LINC01006对肾母细胞瘤细胞增殖、侵袭和凋亡的作用及其机制

赵婷; 刘安生; 王华; 傅蔷

doi:10.16016/j.1000-5404.201912296

您当前的位置：

首页 >

文章列表页 >

LINC01006对肾母细胞瘤细胞增殖、侵袭和凋亡的作用及其机制

临床医学 | 更新时间：2026-01-09

- LINC01006对肾母细胞瘤细胞增殖、侵袭和凋亡的作用及其机制
- LINC01006 inhibits proliferation and invasion and promotes apoptosis of nephroblastoma cells by down-regulating miR-148a-3p to block MEOX2/PI3K/Akt pathway
- 第三军医大学学报 2020年42卷第12期页码：1225-1235
- 作者机构：
  
  710003 西安, 西安市儿童医院血液肿瘤科
- 作者简介：
  
  [赵婷, E-mail:tingzhanghb@sina.com ZHAO Ting, E-mail:tingzhanghb@sina.com
- 基金信息：
- DOI：10.16016/j.1000-5404.201912296
  中图分类号： R394.3;R730.23;R737.11
- 收稿：2019-12-31，
  
  修回：2020-03-13，
  
  纸质出版：2020-06-30
- 稿件说明：
移动端阅览
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.

Ting ZHAO, Ansheng LIU, Hua WANG, 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]. Journal of Third Military Medical University, 2020, 42(12): 1225-1235.
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. DOI： 10.16016/j.1000-5404.201912296.

Ting ZHAO, Ansheng LIU, Hua WANG, 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]. Journal of Third Military Medical University, 2020, 42(12): 1225-1235. DOI： 10.16016/j.1000-5404.201912296.

摘要

目的

段探讨长链非编码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(

0.01)。LINC01006可抑制肾母细胞瘤细胞的活力(

0.01)、体外增殖能力(

0.01)和侵袭能力[(166.00 ± 13.75)

(103.00 ± 9.24)

0.05

]

促进细胞凋亡[(11.30 ±0.24)

(37.25±1.57)

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)

(0.32±0.03)

0.01

]

而抑制miR-148a-3p的表达促进了MEOX2 mRNA和蛋白水平的表达(

0.01)

进而抑制了PI3K/Akt信号通路的活性(

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(

0.01)

促进了PI3K/Akt信号通路的活

性(

0.01)

细胞活力、增殖和侵袭能力均显著提高(

0.01)

细胞凋亡能力降低(

0.01)。此外

过表达LINC01006抑制了裸鼠移植瘤的生长(

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

Objective

To investigate the role of long noncoding RNA LINC01006 in regulating the proliferation

invasion and apoptosis of nephroblastoma cells and explore the molecular mechanism.

Methods

We 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.

Results

The nephroblastoma tissues and cell lines expressed significantly lower level of

LINC01006 than normal tissues and control cell lines (

0.01).LINC01006 over-expression significantly inhibited the viability (

0.01)

proliferation (

0.01) and invasion (166.00±13.75

103.00±9.24

0.05) and promoted apoptosis (11.30 ± 0.24

37.25 ± 1.57

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

0.32±0.03

0.01)

while inhibiting miR-148a-3p promoted the expression of MEOX2 (

0.01)and inhibited the activation of the PI3K /Akt signaling pathway (

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 (

0.01) with enhanced activity of PI3K /Akt signaling pathway (

0.01)

significantly increased cell viability

proliferation and invasion (

0.01)

and decreased cell apoptosis (

0.01).Overexpression of LINC01006 in the tumor cells significantly inhibited the growth of the transplanted tumors in nude mice (

0.01).

Conclusion

LINC01006 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.

关键词

Keywords

references

VERSCHUUR A C, VUJANIC G M, VAN TINTEREN H, et al . Stromal and epithelial predominant Wilms tumours have an excellent outcome: The SIOP 93 01 experience[J ] . Pediatr Blood Cancer, 2010, 55(2): 233-238. DOI: 10.1002/pbc.22496.

GUTSCHNER T, DIEDERICHS S. The hallmarks of cancer: a long non-coding RNA point of view[J]. RNA Biol, 2012, 9(6): 703-719. DOI: 10.4161/rna.20481.

LIU Y F, YAU M S, YERGES-ARMSTRONG L M, et al . Genetic determinants of radiographic knee osteoarthritis in African Americans[J ] . J Rheumatol, 2017, 44(11): 1652-1658. DOI: 10.3899/jrheum.161488.

ZHU X Q , CHEN F, SHAO Y F, et al . Long intergenic non-protein coding RNA 1006 used as a potential novel biomarker of gastric cancer[J ] . Cancer Biomark, 2017, 21(1): 73-80. DOI: 10.3233/CBM-170273.

