LncRNA ANRIL target miR-195 experimental study of radiation sensitivity of HCT116 cells and nude mouse transplant tumors

中华放射肿瘤学杂志 Pub Date : 2019-11-15 DOI:10.3760/CMA.J.ISSN.1004-4221.2019.11.013

Xiaoyan Chen, Che-Hsiang Wu, X. Tian, Xiaoli Gou, Ying-jun Wu, K. Zhao, Ruihui Xie

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Abstract

Objective To investigate the effect and mechanism of LncRNA ANRIL on the radiosensitivity of HCT116 cells line and nude mouse transplant tumors. Methods The expression of LncRNA ANRIL in colorectal cancer cells was detected by qPCR. The negative control siRNA, ANRIL siRNA, miR-NC mimic, miR-195 mimic, miR-NC inhibitor and miR-195 inhibitor were transfected into HCT116 cells, and marked as negative control group, silencing ANRIL group, overexpressing miR-NC group, overexpressing miR-195 group, inhibiting miR-NC group and inhibiting miR-195 group, and the HCT116 cells without any treatment were marked as the blank control group. The clone formation assay was used to detect radiosensitivity of colorectal cancer cells, flow cytometry was used to detect apoptosis. The web site, StarBase, was used to predict the downstream miRNAs of ANRIL and dual luciferase reporter gene assay was used to further verify. Subcutaneous tumor transplantation assay was used to detect the effect of ANRIL on the growth of colorectal cancer cells after irradiation. Results After irradiation with 2, 4, 6 and 8 Gy, the cell survival fraction of silencing ANRIL group was significantly decreased when compared with that of negative control group (P<0.05), and the radiosensitivity ratio was 1.52. The apoptosis rate of the silencing ANRIL+ 4 Gy group was significantly higher than that of the negative control+ 4 Gy group ((27.86±2.78)% vs. (12.06±1.46)%, P<0.05). The results of the experiment on nude mouse transplant tumors showed that the tumor volume in the negative control group was lower than that of the silent ANRIL group on days 13, 16, 19, 22 and 25 ((234±66) mm3, (273±63) mm3, (296±72) mm3, (321±85) mm3 and (403±94) mm3vs. (357±79) mm3, (485±124) mm3, (617±143) mm3, (764±174) mm3 and (985±221) mm3P<0.05). MiR-195 is a target gene of ANRIL, and inhibition of miR-195 can reverse the inhibitory effect of silencing ANRIL on radiosensitivity, apoptosis and xenografts of HCT116 cells. Conclusions LncRNA ANRIL regulates the radiosensitivity of colorectal cancer cells by miR-195, which may provide a new sensitizing target for clinical colorectal cancer radiotherapy. Key words: ANRIL gene; miR-195 gene; HCT116 cell line; Nude mouse transplant tumors; Radiosensitivity

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LncRNA ANRIL靶向miR-195对HCT116细胞和裸鼠移植瘤辐射敏感性的实验研究

目的探讨LncRNA ANRIL对HCT116细胞系和裸鼠移植瘤放射敏感性的影响及其机制。方法采用qPCR方法检测大肠癌细胞中LncRNA ANRIL的表达。将阴性对照siRNA、ANRIL siRNA、miR-NC模拟物、miR-195模拟物、iR-NC抑制剂和miR-195抑制剂转染到HCT116细胞中，并标记为阴性对照组、沉默ANRIL组、过表达miR-NC组、过度表达miR-195组、抑制miR-NC和抑制miR-195，将未经任何处理的HCT116细胞标记为空白对照组。克隆形成法检测大肠癌癌症细胞的放射敏感性，流式细胞术检测细胞凋亡。网站StarBase用于预测ANRIL的下游miRNA，双荧光素酶报告基因测定用于进一步验证。采用皮下肿瘤移植法检测ANRIL对大肠癌癌症细胞照射后生长的影响。结果2、4、6和8Gy照射后，沉默ANRIL组的细胞存活率与阴性对照组相比显著降低（P<0.05），放射敏感性为1.52。沉默ANRIL+4Gy组的细胞凋亡率显著高于阴性对照+4Gy（27.86±2.78）%对（12.06±1.46）%，P<0.05）。裸鼠移植瘤实验结果显示，阴性对照组在第13、16、19、22和25天的肿瘤体积低于沉默ANRIL组（（234±66）mm3、（273±63）mm3，（296±72）mm3、（321±85）mm3和（403±94）mm3vs。MiR-195是ANRIL的靶基因，抑制MiR-195可以逆转沉默ANRIL对HCT116细胞放射敏感性、细胞凋亡和异种移植物的抑制作用。结论LncRNA ANRIL通过miR-195调节结直肠癌癌症细胞的放射敏感性，为临床结直肠癌放疗提供了新的致敏靶点。关键词：ANRIL基因；miR-195基因；HCT116细胞系；裸鼠移植瘤；辐射敏感性

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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.