Silencing of ANKRD12 circRNA induces molecular and functional changes associated with invasive phenotypes

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Abstract

Background: Circular RNAs (circRNAs) that form through non-canonical backsplicing events of pre-mRNA transcripts are evolutionarily conserved and abundantly expressed across species. However, the functional relevance of circRNAs remains a topic of debate. Methods: We identified one of the highly expressed circRNA (circANKRD12) in cancer cell lines and characterized it validated it by Sanger sequencing, Real-Time PCR. siRNA mediated silencing of the circular junction of circANKRD12 was followed by RNA Seq analysis of circANKRD12 silenced cells and control cells to identify the differentially regulated genes. A series of cell biology and molecular biology techniques (MTS assay, Migration analysis, 3D organotypic models, Real-Time PCR, Cell cycle analysis, Western blot analysis, and Seahorse Oxygen Consumption Rate analysis) were performed to elucidate the function, and underlying mechanisms involved in circANKRD12 silenced breast and ovarian cancer cells. Results: In this study, we identified and characterized a circular RNA derived from Exon 2 and Exon 8 of the ANKRD12 gene, termed here as circANKRD12. We show that this circRNA is abundantly expressed in breast and ovarian cancers. The circANKRD12 is RNase R resistant and predominantly localized in the cytoplasm in contrast to its source mRNA. We confirmed the expression of this circRNA across a variety of cancer cell lines and provided evidence for its functional relevance through downstream regulation of several tumor invasion genes. Silencing of circANKRD12 induces a strong phenotypic change by significantly regulating cell cycle, increasing invasion and migration and altering the metabolism in cancer cells. These results reveal the functional significance of circANKRD12 and provide evidence of a regulatory role for this circRNA in cancer progression. Conclusions: Our study demonstrates the functional relevance of circANKRD12 in various cancer cell types and, based on its expression pattern, has the potential to become a new clinical biomarker.

Original languageEnglish
Article number565
JournalBMC Cancer
Volume19
Issue number1
DOIs
Publication statusPublished - 11 Jun 2019

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Phenotype
Neoplasms
Ovarian Neoplasms
Real-Time Polymerase Chain Reaction
Exons
Cell Cycle
Breast Neoplasms
Genes
Smegmamorpha
Cell Line
RNA Precursors
Oxygen Consumption
Small Interfering RNA
Cell Biology
Molecular Biology
Cytoplasm
Biomarkers
Western Blotting
RNA
Messenger RNA

Keywords

  • Breast cancer
  • Cancer invasion
  • Circular RNA
  • OCR
  • OXPHOS
  • RNA-seq
  • siRNA

ASJC Scopus subject areas

  • Oncology
  • Genetics
  • Cancer Research

Cite this

@article{94292bb9be9c44e7883df4d0527fd822,
title = "Silencing of ANKRD12 circRNA induces molecular and functional changes associated with invasive phenotypes",
abstract = "Background: Circular RNAs (circRNAs) that form through non-canonical backsplicing events of pre-mRNA transcripts are evolutionarily conserved and abundantly expressed across species. However, the functional relevance of circRNAs remains a topic of debate. Methods: We identified one of the highly expressed circRNA (circANKRD12) in cancer cell lines and characterized it validated it by Sanger sequencing, Real-Time PCR. siRNA mediated silencing of the circular junction of circANKRD12 was followed by RNA Seq analysis of circANKRD12 silenced cells and control cells to identify the differentially regulated genes. A series of cell biology and molecular biology techniques (MTS assay, Migration analysis, 3D organotypic models, Real-Time PCR, Cell cycle analysis, Western blot analysis, and Seahorse Oxygen Consumption Rate analysis) were performed to elucidate the function, and underlying mechanisms involved in circANKRD12 silenced breast and ovarian cancer cells. Results: In this study, we identified and characterized a circular RNA derived from Exon 2 and Exon 8 of the ANKRD12 gene, termed here as circANKRD12. We show that this circRNA is abundantly expressed in breast and ovarian cancers. The circANKRD12 is RNase R resistant and predominantly localized in the cytoplasm in contrast to its source mRNA. We confirmed the expression of this circRNA across a variety of cancer cell lines and provided evidence for its functional relevance through downstream regulation of several tumor invasion genes. Silencing of circANKRD12 induces a strong phenotypic change by significantly regulating cell cycle, increasing invasion and migration and altering the metabolism in cancer cells. These results reveal the functional significance of circANKRD12 and provide evidence of a regulatory role for this circRNA in cancer progression. Conclusions: Our study demonstrates the functional relevance of circANKRD12 in various cancer cell types and, based on its expression pattern, has the potential to become a new clinical biomarker.",
keywords = "Breast cancer, Cancer invasion, Circular RNA, OCR, OXPHOS, RNA-seq, siRNA",
author = "{Karedath Abdul Azis}, Thasni and Ikhlak Ahmed and {Al Ameri}, Wafa and Al-Dasim, {Fatima M.} and Andrews, {Simeon S.} and {Mathews Samuel}, Samson and {Al Azwani}, Iman and {Ali Mohamoud}, Yasmin and Tabrizi, {Arash Rafii} and Joel Malek",
year = "2019",
month = "6",
day = "11",
doi = "10.1186/s12885-019-5723-0",
language = "English",
volume = "19",
journal = "BMC Cancer",
issn = "1471-2407",
publisher = "BioMed Central",
number = "1",

}

TY - JOUR

T1 - Silencing of ANKRD12 circRNA induces molecular and functional changes associated with invasive phenotypes

AU - Karedath Abdul Azis, Thasni

AU - Ahmed, Ikhlak

AU - Al Ameri, Wafa

AU - Al-Dasim, Fatima M.