ISRAELS T, PIDINI D, BORGSTEIN E, et al . Survival of children with a wilms tumor in Blantyre, Malawi[J ] . Pediatr Hematol Oncol, 2018, 35(3): 196-202. DOI: 10.1080/08880018.2018.1498564.

VUJANIC′ G M, D'HOOGHE E, POPOV S D, et al . The effect of preoperative chemotherapy on histological subtyping and staging of Wilms tumors: The United Kingdom Children's Cancer Study Group (UKCCSG) Wilms tumor trial 3 (UKW3) experience[J ] . Pediatr Blood Cancer, 2019, 66(3): e27549. DOI: 10.1002/pbc.27549.

PONTING C P, OLIVER P L, REIK W. Evolution and functions of long noncoding RNAs[J]. Cell, 2009, 136(4): 629-641. DOI: 10.1016/j.cell.2009.02.006.

QUINN J J, CHANG H Y. Unique features of long non-coding RNA biogenesis and function[J]. Nat Rev Genet, 2016, 17(1): 47-62. DOI: 10.1038/nrg.2015.10.

WU Y L, WANG W J, JIANG W J, et al . An investigation of obesity susceptibility genes in Northern Han Chinese by targeted resequencing[J ] . Medicine (Baltimore), 2017, 96(7): e6117. DOI: 10.1097/MD.0000000000006117.

ZHANG L Y, WANG Y J, ZHANG L, et al . LINC01006 promotes cell proliferation and metastasis in pancreatic cancer via miR-2682-5p/HOXB8 Axis[J ] . Cancer Cell Int, 2019, 19: 320. DOI: 10.1186/s12935-019-1036-2.

TAY Y, RINN J, PANDOLFI P P. The multilayered complexity of Cerna crosstalk and competition[J]. Nature, 2014, 505(7483): 344-352. DOI: 10.1038/nature12986.

HA M J, KIM V N. Regulation of microRNA biogenesis[J]. Nat Rev Mol Cell Biol, 2014, 15(8): 509-524. DOI: 10.1038/nrm3838.

BARTEL D P. MicroRNAs: target recognition and regulatory functions[J]. Cell, 2009, 136(2): 215-233. DOI: 10.1016/j.cell.2009.01.002.

HEZOVA R, KOVARIKOVA A, SROVNAL J, et al . Diagnostic and prognostic potential of miR-21, miR-29c, miR-148 and miR-203 in adenocarcinoma and squamous cell carcinoma of esophagus[J ] . Diagn Pathol, 2015, 10: 42. DOI: 10.1186/s13000-015-0280-6.

ZHOU X, ZHAO F, WANG Z N, et al . Altered expression of miR-152 and miR-148a in ovarian cancer is related to cell proliferation[J ] . Oncol Rep, 2012, 27(2): 447-454. DOI: 10.3892/or.2011.1482.

SAKAMOTO N, NAITO Y, OUE N, et al . MicroRNA-148a is downregulated in gastric cancer, targets MMP7, and indicates tumor invasiveness and poor prognosis[J ] . Cancer Sci, 2014, 105(2): 236-243. DOI: 10.1111/cas.12330.

WANG S H, LIX, ZHOU L S, et al . MicroRNA-148a suppresses human gastric cancer cell metastasis by reversing epithelial-to-mesenchymal transition[J ] . Tumour Biol, 2013, 34(6): 3705-3712. DOI: 10.1007/s13277-013-0954-1.

YU B Q, LV X, SU L P, et al . MiR-148a functions as a tumor suppressor by targeting CCK-BR via inactivating STAT3 and akt in human gastric cancer[J ] . PLoS ONE, 2016, 11(8): e0158961. DOI: 10.1371/journal.pone.0158961.

HUANG Y S, DAI Y, YU X F, et al . Microarray analysis of microRNA expression in hepatocellular carcinoma and non-tumorous tissues without viral hepatitis[J ] . J Gastroenterol Hepatol, 2008, 23(1): 87-94. DOI: 10.1111/j.1440-1746.2007.05223.x.

BAYRAK O F, GULLUOGLU S, AYDEMIR E, et al . MicroRNA expression profiling reveals the potential function of microRNA-31 in chordomas[J ] . J Neurooncol, 2013, 115(2): 143-151. DOI: 10.1007/s11060-013-1211-6.

WU Y, YU J, MA Y N, et al . MiR-148a and miR-375 may serve as predictive biomarkers for early diagnosis of laryngeal carcinoma[J ] . Oncol Lett, 2016, 12(2): 871-878. DOI: 10.3892/ol.2016.4707.

ZHANG H, WANG Y, XU T M, et al . Increased expression of microRNA-148a in osteosarcoma promotes cancer cell growth by targeting PTEN[J ] . Oncol Lett, 2016, 12(5): 3208-3214. DOI: 10.3892/ol.2016.5050.

