MEDLINE/PubMed Journal Browser Search

Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

Title: [Effects of miR-144 on proliferation, apoptosis and cisplatin resistance by targeting MYCN in pediatric neuroblastoma].
Author: Liu HJ, Li GL, Lei PC.
Journal: Zhonghua Zhong Liu Za Zhi; 2019 Jul 23; 41(7):516-521. PubMed ID: 31357838.
Abstract:
Objective: To investigate the effects and mechanisms of miR-144 on proliferation, apoptosis and cisplatin (DDP) resistance of neuroblastoma cells. Methods: Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the mRNA expressions of miR-144 and MYCN in neuroblastoma cell lines, including SH-SY5Y and SK-N-SH, and human umbilical vein endothelial cells HUVEC. The miR-negative control, miR-144 mimics, si-negative control, si-MYCN, miR-144 mimics and pcDNA, miR-144 mimics and pcDNA-MYCN co-transfected SH-SY5Y cells were described as miR-NC, miR-144, si-NC, si-MYCN, miR-144+ pcDNA and miR-144+ pcDNA-MYCN group, respectively. The half maximal inhibitory concentration (IC(50)) and cell proliferation were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. The protein expressions of MYCN, p21, cyclin D1, Bax, Bcl-2 were analyzed by western blot. Cell apoptosis was detected by flow cytometry. The cell fluorescence activity was detected by double luciferase reporter gene assay. Results: Compared with HUVEC cells, the expressions of miR-144 in neuroblastoma cells SH-SY5Y and SK-N-SH significantly decreased, while the mRNA and protein expression of MYCN significantly increased. The IC(50) of DDP was 9.16 μg/ml in SH-SY5Y cells. The absorbance value in 490nm (A(490) value) of miR-144 group was 0.30±0.03, significantly lower than 0.46±0.03 of miR-NC group. The cell apoptotic rate of miR-144 group was 26.94%±2.01%, significantly higher than 9.68%±0.52% of miR-NC group. The IC(50) value of DDP in miR-144 group was 2.95±0.26, significantly lower than 9.23±0.61 of miR-NC group. The expressions of p21, cyclin D1, Bax, Bcl-2 in miR-NC and miR-144 group were 2.67±0.19, 0.41±0.04, 2.12±0.21, 0.18±0.01 and 1.01±0.07, 1.00±0.06, 1.00±0.05, 1.00±0.06, respectively, with statistical significance (all P<0.05). Knockdown of MYCN showed the similar effects with those of miR-144 overexpression in SH-SYSY cells. MiR-144 significantly inhibited the fluorescence activity of ectopic MYCN expressing cells and negatively regulated the expression of MYCN. Overexpression of MYCN can reverse the effects of miR-144 on proliferation inhibition, apoptosis promotion and sensitization of SH-SY5Y cells to DDP. Conclusion: MiR-144 inhibits proliferation, promotes apoptosis and enhances the sensitivity of neuroblastoma cells to DDP through targeting MYCN, which provides a potential treatment for neuroblastoma. 目的： 探讨miR-144对神经母细胞瘤细胞增殖凋亡和顺铂耐药的影响及其机制。 方法： 采用实时荧光定量PCR法检测神经母细胞瘤细胞SH-SY5Y、SK-N-SH和人脐静脉内皮细胞HUVEC中miR-144、MYCN mRNA的表达。将miR-NC、miR-144 mimics、si-NC、si-MYCN、miR-144 mimics＋pcDNA和miR-144 mimics＋pcDNA-MYCN转染至SH-SY5Y细胞，四甲基偶氮唑蓝法检测细胞的半数抑制率(IC(50))和增殖情况，Western blot检测细胞中MYCN、p21、Cyclin D1、Bax和Bcl-2蛋白的表达，流式细胞术检测细胞的凋亡，双荧光素酶报告基因检测实验检测细胞的荧光活性。 结果： 与人脐静脉内皮细胞HUVEC(1.01±0.03)比较，神经母细胞瘤细胞SH-SY5Y中miR-144的表达(0.32±0.03)降低，MYCN mRNA和蛋白的表达水平(分别为2.42±0.18和1.98±0.15)升高，顺铂对SH-SY5Y细胞的IC(50)为9.16 μg/ml。miR-144组和miR-NC组SH-SY5Y细胞在490 nm波长处的吸光度值分别为0.30±0.03和0.46±0.03，凋亡率分别为(26.94±2.01)%和(9.68±0.52)%。顺铂的IC(50)分别为2.95±0.26和9.23±0.61，差异均有统计学意义(均P>0.05)。miR-NC组和miR-144组SH-SY5Y细胞中p21蛋白的表达水平分别为2.67±0.19和1.01±0.07，Cyclin D1蛋白的表达水平分别为0.41±0.04和1.00±0.06，Bax蛋白的表达水平分别为2.12±0.21和1.00±0.05，Bcl-2蛋白的表达水平分别为0.18±0.01和1.00±0.06，差异均有统计学意义(均P<0.05)。敲减原癌基因MYCN与过表达miR-144对SH-SY5Y细胞的作用类似。miR-144可明显抑制野生型MYCN细胞的荧光活性，并负向调控MYCN的表达；过表达MYCN可逆转miR-144对SH-SY5Y细胞的增殖抑制，促进凋亡及对顺铂的增敏作用。 结论： miR-144可抑制神经母细胞瘤细胞的增殖，促进凋亡，增强对顺铂的敏感性，其机制可能与靶向MYCN有关，可为神经母细胞瘤的治疗提供依据。.

[Abstract] [Full Text] [Related] [New Search]