AU - Andrews, Simeon S.

AU - Mathews Samuel, Samson

AU - Al Azwani, Iman

AU - Ali Mohamoud, Yasmin

AU - Tabrizi, Arash Rafii

AU - Malek, Joel

PY - 2019/6/11

Y1 - 2019/6/11

N2 - Background: Circular RNAs (circRNAs) that form through non-canonical backsplicing events of pre-mRNA transcripts are evolutionarily conserved and abundantly expressed across species. However, the functional relevance of circRNAs remains a topic of debate. Methods: We identified one of the highly expressed circRNA (circANKRD12) in cancer cell lines and characterized it validated it by Sanger sequencing, Real-Time PCR. siRNA mediated silencing of the circular junction of circANKRD12 was followed by RNA Seq analysis of circANKRD12 silenced cells and control cells to identify the differentially regulated genes. A series of cell biology and molecular biology techniques (MTS assay, Migration analysis, 3D organotypic models, Real-Time PCR, Cell cycle analysis, Western blot analysis, and Seahorse Oxygen Consumption Rate analysis) were performed to elucidate the function, and underlying mechanisms involved in circANKRD12 silenced breast and ovarian cancer cells. Results: In this study, we identified and characterized a circular RNA derived from Exon 2 and Exon 8 of the ANKRD12 gene, termed here as circANKRD12. We show that this circRNA is abundantly expressed in breast and ovarian cancers. The circANKRD12 is RNase R resistant and predominantly localized in the cytoplasm in contrast to its source mRNA. We confirmed the expression of this circRNA across a variety of cancer cell lines and provided evidence for its functional relevance through downstream regulation of several tumor invasion genes. Silencing of circANKRD12 induces a strong phenotypic change by significantly regulating cell cycle, increasing invasion and migration and altering the metabolism in cancer cells. These results reveal the functional significance of circANKRD12 and provide evidence of a regulatory role for this circRNA in cancer progression. Conclusions: Our study demonstrates the functional relevance of circANKRD12 in various cancer cell types and, based on its expression pattern, has the potential to become a new clinical biomarker.

AB - Background: Circular RNAs (circRNAs) that form through non-canonical backsplicing events of pre-mRNA transcripts are evolutionarily conserved and abundantly expressed across species. However, the functional relevance of circRNAs remains a topic of debate. Methods: We identified one of the highly expressed circRNA (circANKRD12) in cancer cell lines and characterized it validated it by Sanger sequencing, Real-Time PCR. siRNA mediated silencing of the circular junction of circANKRD12 was followed by RNA Seq analysis of circANKRD12 silenced cells and control cells to identify the differentially regulated genes. A series of cell biology and molecular biology techniques (MTS assay, Migration analysis, 3D organotypic models, Real-Time PCR, Cell cycle analysis, Western blot analysis, and Seahorse Oxygen Consumption Rate analysis) were performed to elucidate the function, and underlying mechanisms involved in circANKRD12 silenced breast and ovarian cancer cells. Results: In this study, we identified and characterized a circular RNA derived from Exon 2 and Exon 8 of the ANKRD12 gene, termed here as circANKRD12. We show that this circRNA is abundantly expressed in breast and ovarian cancers. The circANKRD12 is RNase R resistant and predominantly localized in the cytoplasm in contrast to its source mRNA. We confirmed the expression of this circRNA across a variety of cancer cell lines and provided evidence for its functional relevance through downstream regulation of several tumor invasion genes. Silencing of circANKRD12 induces a strong phenotypic change by significantly regulating cell cycle, increasing invasion and migration and altering the metabolism in cancer cells. These results reveal the functional significance of circANKRD12 and provide evidence of a regulatory role for this circRNA in cancer progression. Conclusions: Our study demonstrates the functional relevance of circANKRD12 in various cancer cell types and, based on its expression pattern, has the potential to become a new clinical biomarker.

KW - Breast cancer

KW - Cancer invasion

KW - Circular RNA

KW - OCR

KW - OXPHOS

KW - RNA-seq

KW - siRNA

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U2 - 10.1186/s12885-019-5723-0

DO - 10.1186/s12885-019-5723-0

M3 - Article

VL - 19

JO - BMC Cancer

JF - BMC Cancer

SN - 1471-2407

IS - 1

M1 - 565

ER -