[1]赵婷,刘安生,王华,等.LINC01006对肾母细胞瘤细胞增殖、侵袭和凋亡的作用及其机制[J].第三军医大学学报,2020,42(12):1225-1235.
 HAO Ting,LIU Ansheng,WANG Hua,et al.LINC01006 inhibits proliferation and invasion and promotes apoptosis of nephroblastoma cells by down-regulating miR-148a-3p to block MEOX2/PI3K/Akt pathway[J].J Third Mil Med Univ,2020,42(12):1225-1235.
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LINC01006对肾母细胞瘤细胞增殖、侵袭和凋亡的作用及其机制(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
42卷
期数:
2020年第12期
页码:
1225-1235
栏目:
临床医学
出版日期:
2020-06-30

文章信息/Info

Title:
LINC01006 inhibits proliferation and invasion and promotes apoptosis of nephroblastoma cells by down-regulating miR-148a-3p to block MEOX2/PI3K/Akt pathway
作者:
赵婷刘安生王华傅蔷
西安市儿童医院血液肿瘤科
 
Author(s):
HAO Ting LIU Ansheng WANG Hua FU Qiang

Department of Hematology, Xi’an Children’s Hospital, Xi’an, Shaanxi Province, 710003, China

关键词:
肾母细胞瘤LINC01006miR-148a-3pMEOX2/PI3K/Akt通路增殖与侵袭凋亡
Keywords:
nephroblastoma LINC01006 miR-148a-3p MEOX2/PI3K/Akt pathway proliferation and invasion apoptosis
分类号:
R394.3;R730.23;R737.11
文献标志码:
A
摘要:

目的探讨长链非编码RNA LINC01006对肾母细胞瘤细胞增殖、侵袭和凋亡的影响及分子机制。方法通过实时定量PCR检测肾母细胞瘤组织、细胞系中LINC01006的表达。在人肾母细胞瘤细胞中转染LINC01006的腺病毒过表达载体和siRNA,分别采用CCK-8、5-溴脱氧尿嘧啶核苷(5-bromo-2-deoxyuridine, BrdU)细胞增殖检测试剂盒、Transwell侵袭实验、流式细胞术检测细胞活力、增殖、侵袭及凋亡能力的变化。通过生物信息学分析预测LINC01006的靶基因, 双荧光素酶报告基因分析验证预测结果;检测过表达和敲低LINC01006后miR-148a-3p及其靶基因中胚层同源盒基因(mesoderm homoobox2, MEOX2)表达水平的变化。随后,分别在裸鼠皮下注射转染腺病毒空载体和LINC01006过表达载体的肾母细胞瘤细胞系,建立小鼠移植瘤模型,测定移植瘤的体积及质量,以及肿瘤组织中LINC01006、miR-148a-3p、MEOX2和磷酸化的磷脂酰肌醇-3激酶(phosphorylated-phosphatidylinositol-3 kinase, p-PI3K)、磷酸化的丝氨酸/苏氨酸激酶,即蛋白激酶B(phosphorylated-protein kinase B, p-PKB/p-Akt)的表达。结果肾母细胞瘤组织和细胞系中LINC01006的表达水平显著低于对照组织和细胞(P<0.01)。LINC01006可抑制肾母细胞瘤细胞的活力(P<0.01)、体外增殖能力(P<0.01)和侵袭能力[(166.00±13.75) vs (103.00±9.24), P<0.05],促进细胞凋亡[(11.30±0.24) vs (37.25±1.57), P<0.01]。双荧光素酶报告基因实验证实LINC01006与miR-148a-3p直接结合,而MEOX2是miR-148a-3p的靶基因。过表达LINC01006抑制了miR-148a-3p的表达[(1.07±0.02) vs (0.32±0.03), P<0.01],而抑制miR-148a-3p的表达促进了MEOX2 mRNA和蛋白水平的表达(P<0.01),进而抑制了PI3K/Akt信号通路的活性(P<0.05)。与转染pcDNA-LINC01006组相比,共转染LINC01006和miR-148a-3p mimic组中MEOX2的表达下调(P<0.01),促进了PI3K/Akt信号通路的活性(P<0.01),细胞活力、增殖和侵袭能力均显著提高(P<0.01),细胞凋亡能力降低(P<0.01)。此外,过表达LINC01006抑制了裸鼠移植瘤的生长(P<0.01)。结论LINC01006可直接与miR-148a-3相互作用并下调其表达水平,从而阻断MEOX2/PI3K/Akt通路的活性,抑制肾母细胞瘤细胞的增殖和侵袭,促进细胞凋亡,抑制肿瘤的生长。

Abstract:

ObjectiveTo investigate the role of long noncoding RNA LINC01006 in regulating the proliferation, invasion and apoptosis of nephroblastoma cells and explore the molecular mechanism. MethodsWe detected the expression of LINC01006 in nephroblastoma tissues and cell lines using real-time quantitative PCR (RT-qPCR). We subsequently tested the effects of transfection with an adenovirus vector for LINC01006 overexpression or a siRNA targeting LINC01006 on cell viability, proliferation, invasion and apoptosis in human nephroblastoma cells using cell counting kit-8 (CCK-8), 5-bromo-2-deoxyuridine (BrdU) cell proliferation assay kit, Transwell assay or flow cytometry as appropriate. Bioinformatics analysis was conducted to identify the target gene of LINC01006, and the results were verified by luciferase reporter gene analysis. The expression of miR-148a-3p and its target gene mesoderm homeobox2 (MEOX2) was detected in human nephroblastoma cells with overexpression and knockdown of LINC01006. The tumor cell lines transfected with an empty adenovirus vector or a LINC01006-overexpressing vector were injected subcutaneously into nude mice to establish mouse models bearing transplanted nephroblastoma, in which the tumor growth dynamics were observed. The expression levels of LINC01006, miR-148a-3p, MEOX2, phosphorylated-phosphatidylinositol-3 kinase (p-PI3K), and phosphorylated-protein kinase B (p-PKB/p-Akt) were examined in the tumor tissues. ResultsThe nephroblastoma tissues and cell lines expressed significantly lower level of LINC01006 than normal tissues and control cell lines (P<0.01). LINC01006 over-expression significantly inhibited the viability (P<0.01), proliferation (P<0.01) and invasion (166.00±13.75 vs 103.00±9.24, P<0.05) and promoted apoptosis (11.30±0.24 vs 37.25±1.57, P<0.01) of nephroblastoma cells in vitro. Luciferase reporter gene analysis confirmed that LINC01006 bound directly to miR-148a-3p, which targets MEOX2 gene. Overexpression of LINC01006 obviously inhibited the expression of miR-148a-3p (1.07±0.02 vs 0.32±0.03, P<0.01), while inhibiting miR-148a-3p promoted the expression of MEOX2 (P<0.01) and inhibited the activation of the PI3K/Akt signaling pathway (P<0.05). Compared with the cells transfected with pcDNA-LINC01006, the cells co-transfected with LINC01006 and miR-148a-3p exhibited a lowered expression of MEOX2 (P<0.01) with enhanced activity of PI3K/Akt signaling pathway (P<0.01), significantly increased cell viability, proliferation and invasion (P<0.01), and decreased cell apoptosis (P<0.01). Overexpression of LINC01006 in the tumor cells significantly inhibited the growth of the transplanted tumors in nude mice (P<0.01). ConclusionLINC01006 can directly interact with miR-148a-3p to down-regulate the expression of the latter, thus blocking the activity of MEOX2/PI3K/Akt pathway to inhibit the proliferation and invasion and promote apoptosis of nephroblastoma cells.

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更新日期/Last Update: 2020-06-19