miR-142-3p调控高迁移率族蛋白1及对神经母细胞瘤顺铂化疗敏感性的影响

MiR-142-3p regulates CDDP resistance of neuroblastoma cell by targeting HMGB1

目的 研究miR-142-3p靶向调控高迁移率族蛋白1（high mobility group box-1 protein,HMGB1）及其对神经母细胞瘤细胞顺铂（CDDP）化疗敏感性影响的分子机制.方法 ①构建神经母细胞瘤耐药细胞株SH-SY5 Y/CDDP,RT-PCR检测细胞内miR-142-3p的表达水平;②CCK8和Elisa检测过表达miR-142-3p对耐药细胞株SH-SY5 Y/CDDP的凋亡和IC50水平的影响;③构建HMGB1 3＇UTR区荧光素酶报告载体,验证miR-142-3p对HMGB1的靶向调控作用;④Westernblot检测转染miR-142-3p后SH-SY5Y细胞中HMGB1的表达水平;⑤利用siRNA敲减耐药细胞株SH-SY5 Y/CDDP中HMGB1的表达,Western-blot检测siRNA的敲减效果,CCK8和Elisa方法检测SH-SY5 Y/CDDP细胞的IC50和凋亡情况.结果 ①构建神经母细胞瘤耐药细胞株SH-SY5 Y/CDDP成功,耐药细胞株IC50由原来的（4.57±0.65） μg/ml增加到（23.53±3.87） μg/ml,比非耐药细胞株提高了5.15倍;RT-PCR结果显示耐药的SH-SY5Y/CDDP细胞株中miR-142-3p表达水平显著低于正常的SH-SY5Y细胞,差异有统计学意义（P＜0.01）.②Elisa结果显示,过表达miR-142-3p后,与转染Control mimics组相比,耐药细胞株SH SY5Y/CDDP细胞用CDDP处理后凋亡水平升高,IC50显著降低（7.02±1.38 vs 24.27±4.13）μg/ml,差异有统计学意义（P＜0.05）;③荧光素酶活性检测表明miR-142-3p能够显著抑制HMGB1的荧光素酶活性（P＜0.01）;④Western-blot结果显示,在SH-SY5Y细胞中过表达miR-142-3p后,细胞中HMGB1蛋白水平明显下调（P＜0.01）,miR-142-3p与HMGB1的表达呈负相关;⑤两条siRNA敲减HMGB1后,SH-SY5 Y/CDDP耐药细胞株用CDDP处理后凋亡水平升高,细胞的IC50由对照组的（24.61±4.07） μg/ml,分别下降到（9.65±1.39）μg/ml和（8.43±1.76）μg/ml,与对照组比较,差异有统计学意义（P＜0.05）.结论 miR-142-3p的靶基因为HMGB1,miR-142-3p能够通过靶向调控HMGB1的表达而增加神经母细胞瘤细胞对CDDP化疗的敏感性.

Objective To explore the effects of miR-142 3p on CDDP resistance of neuroblastoma cells by targeting high mobility group box-1 protein （HMGB1）.Methods The CDDP resistance neuroblastoma cell line SH-SY5Y/CDDP was constructed.And the expression level of miR-142 3p was detected by real-time polymerase chain reaction （PCR）.Then the apoptosis level and IC50 value of SH-SY5Y/CDDP were quantified by CCK8 and enzyme-linked immunosorbent assays after a transfection of miR-142-3pmimic.The 3＇UTR of HMGB1 was cloned into luciferase reporter vector to verify that miR-142-3p could target HMGB1.The post-transfection expression level of HMGB1 in SH-SY5Y cell was detected by Western-blot.And the IC50 value and apoptosis level of SH-SY5Y/CDDP cells after a knockdown of HMGB1 in SH-SY5Y/CDDP cell line by siRNA.Results The CDDP-resistance cell line SH-SY5Y/CDDP was successfully constructed.The IC50 value of SH-SY5Y/CDDP increased from （4.57 ± 0.65） to （23.53 ± 3.87） μg/ml.And it was 5.15 times higher than parental cell line SH-SY5Y （P＜0.01）.And the expression level of miR-142-3p decreased remarkably in SH-SY5Y/CDDP cell than SH SY5Y cell.The apoptosis level of SH-SY5Y/CDDP increased,IC50 value of CDDP decreased（7.02 ± 1.38 vs 24.27 ± 4.13） μg/ml while the expression of miR-142-3p was up-regulated（P＜0.05）.MiR-142-3p could inhibit the enzymatic activity of luciferase reporter vector of HMGB1 （P＜0.01）.The expression level of HMGB1 decreased and there was an over-expression of miR-142-3p in SH-SY5Y cell.And the expression levels of HMGB1 and miR-142-3p were negatively correlated in SH-SY5Y cell （P＜0.01）.The apoptosis level of SH-SY5Y/CDDP increased and IC50 value of CDDP decreased from（24.61 ± 4.07）to（9.65 ± 1.39）and（8.43 ± 1.76） μg/ml respectively after a siRNA knockdown of HMGB1 in SH-SY5Y/CDDP cell line （P＜ 0.05）.Conclusions MiR-142-3p can increase the CDDP resistance of neuroblastoma cell by targeting HMGB1.