KIM J, ZHANG Y, SKALSKI M, et al . MicroRNA-148a is a prognostic oncomiR that targets MIG6 and BIM to regulate EGFR and apoptosis in glioblastoma[J ] . Cancer Res, 2014, 74(5): 1541-1553. DOI: 10.1158/0008-5472.CAN-13-1449.

CUI D M, SAJANP, SHI J L, et al . MiR-148a increases glioma cell migration and invasion by downregulating GADD45A in human gliomas with IDH1 R132H mutations[J ] . Oncotarget, 2017, 8(15): 25345-25361. DOI: 10.18632/oncotarget.15867.

CAO G, HUANG B, LIU Z, et al . Intronic miR-301 feedback regulates its host gene, ska2, in A549 cells by targeting MEOX2 to affect ERK/CREB pathways[J ] . Biochem Biophys Res Comm, 2010, 396(4): 978-982. DOI: 10.1016/j.bbrc.2010.05.037.

TIAN L, TAO Z Z, YE H P, et al . Over-expression of MEOX2 promotes apoptosis through inhibiting the PI3K/Akt pathway in laryngeal cancer cells[J ] . Neoplasma, 2018, 65(5): 745-752. DOI: 10.4149/neo_2018_171218N824.

OHSHIMA J, HARUTA M, ARAI Y, et al . Two candidate tumor suppressor genes, MEOX2 and SOSTDC1, identified in a 7p21 homozygous deletion region in a Wilms tumor[J ] . Gene Chromosome Cancer, 2009, 48(12): 1037-1050. DOI: 10.1002/gcc.20705.

LIU P, KONG F, WANG J, et al . Involvement of IGF-1 and MEOX2 in PI3K/Akt1/2 and ERK1/2 pathways mediated proliferation and differentiation of perivascular adipocytes[J ] . Exp Cell Res, 2015, 331(1): 82-96. DOI: 10.1016/j.yexcr.2014.09.011.

KWAK D H, LEE J H, KIM D G, et al . Inhibitory effects of hwangryunhaedok-Tang in 3T3-L1 adipogenesis by regulation of raf/MEK1/ERK1/2 pathway and PDK1/akt phosphorylation[J ] . Evid Based Complement Alter nat Med, 2013, 2013: 413906. DOI: 10.1155/2013/413906.

CLEVELAND-DONOVAN K, MAILE L A, TSIARAS W G, et al . IGF-I activation of the AKT pathway is impaired in visceral but not subcutaneous preadipocytes from obese subjects[J ] . Endocrinology, 2010, 151(8): 3752-3763. DOI: 10.1210/en.2010-0043.

TSUCHIYA A, KANNO T, NISHIZAKI T. PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway[J]. J Endocrinol, 2014, 220(1): 49-59. DOI: 10.1530/JOE-13-0172.

CHIAPPINI P B O, DE MEDEIROS I U D, LIMA L G C, et al . Prognostic implications of phosphatidylinositol 3-kinase/AKT signaling pathway activation in gastric carcinomas[J ] . Arch Med Sci, 2017, 13(6): 1262-1268. DOI: 10.5114/aoms.2016.60394.

LIU Z, WANG F, ZHOU Z W, et al . Alisertib induces G2/M arrest, apoptosis, and autophagy via PI3K/Akt/mTOR- and p38 MAPK-mediated pathways in human glioblastoma cells[J ] . Am J Transl Res, 2017, 9(3): 845-873.

WANG H, DENG X B, ZHANG J S, et al . Elevated expression of zinc finger protein 703 promotes cell proliferation and metastasis through PI3K/AKT/GSK-3β signalling in oral squamous cell carcinoma[J ] . Cell Physiol Biochem, 2017, 44(3): 920-934. DOI: 10.1159/000485360.

GARCÍA-CARRACEDO D, VILLARONGA M Á, ÁLVAREZ-TEIJEIRO S, et al . Impact of PI3K/AKT/mTOR pathway activation on the prognosis of patients with head and neck squamous cell carcinomas[J ] . Oncotarget, 2016, 7(20): 29780-29793. DOI: 10.18632/oncotarget.8957.

浏览量

下载量

CSCD

文章被引用时，请邮件提醒。

提交

工具集

关联资源

丙戊酸钠通过激活miR-148a-3p/Mcl-1通路促进SH-SY5Y细胞凋亡

MiR-616在骨肉瘤中的表达及对其细胞增殖、凋亡、迁移侵袭的作用

Gm49394对胰岛β细胞凋亡的调控及机制研究

Kallistatin诱导SIRT1表达抑制人髓核细胞凋亡及细胞外基质降解

千金藤素协同多柔比星逆转K562/ADR耐药的分子机制研究