上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2022 , Vol. 42 >Issue 5: 591 - 601

DOI: https://doi.org/10.3969/j.issn.1674-8115.2022.05.006

论著 · 基础研究

lncRNA GK-IT1通过调控醛缩酶A影响非小细胞肺癌细胞的恶性进展

  • 刘子杨 ,
  • 王小文 ,
  • 陈力
展开
  • 重庆医科大学附属第一医院胸心外科，重庆 400016
刘子杨（1996—），男，硕士生；电子信箱：327912537@qq.com。
陈力，电子信箱：chenli@hospital.cqmu.edu.cn。

收稿日期: 2022-02-23

  录用日期: 2022-05-06

  网络出版日期: 2022-05-28

基金资助

国家自然科学基金(81700320);重庆市自然科学基金(CSTC2019JCYJ-MSXMX0827)

收起

lncRNA GK-IT1 influences the carcinogenesis of non-small cell lung cancer cells through regulating aldolase A

  • Ziyang LIU ,
  • Xiaowen WANG ,
  • Li CHEN
Expand
  • Department of Cardiothoracic Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
CHEN Li, E-mail: chenli@hospital.cqmu.edu.cn.

Received date: 2022-02-23

  Accepted date: 2022-05-06

  Online published: 2022-05-28

Supported by

National Natural Science Foundation of China(81700320);Natural Science Foundation of Chongqing(CSTC2019JCYJ-MSXMX0827)

Fold

摘要

目的·探讨长链非编码RNA（long non-coding RNA，lncRNA）GK-IT1通过调控醛缩酶A（aldolase A，ALDOA）对非小细胞肺癌（non-small-cell lung cancer，NSCLC）细胞A549及H358恶性进展的影响。方法·应用实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）检测42对NSCLC组织和癌旁组织以及NSCLC细胞系中GK-IT1的表达。荧光原位杂交（fluorescence in situ hybridization，FISH）实验检测GK-IT1在A549及H358中的亚细胞定位。RNA免疫共沉淀（RNA immunoprecipitation，RIP）检测GK-IT1与ALDOA的相互作用关系。以2条GK-IT1的小干扰RNA序列（Si-GK-IT1 #1和Si-GK-IT1 #2）及空白对照（Si-NC）转染A549及H358细胞，再以Si-GK-IT1 #2和ALDOA过表达质粒共转染上述细胞系。设置Si-NC组、Si-GK-IT1 #1及Si-GK-IT1 #2组，采用CCK-8及EdU实验检测细胞增殖能力，Transwell侵袭实验与细胞划痕实验检测细胞侵袭能力及迁移能力。Western blotting检测各实验组内波形蛋白（vimentin）、E钙黏蛋白（E-cadherin）、N钙黏蛋白（N-cadherin）及ALDOA的表达水平。结果·与正常组织相比，NSCLC组织中GK-IT1的表达量明显升高；与正常支气管上皮细胞BEAS-2B相比，H358、A549细胞中GK-IT1的表达量明显升高（均P<0.05）。FISH实验表明GK-IT1主要位于细胞质；RIP实验及生物信息学分析表明GK-IT1可能与ALDOA蛋白结合；CCK-8实验结果显示，Si-GK-IT1 #1及Si-GK-IT1 #2组较Si-NC组细胞增殖能力明显受到抑制（均P<0.05）；EdU实验结果显示，Si-GK-IT1 #1及Si-GK-IT1 #2组较Si-NC组细胞EdU阳性率显著降低（均P<0.05）；Transwell侵袭实验结果显示，与对照组相比，Si-GK-IT1 #1及Si-GK-IT1 #2组细胞侵袭能力明显受到抑制（均P=0.000）；细胞划痕实验结果显示，Si-GK-IT1 #1及Si-GK-IT1 #2组较Si-NC组细胞迁移率明显降低（均P=0.000）。敲减GK-IT1后，ALDOA、vimentin表达水平降低，E-cadherin表达水平升高。过表达ALDOA可逆转敲低GK-IT1对NSCLC细胞增殖、侵袭、迁移及蛋白表达水平的影响（均P<0.05）。结论·GK-IT1可通过调控ALDOA蛋白促进NSCLC细胞的增殖、侵袭与迁移能力。

关键词： 长链非编码RNA; GK-IT1; 醛缩酶A; 非小细胞肺癌; 细胞侵袭; 细胞迁移

本文引用格式

刘子杨 , 王小文 , 陈力 . lncRNA GK-IT1通过调控醛缩酶A影响非小细胞肺癌细胞的恶性进展[J]. 上海交通大学学报（医学版）, 2022 , 42(5) : 591 -601 . DOI: 10.3969/j.issn.1674-8115.2022.05.006

Abstract

Objective

·To investigate the effects of long non-coding RNA (lncRNA) GK-IT1 on the carcinogenesis of non-small-cell lung cancer (NSCLC) A549 and H358 cells by regulating aldolase A (ALDOA).

