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RUNX1-activated upregulation of lncRNA RNCR3 promotes cell proliferation, invasion, and suppresses apoptosis in colorectal cancer via miR-1301-3p/AKT1 axis in vitro and in vivo

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Abstract

Purpose

Long non-coding RNAs (lncRNAs) have participated in progression of colorectal cancer. This study aims to study the role of RUNX1/RNCR3/miR-1301-3p/AKT1 axis in colorectal cancer.

Methods

The cancer tissues were from patients with colorectal cancer. The qRT-PCR was used to determine expression of lncRNA RNCR3, miR-1301-3p, and AKT1. Both dual-luciferase reporter assay and ChIP assay were conducted to investigate the binding sites of RUNX1 on RNCR3 promoter. Western blot was performed to analyze expression of AKT1 protein. Both dual-luciferase reporter assay and RIP assay were performed to detect the interacting sites between RNCR3 and miR-1301-3p. The CCK-8 assay, soft agar assay, transwell assay, and annexin-V-FITC/PI staining were applied to analyze the cell growth, invasion, and apoptosis, respectively.

Results

The data demonstrated that RNCR3 was elevated in colorectal cancer, and it was negatively correlated with expression of miR-1301-3p which was decreased in cancers. Then, RNCR3 could interact with and suppress miR-1301-3p expression in HCT116 and SW480. Knockdown of RNCR3 or miR-1301-3p overexpression significantly inhibited cell growth, invasion, and increased apoptosis through suppressing expression of Cyclin A1, PCNA, N-cadherin, Bcl-2, and promoting expression of E-cadherin, Bax in vitro and in vivo. RUNX1 was directly bound to RNCR3 promoter to activate RNCR3 expression. Furthermore, overexpression of RNCR3 blocked tumor inhibitory effects of miR-1301-3p on proliferation, colony formation, invasion, and apoptosis in vitro and in vivo. Additionally, RNCR3 and miR-1301-3p synergistically modulated AKT1 expression.

Conclusion

RUNX1-activated upregulation of RNCR3 promoted colorectal cancer progression by sponging miR-1301-3p to elevate AKT1 levels in vitro and in vivo.

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Abbreviations

lncRNAs:

Long non-coding RNAs

CRC:

Colorectal cancer

ncRNAs:

Non-coding RNAs

lncRNAs:

Long non-coding RNAs

miRNAs:

MicroRNAs

NBR2:

Neighbor of BRCA1 lncRNA 2

TTN-AS1:

TTN antisense RNA 1

KLF15:

Kruppel-like factor 15

ROR1-AS1:

LncRNA ROR1 antisense RNA 1

MIR17HG:

MiR-17-92a-1 cluster host gene

GAS5:

Growth arrest specific 5

SLCO4A1-AS1:

SLCO4A1 antisense RNA 1

PARD3:

Par-3 family cell polarity regulator

NEAT1:

Nuclear paraspeckle assembly transcript 1

CDK6:

Cyclin-dependent kinase 6

RNCR3:

LncRNA retinal non-coding RNA3

KLF16:

Kruppel-like factor 16

qRT-PCR:

Quantitative real-time polymerase chain reaction

RUNX1:

RUNX family transcription factor 1

AKT1:

AKT serine/threonine kinase 1

ATCC:

American type culture collection

DMEM:

Dulbecco's modified eagle medium

siRNAs:

Small interfering RNAs

shRNA:

Short hairpin RNA

WT:

Wild type

NC:

Negative controls

FL:

Full length of wild type

UTR:

Untranslated regions

RIP:

RNA immunoprecipitation

anti-Ago2:

Anti-Argonaute-2

CCK-8:

Cell Counting Kit-8

Annexin-V-FITC/PI staining:

Annexin-V/fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining

RIPA:

Radio immunoprecipitation assay

SDS-PAGE:

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis

PVDF:

Polyvinylidene difluoride

PBS:

Phosphate-buffered saline

HRP:

Horseradish peroxidase

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

qRT-PCR:

Quantitative real-time polymerase chain reaction

cDNA:

Complementary DNA

ChIP:

Chromatin immunoprecipitation

S.D.:

Standard deviation

ANOVA:

One-way analysis of variance

TCF3:

Transcription factor 3

STAT3:

Signal transducer and activator of transcription 3

LUCAT1:

Lung cancer-associated transcript 1

SP1:

Sp1 transcription factor

RUNX1:

Runt-related transcription factor 1

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Acknowledgements

We thank our team for technical cooperation.

Funding

This study received no financial supporting from government subjects.

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Authors and Affiliations

  1. Department of Surgical Oncology, The First Affiliated Hospital of **’an Jiao Tong University, 277 West Yan-ta Road, ** ’an, 710061, Shaanxi, People’s Republic of China

    G. Xu, H. Wang, D. Yuan, J. Yao, L. Meng, K. Li, Y. Zhang, C. Dang & K. Zhu

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  1. G. Xu
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  2. H. Wang
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  3. D. Yuan
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Contributions

GX designed the project. H-nW, D-wY, JY, LM, KL, YZ, C-xD, and KZ performed the animal experiments, and they cultured the cell lines and conducted the cell experiments. GX, H-nW, and D-wY analyzed the experimental data. GX, H-nW, and D-w wrote the manuscript, and GX offered discussion and suggestions. GXX provided the financial support.

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Correspondence to G. Xu.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in our studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

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Xu, G., Wang, H., Yuan, D. et al. RUNX1-activated upregulation of lncRNA RNCR3 promotes cell proliferation, invasion, and suppresses apoptosis in colorectal cancer via miR-1301-3p/AKT1 axis in vitro and in vivo. Clin Transl Oncol 22, 1762–1777 (2020). https://doi.org/10.1007/s12094-020-02335-5

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