疫组织化学法检测转染及照射后细胞中hMSH2的表达。采用定量实时聚合酶链反应（qRT-PCR）和蛋白质印迹法评估转染及照射后A549细胞中miR-21-5p和hMSH2的表达。采用集落形成试验进行细胞存活分析。通过荧光素酶报告基因试验验证miR-21-5p与hMSH2之间的关系。采用MTT（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐）法检测细胞活力，通过流式细胞术评估细胞凋亡情况。建立NSCLC裸鼠模型，并记录肿瘤体积和肿瘤重量。
放射敏感组患者的miR-21-5p表达低于放射不敏感组，但hMSH2表达高于放射不敏感组。对于照射后的A549细胞，与0、2和4 Gy相比，6和8 Gy时细胞存活率降低、凋亡增加、miR-21-5p表达增加而hMSH2表达降低；与6 Gy相比，8 Gy时细胞存活率和hMSH2表达降低，凋亡和miR-21-5p表达增加。此外，发现miR-21-5p靶向hMSH2。与对照组相比，miR-21-5p抑制剂组细胞存活率较低而凋亡率较高，而miR-21-5p模拟物组和sihMSH2组则相反。对于小鼠模型，与对照组相比，miR-21-5p抑制剂组、放疗组、hMSH2过表达组、miR-21-5p抑制剂+放疗组和hMSH2过表达+放疗组的肿瘤体积和肿瘤重量减小，hMSH2表达增加。此外，与单纯放疗组相比，miR-21-5p抑制剂+放疗组和hMSH2过表达+放疗组的肿瘤体积和肿瘤重量减小，hMSH2表达增加。
这些发现表明，抑制miR-21可通过靶向hMSH2促进NSCLC的放射敏感性。