Methods

·The expression of GK-IT1 in 42 pairs of NSCLC tissues and adjacent tissues, and NSCLC cell lines were tested by quantitative real-time PCR (qRT-PCR). The subcellular localization of GK-IT1 in A549 and H358 cells was tested by fluorescence in situ hybridization (FISH). RNA immunoprecipitation (RIP) was used to assess the interaction of GK-IT1 and ALDOA. A549 and H358 cells were transfected with GK-IT1 small interfering RNA sequences (Si-GK-IT1 #1 and Si-GK-IT1 #2) and negative control (Si-NC), and then the two cell lines were also cotransfected with Si-GK-IT1 #2 and ALDOA overexpression plasmid. The Si-NC, Si-GK-IT1 #1, Si-GK-IT1 #2 groups were set. CCK-8 and EdU assay were used to detect cell proliferation. Transwell invasion and scratch assay were used to observe cell invasion and migration. Western blotting was carried out to verify the expression of vimentin, E-cadherin, N-cadherin and ALDOA in each experimental group.

Results

·Compared with the normal tissues, the relative expression of GK-IT1 was significantly higher in NSCLC tissues. Compared with BEAS-2B cells, the expression of GK-IT1 in H358 and A549 cells was significantly higher (P<0.05). FISH assay indicated that GK-IT1 was mainly located in the cytoplasm. RIP and bioinformatics analysis suggested that GK-IT1 might interact with ALDOA. Compared with the Si-NC group, the results of CCK-8 assay showed that the cell proliferation of the Si-GK-IT1 #1 and Si-GK-IT1 #2 group was significantly inhibited (P<0.05). The results of EdU assay showed that the ratio of EdU positive cells in the Si-GK-IT1 #1 and Si-GK-IT1 #2 group was significantly lower than that in the Si-NC group (P<0.05). Compared with the Si-NC group, the results of Transwell invasion assay indicated that the invasion of the Si-GK-IT1 #1 and Si-GK-IT1 #2 group was significantly inhibited (all P=0.000). The results of cell scratch assay showed that the healed wound ratio in the Si-GK-IT1 #1 and Si-GK-IT1 #2 group was remarkably lower than that in Si-NC group (all P=0.000). GK-IT1 knockdown significantly decreased the expression of ALDOA and vimentin, while the expression of E-cadherin was increased. Overexpression of ALDOA reversed the effects of GK-IT1 silencing on cell proliferation, invasion, migration and protein expression of NSCLC cells (all P<0.05).

Conclusion

·GK-IT1 could promote the proliferation, invasion and migration of NSCLC cells via regulating ALDOA.

Key words： long non-coding RNA (lncRNA); GK-IT1; aldolase A (ALDOA); non-small cell lung cancer (NSCLC); cell invasion; cell migration

