LncRNA ANRIL靶向miR-195对HCT116细胞及裸鼠移植瘤放射增敏实验研究

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

目的探究LncRNA ANRIL对结直肠癌HCT116细胞体外体内放射增敏作用及机制。方法qPCR检测ANRIL表达。将阴性对照siRNA、ANRIL siRNA、miR-NC mimic、miR-195 mimic、miR-NC inhibitor、miR-195 inhibitor转染至HCT116细胞中分别记为阴性对照、沉默ANRIL、过表达miR-NC、过表达miR-195、抑制miR-NC、抑制miR-195组,以不做任何处理的HCT116细胞为空白对照组。克隆形成实验检测放射敏感性,流式细胞术检测细胞凋亡,StarBase预测ANRIL下游miRNAs,双荧光素酶报告基因实验进一步验证。裸鼠皮下移植瘤实验检测ANRIL对照射后移植瘤生长影响。结果沉默ANRIL组细胞存活分数较阴性对照组降低(P<0.05),其放射增敏比为1.52。沉默ANRIL+4 Gy组细胞凋亡率较阴性对照+4 Gy组增加[(27.86±2.78)%︰(12.06±1.46)%,P<0.05]。裸鼠皮下移植瘤实验结果显示在13、16、19、22、25天时阴性对照组肿瘤体积比沉默ANRIL组降低[(234±66)、(273±63)、(296±72)、(321±85)、(403±94)mm^3与(357±79)、(485±124)、(617±143)、(764±174)、(985±221)mm^3,P<0.05]。miR-195是ANRIL靶基因,抑制miR-195可逆转沉默ANRIL对HCT116细胞放射增敏和凋亡促进作用及移植瘤生长抑制作用。结论LncRNA ANRIL通过调控miR-195表达来调节HCT116细胞放射敏感性,可能为临床结直肠癌放疗提供一个新的增敏靶点。

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)mm^3,(273±63)mm^3,(296±72)mm^3,(321±85)mm^3 and(403±94)mm^3vs.(357±79)mm^3,(485±124)mm^3,(617±143)mm^3,(764±174)mm^3 and(985±221)mm^3P<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.