Silencing long non-coding RNA HCP5 increases the radiosensitivity of glioma cells by up-regulating miR-508-3p expression

中华行为医学与脑科学杂志 Pub Date : 2020-03-20 DOI:10.3760/CMA.J.CN371468-20191023-00765

Xueyuan Li, Qiankun Liu, Shanpeng Yuan, T. Liang, Wenzheng Luo, Y. Zhen, Lixin Wu, Kang Wang, Dongming Yan

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引用次数: 0

Abstract

Objective To investigate the effect of long non-coding (lnc) RNA HCP5 on the radiation sensitivity of glioma cells and underlying mechanism. Methods The glioma cells U251 and U87 were irradiated with 0, 2, 4, 6, and 8 Gy rays as different doses.si-Con, si-HCP5, pcDNA, and pcDNA-HCP5 were transfected into cells U251 and U87, recorded as si-con group, si-HCP5 group, pcDNA group, and pcDNA-HCP5 group.si-Con and si-HCP5 were transfected into cells U251 and U87, and then irradiated with 4 Gy rays, respectively, recorded as IR+ si-con group and IR+ si-HCP5 group, the cells only irradiated with 4 Gy rays were recorded as IR group.After si-HCP5 with anti-miR-con and anti-miR-508-3p was co-transfected into cell U251 and U87, respectively, irradiated with 4 Gy rays, recorded as IR+ si-HCP5+ anti-miR-con group and IR+ si-HCP5+ anti-miR-508-3p group, respectively, the transfection was performed by liposome method.RT-qPCR was used to detect the expression of miR-508-3p and HCP5.Cell clone formation assay was used to detect the radiosensitivity of glioma cells.Flow cytometry was used to detect apoptosis, dual luciferase Reporter gene detection experiments detects fluorescence activity. Results HCP5 was highly expressed in radiation-treated glioma cells, and miR-508-3p was lowly expressed.After silenced HCP5, U251 and U87 cells had enhanced radiosensitivity and apoptotic rate((16.67±1.68) vs (3.58±0.62), t=21.929, P<0.05; (12.32±1.08) vs (4.48±0.71), t=18.198, P<0.05) was increased, and γ-H2AX( (0.45±0.04) vs (0.23±0.05), t=10.307, P<0.05; (0.38±0.04) vs (0.24±0.03), t=8.400, P<0.05), Cleaved caspase-3((0.37±0.04) vs (0.16±0.03), t=12.600, P<0.05; (0.38±0.04) vs (0.22±0.03), t=9.600, P<0.05) expressions were increased.Compared with silencing HCP5 or radiation treatment alone, silencing HCP5 and radiation treatment of U251 cells simultaneously, the apoptosis rate ((25.34±1.54) vs (16.67±1.68), t=11.413, P<0.05; (25.34±1.54) vs (11.13±1.06), t=22.802, P<0.05) was significantly increased, and γ-H2AX((0.69±0.05) vs (0.45±0.04), t=11.245, P<0.05; (0.69±0.05) vs (0.31±0.04), t=17.804, P<0.05), Cleaved caspase-3 ((0.52±0.06/0.37±0.04, t=6.240, P<0.05) (0.52±0.06/0.34±0.04, t=7.488, P<0.05) expressions were increased.The expressions of p-PI3K ((0.21±0.02) vs (0.52±0.04), t=20.795, P<0.05; (0.26±0.23 ), ( 0.67±0.07), t=5.116, P<0.05), p- AKT ((0.22±0.03) vs (0.66±0.07), t=17.332, P<0.05; (0.23±0.04) vs (0.71±0.03), t=28.800, P<0.05) in U251 and U87 cells were decreased.HCP5 can target the regulation of miR-508-3p expression; interfering with miR-508-3p reversed the effects of silent HCP5 and radiation on the radiation sensitization and apoptosis of U251 and U87 cells.It reduced the expression levels of reducing γ-H2AX and Cleaved caspase-3, while increased the expression levels of p-PI3K and p-AKT. Conclusion Silencing lncRNA HCP5 can enhance the radiation sensitivity of glioma cells and promote apoptosis.The mechanism may be related with the miR-508-3p and PI3K/Akt signaling pathway, which will provide new targets and new ideas for glioma treatment. Key words: LncRNA HCP5; MicroRNA-508-3p; Glioma; Radiosensitivity; Apoptosis

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沉默长非编码RNA HCP5通过上调miR-508-3p表达增加胶质瘤细胞的放射敏感性

