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LINC00858 knockdown inhibits gastric cancer cell growth and induces apoptosis through reducing WNK2 promoter methylation

Cellular Oncology volume 43, pages 709–723 (2020)Cite this article

A Correction to this article was published on 19 November 2020

This article has been updated

Abstract

Background

Emerging evidence indicates a regulatory role of long non-coding RNAs (lncRNAs) in the development of gastric cancer (GC), but the mechanisms underlying their function have remained largely unknown. Recent microarray-based expression profiling has led to the identification of a novel differentially expressed lncRNA, LINC00858, in GC. Subsequently, LINC00858 was found to be highly expressed in GC tissues and cells. This study was designed to clarify the functional role of LINC00858 in GC, including its effect on methylation of the WNK2 gene promoter and its downstream MAPK signaling pathway.

Methods

After exogenous over-expression and knockdown of LINC00858 and the addition of a MAPK pathway inhibitor in GC cells, we explored the effects of LINC00858 and the MAPK signaling pathway on GC cell behavior using various in vitro and in vivo assays.

Results

LINC00858 was found to negatively regulate WNK2 expression by enhancing its promoter methylation and to activate the MAPK signaling pathway. Moreover, we found that knockdown of LINC00858 or inhibition of the MAPK signaling pathway resulted in decreased GC cell growth, migration and invasion, as well as decreased cell cycle progression, along with increased apoptosis and decreased tumorigenicity.

Conclusions

Together, these findings indicate that silencing of LINC00858 increases WNK2 expression and inhibits the MAPK signaling pathway, thereby inhibiting GC growth and development. Our data highlight LINC00858 as a potential target in GC therapy.

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Change history

  • 19 November 2020

    A Correction to this paper has been published: <ExternalRef><RefSource>https://doi.org/10.1007/s13402-020-00574-w</RefSource><RefTarget Address="10.1007/s13402-020-00574-w" TargetType="DOI"/></ExternalRef>

Abbreviations

GC:

gastric cancer

WNK2:

WNK lysine deficient protein kinase 2

MAPK:

mitogen-activated protein kinase

lncRNAs:

long non-coding RNAs

ERK:

extracellular signal-regulated kinase

JNK:

Jun amino-terminal kinase

CASC2:

cancer susceptibility 2

GEO:

Gene Expression Omnibus

OS:

overall survival

ATCC:

American Type Culture Collection

RPMI:

Roswell Park Memorial Institute

RT-qPCR:

reverse transcription quantitative polymerase chain reaction

shRNA:

short hairpin RNAs

NC:

negative control

cDNA:

complementary DNA

WT:

wild-type

MUT:

mutant

UV:

ultraviolet-visible

MSP:

methylation-specific polymerase chain reaction

EB:

ethidium bromide

SDS:

sodium dodecyl sulfate

PVDF:

polyvinylidene fluoride

IgG:

immunoglobulin G

ECL:

enhanced chemiluminescence

GAPDH:

glyceraldehyde-3-phosphate dehydrogenase

MTT:

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide

OD:

optical density

PI:

propidium iodide

EDTA:

ethylenediaminetetraacetic acid

FITC:

fluorescein isothiocyanate

PBS:

phosphate-buffered saline

DMEM:

Dulbecco’s modified Eagle’s medium

ECM:

extracellular matrix

ANOVA:

analysis of variance

NCBI:

National Center for Biotechnology Information

UCSC:

University of California Santa Cruz.

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Acknowledgements

We acknowledge and appreciate our colleagues for their valuable suggestions and technical assistance for this study.

Funding

This study was supported by a China Postdoctoral Science Foundation Grant (2019 M661171).

Author information

Affiliations

  1. Department of Pathology, The First Affiliated Hospital and College of Basic Medical Science, China Medical University, Shenyang, 110001, People’s Republic of China

    Jiang Du, Ji Li, Jin-Ming Zhao & Xu-Yong Lin

  2. Medical Oncology Department of Thoracic Cancer (2), Cancer Hospital of China Medical University, Liaoning Cancer Hospital & Institute, Shenyang, 110042, People’s Republic of China

    Yuan Liang

Authors
  1. Jiang Du
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  3. Ji Li
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Correspondence to Xu-Yong Lin.

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Electronic supplementary material

Supplementary Fig. 1
figure 10

Silencing of LINC00858 blocks activation of the MAPK signaling pathway in KATO III cells. A, protein bands of WNK2, ERK1/2, p-ERK1/2, p38 and p-p38 in KATO III cells determined by Western blot analysis. B, protein levels of WNK2 in KATO III cells determined by Western blot analysis. C, protein levels of ERK1/2 in KATO III cells determined by Western blot analysis. D, extent of ERK1/2 phosphorylation in KATO III cells determined by Western blot analysis. E, protein levels of p38 in KATO III cells determined by Western blot analysis. F, extent of p38 phosphorylation in KATO III cells determined by Western blot analysis. Data were expressed as mean ± standard deviation from three independent experiments, and analyzed using one-way ANOVA. *p < 0.05 vs. the un-transduced KATO III cells (PNG 1173 kb)

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Supplementary Fig. 2
figure 11

LINC00858 silencing delays KATO III cell growth in GC via inhibition of the MAPK signaling pathway. A, OD values in KATO III cells at 0, 24, 48, and 72 h post culture measured by MTT assay. B, representative images of colony formation images of KATO III cells. C, quantitative analysis for the number of cell colonies. Data were expressed as mean ± standard deviation from three independent experiments. Data among multiple groups were compared by one-way ANOVA while those at different time points were compared by repeated measures ANOVA. MTT assay was used to measure the OD value at a wavelength of 490 nm at 0, 24, 48, and 72 h of KATO III cells in each group. *p < 0.05 vs. the un-transduced KATO III cells (PNG 525 kb)

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Supplementary Fig. 3
figure 12

LINC00858 silencing promotes apoptosis and cell cycle arrest of KATO III cells via inhibition of the MAPK signaling pathway. A and B, the apoptosis conditions and quantitation of apoptosis rates of KATO III cells measured by flow cytometry. C and D, KATO III cell cycle progression detected using PI single staining. Data were expressed as mean ± standard deviation from three independent experiments and analyzed by one-way ANOVA. *p < 0.05 vs. un-transduced KATO III cells (PNG 1711 kb)

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Supplementary Fig. 4
figure 13

LINC00858 silencing attenuates migration and invasion of KATO III cells via inhibition of the MAPK signaling pathway. A, the number of KATO III cells migrated to the basolateral chamber assessed by transwell assay. B, the number of KATO III cells invaded to the basolateral chamber assessed by transwell assay. Data were expressed as mean ± standard deviation from three independent experiments and tested by one-way ANOVA; *p < 0.05 vs. un-transduced KATO III cells (PNG 1304 kb)

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Du, J., Liang, Y., Li, J. et al. LINC00858 knockdown inhibits gastric cancer cell growth and induces apoptosis through reducing WNK2 promoter methylation. Cell Oncol. 43, 709–723 (2020). https://doi.org/10.1007/s13402-020-00518-4

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  • DOI: https://doi.org/10.1007/s13402-020-00518-4

Keywords

  • Gastric cancer
  • Long non-coding RNA
  • LINC00858
  • WNK lysine deficient protein kinase 2
  • MAPK signaling pathway