参考文献

1 CHEN Z Y, LEI T Y, CHEN X, et al. Long non-coding RNA in lung cancer[J]. Clin Chim Acta, 2020, 504: 190-200.
2 刘宗超, 李哲轩, 张阳, 等. 2020全球癌症统计报告解读[J]. 肿瘤综合治疗电子杂志, 2021, 7(2): 1-14.
2 LIU Z C, LI Z X, ZHANG Y, et al. Interpretation on the report of Global Cancer Statistics 2020[J]. J Multidiscip Cancer Manag (Electron Version), 2021, 7(2): 1-14.
3 LU W, ZHANG H H, NIU Y Q, et al. Long non-coding RNA linc00673 regulated non-small cell lung cancer proliferation, migration, invasion and epithelial mesenchymal transition by sponging miR-150-5p[J]. Mol Cancer, 2017, 16(1): 118.
4 CHANG Y C, YANG Y C, TIEN C P, et al. Roles of aldolase family genes in human cancers and diseases[J]. Trends Endocrinol Metab, 2018, 29(8): 549-559.
5 ZHANG F, LIN J D, ZUO X Y, et al. Elevated transcriptional levels of aldolase A (ALDOA) associates with cell cycle-related genes in patients with NSCLC and several solid tumors[J]. BioData Min, 2017, 10: 6.
6 CUI K, JIN S L, DU Y B, et al. Long noncoding RNA DIO3OS interacts with miR-122 to promote proliferation and invasion of pancreatic cancer cells through upregulating ALDOA[J]. Cancer Cell Int, 2019, 19: 202.
7 SUN J, HE D, FU Y B, et al. A novel lncRNA ARST represses glioma progression by inhibiting ALDOA-mediated actin cytoskeleton integrity[J]. J Exp Clin Cancer Res, 2021, 40(1): 187.
8 XIAO Y H, SU M, OU W, et al. Involvement of noncoding RNAs in epigenetic modifications of esophageal cancer[J]. Biomed Pharmacother, 2019, 117: 109192.
9 SUZUKI H, MARUYAMA R, YAMAMOTO E, et al. Relationship between noncoding RNA dysregulation and epigenetic mechanisms in cancer[J]. Adv Exp Med Biol, 2016, 927: 109-135.
10 KHORKOVA O, HSIAO J, WAHLESTEDT C. Basic biology and therapeutic implications of lncRNA[J]. Adv Drug Deliv Rev, 2015, 87: 15-24.
11 DOU Q R, XU Y, ZHU Y Y, et al. lncRNA FAM83H-AS1 contributes to the radioresistance, proliferation, and metastasis in ovarian cancer through stabilizing HuR protein[J]. Eur J Pharmacol, 2019, 852: 134-141.
12 SHEN L, HAN J, WANG H Y, et al. Cachexia-related long noncoding RNA, CAAlnc1, suppresses adipogenesis by blocking the binding of HuR to adipogenic transcription factor mRNAs[J]. Int J Cancer, 2019, 145(7): 1809-1821.
13 YANG Y X, CHEN D, LIU H, et al. Increased expression of lncRNA CASC9 promotes tumor progression by suppressing autophagy-mediated cell apoptosis via the AKT/mTOR pathway in oral squamous cell carcinoma[J]. Cell Death Dis, 2019, 10(2): 41.
14 杨鑫, 陈力. 敲低长链非编码RNA GK-IT1抑制食管鳞状癌细胞增殖和促进凋亡[J]. 第三军医大学学报, 2021, 43(2): 146-155.
14 YANG X, CHEN L. Knock-down of long non-coding RNA GK-IT1 inhibits proliferation and promotes apoptosis in esophageal carcinoma cells[J]. J Army Med Univ, 2021, 43(2): 146-155.
15 PORTO F W, DAULATABAD S V, JANGA S C. Long non-coding RNA expression levels modulate cell-type-specific splicing patterns by altering their interaction landscape with RNA-binding proteins[J]. Genes, 2019, 10(8): 593.
16 CHEN J, WANG R, ZHANG K, et al. Long non-coding RNAs in non-small cell lung cancer as biomarkers and therapeutic targets[J]. J Cell Mol Med, 2014, 18(12): 2425-2436.
17 LI L, ZHANG K J, LU C H, et al. Caveolin-1-mediated STAT3 activation determines electrotaxis of human lung cancer cells[J]. Oncotarget, 2017, 8(56): 95741-95754.
18 LI J, HE M R, XU W, et al. LINC01354 interacting with hnRNP-D contributes to the proliferation and metastasis in colorectal cancer through activating Wnt/β-catenin signaling pathway[J]. J Exp Clin Cancer Res, 2019, 38(1): 161.
19 FU H L, GAO H J, QI X Y, et al. Aldolase A promotes proliferation and G1/S transition via the EGFR/MAPK pathway in non-small cell lung cancer[J]. Cancer Commun (Lond), 2018, 38(1): 18.
21 DU S, GUAN Z Z, HAO L H, et al. Fructose-bisphosphate aldolase A is a potential metastasis-associated marker of lung squamous cell carcinoma and promotes lung cell tumorigenesis and migration[J]. PLoS One, 2014, 9(1): e85804.
22 LU Y J, LIU X, XIE M H, et al. The NF-κB-responsive long noncoding RNA FIRRE regulates posttranscriptional regulation of inflammatory gene expression through interacting with hnRNPU[J]. J Immunol, 2017, 199(10): 3571-3582.
23 TULCHINSKY E, DEMIDOV O, KRIAJEVSKA M, et al. EMT: a mechanism for escape from EGFR-targeted therapy in lung cancer[J]. Biochim Biophys Acta Rev Cancer, 2019, 1871(1): 29-39.
25 HE R, ZHANG F H, SHEN N. lncRNA FEZF1-AS1 enhances epithelial-mesenchymal transition (EMT) through suppressing E-cadherin and regulating WNT pathway in non-small cell lung cancer (NSCLC)[J]. Biomed Pharmacother, 2017, 95: 331-338.
26 LIU B, Sun L J, LIU Q, et al. A cytoplasmic NF-κB interacting long noncoding RNA blocks IκB phosphorylation and suppresses breast cancer metastasis[J]. Cancer Cell, 2015, 27(3):370-381.
27 YANG F, ZHANG H F, MEI Y D, et al. Reciprocal regulation of HIF-1α and lincRNA-p21 modulates the Warburg effect[J]. Mol Cell, 2014, 53(1): 88-100.
Options
文章导航

/

〈 〉