目的探讨长非编码核糖核酸HCP5对胶质瘤细胞辐射敏感性的影响及其机制。方法分别用0、2、4、6、8Gy射线照射胶质瘤细胞U251和U87，将si-Con、si-HCP5、pcDNA和pcDNA-HCP5转染U251和U27细胞，分别记为si-Con组、si-HCP5组、pcDNA组和pcDNA-HCP5组，记录为IR+si-con组和IR+si-HCP5组。将具有抗-miR-con和抗-miR-508-3p的si-HCP5分别共转染到U251和U87细胞中，用4Gy射线照射，分别记录为IR+si-HCP5+抗-miR-con组和IR+si-HCCP5+抗-miR-508-3p组，然后通过脂质体法进行转染。RT-qPCR检测miR-508-3p和HCP5的表达。细胞克隆形成法检测胶质瘤细胞的放射敏感性。流式细胞术检测细胞凋亡，双荧光素酶报告基因检测实验检测荧光活性。结果HCP5在放射治疗的胶质瘤细胞中高表达，miR-508-3p低表达。在HCP5沉默后，U251和U87细胞的放射敏感性和凋亡率分别为（16.67±1.68）和（3.58±0.62），t=21.929，P<0.05；γ-H2AX（（0.45±0.04）vs（0.23±0.05），t=10.307，P<0.05；（0.38±0.04）vs（0.24±0.03），t=8.400，P<0.05），裂解胱天蛋白酶-3（（0.37±0.04vs（0.16±0.03），t=12.600，P<0.05；（0.38±0.04）vs（0.22±0.03），t=9.600，P<0.05）表达增加。与单独沉默HCP5或放射线处理、同时沉默HCP5和放射线处理U251细胞相比，凋亡率（（25.34±1.54）vs（16.67±1.68），t=11.413，P<0.05；γ-H2AX（（0.69±0.05）vs（0.45±0.04），t=11.245，P<0.05；（0.69±0.05）vs（0.31±0.04），t=17.804，P<0.05），裂解胱天蛋白酶-3（（0.52±0.06/0.37±0.04，t=6.240，P<0.05）（0.52±06/0.34±0.04，t=7.488，P<0.05）表达增加。p-PI3K的表达（0.21±0.02）vs（0.52±0.04），t=20.795，p<0.05；（0.26±0.23），（0.67±0.07），t=5.116，P<0.05），P-AKT（（0.22±0.03）vs（0.66±0.07，t=17.332，P<0.05）；（0.23±0.04）vs（0.71±0.03），t=28.800，P<0.05）。HCP5可以靶向调节miR-508-3p的表达；干扰miR-508-3p逆转了沉默HCP5和辐射对U251和U87细胞辐射增敏和凋亡的影响。它降低了还原γ-H2AX和裂解胱天蛋白酶-3的表达水平，同时增加了p-PI3K和p-AKT的表达水平。结论沉默lncRNA HCP5可增强胶质瘤细胞的辐射敏感性，促进细胞凋亡。其机制可能与miR-508-3p和PI3K/Akt信号通路有关，这将为神经胶质瘤的治疗提供新的靶点和新的思路。关键词：LncRNA HCP5；MicroRNA-508-3p；胶质瘤；放射敏感性；细胞凋亡

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来源期刊

中华行为医学与脑科学杂志

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8131

期刊介绍： "Chinese Journal of Behavioral Medicine and Brain Science" (CN 37-1468/R, ISSN 1674-6554) is a national academic journal under the supervision of the National Health Commission, sponsored by the Chinese Medical Association and Jining Medical College. The journal was founded in June 1992 and was formerly known as "Chinese Journal of Behavioral Medicine" (1992-1993) and "Chinese Behavioral Medical Science" (1994-2008). In 2009, it was renamed "Chinese Journal of Behavioral Medicine and Brain Science" with the approval of the State Administration of Press, Publication, Radio, Film and Television. The purpose of "Chinese Journal of Behavioral Medicine and Brain Science" is to implement the health and health policies of the Party and the State, implement the principle of combining theory with practice and popularization and improvement, and reflect the major progress in the theory and practical application of behavioral medicine and brain science in my country. It publishes academic papers and scientific research results in the field of behavioral medicine and brain science in my country, and has columns such as monographs/reviews, basic research, clinical research, health prevention, methods and techniques, psychological behavior and evaluation, and systematic evaluation.