Reciprocal interplay of miR-497 and MALAT1 promotes tumourigenesis of adrenocortical cancer

in Endocrine-Related Cancer
Correspondence should be addressed to S B Sidhu: stansidhu@nebsc.com.au
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Adrenocortical carcinoma (ACC) has high recurrence rates and poor prognosis with limited response to conventional cancer therapy. Recent contributions of high-throughput transcriptomic profiling identified microRNA-497 (miR-497) as significantly underexpressed, while lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) as overexpressed in ACC. miR-497 is located in the chromosomal region 17p13.1, in which there is a high frequency of loss of heterozygosity in ACC. We aim to investigate the interaction of miR-497 and MALAT1 in ACC and its functional roles in the process of tumourigenesis. In this study, we demonstrated miR-497 post-transcriptionally repressed MALAT1 while MALAT1 also competes for miR-497 binding to its molecular target, EIF4E (eukaryotic translation initiation factor 4E). We showed that overexpression of miR-497 and silencing of MALAT1 suppressed cellular proliferation and induced cell cycle arrest through downregulation of EIF4E expression. Furthermore, MALAT1 directly binds to SFPQ (splicing factor proline and glutamine rich) protein, indicating its multifaceted roles in ACC pathophysiology. This is the first study to identify the feedback axis of miR-497-MALAT1/EIF4E in ACC tumourigenesis, providing novel insights into the molecular functions of noncoding RNAs in ACC.

Downloadable materials

  • Supplementary Figure 1: miR-497 expression in H295R cells three days after the transfection. RNU48 reference gene; Error bars show SEM, n=3. * indicates P < 0.05.
  • Supplementary Figure 2: Putative miR-497 seed binding sites on 3′UTR of targets in eIF4E.
  • Supplementary Table 1: Selected predicted targets for experimental validation

 

      Society for Endocrinology

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    miR-497 exerts tumour suppressive roles in ACC. (A) miR-497 is underexpressed in ACC tumour samples compared to NAC. There is also a reduction of miR-497 expression in ACC H295R cell line (n = 3). Data are presented as Tukey box plot; median expression is represented by the solid line within the box and the outliers are represented by the dots outside the boxes. (B) miR-497 replacement had growth inhibition in H295R cells. (C) Extra 5% of cellular events underwent early apoptosis post transfection of miR-497 when compared to miR-NC. (D) miR-497 increased cell percentage in the G1 phase by 6% cells. miR-497 decreased cell percentage in the S phase by 15%. (E) Significant decrease in cell invasion by 44% following miR-497 replacement. (F) miR-497 reduced migration by 31% compared to miR-NC. Error bars show s.d., n = 3. *P < 0.05, ****P < 0.0001.

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    miR-497 targets lncRNA MALAT1. (A) Putative miR-497 seed-binding sites of targets in lncRNA MALAT1. (B) Co-transfection of the luciferase reporter vector containing potential binding region for of MALAT1, respectively along with miR-497 mimics suppressed luciferase activity compared to miR-NC. (C) Following miR-497 replacement in H295R cells, reduced mRNA expression of MALAT1 was detected compared to miR-NC-treated cells. (D) Following MALAT1 knockdown in H295R cells, changes in miR-497 was increased by a fold change of ~4. Error bars show s.d., n = 3. *P < 0.05.

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    MALAT1 knockdown induced growth inhibition and cell cycle arrest in ACC. (A) MALAT1 transcript was reduced by ~80% following the transfection of MALAT1 LNA™ GapmeRs, detected by RT-qPCR. (B) Silence of MALAT1 caused a significant growth inhibition in H295R cells. (C) MALAT1 knockdown induced cell cycle arrest in H295R cell line with ~10% reduction in cells going through G1 phase and an increase of ~35% cells entering the G2 phase. Error bars show s.d., n = 3. *P < 0.05, **P < 0.01.

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    Functional effects of miR-497 and MALAT1 on cancer-related genes. (A) SFPQ is underexpressed in ACC tissues compared to NAC. There is also a reduction of SFPQ expression in ACC H295R cells (n = 3). The data are presented as Tukey box plot; median expression is represented by the solid line within the box. (B) The expression of SFPQ mRNA transcript was increased by ~1.8-folds in H295R cells following MALAT1 knockdown. (C) RNA immunoprecipitation with SFPQ antibody followed by RT-qPCR to examine MALAT1 expression. Data were normalized to mouse IgG as negative control (MALAT1 expression equals to 1). (D) miR-497 overexpression reduced the levels of EIF4E by ~78%. (E) Co-transfection of the luciferase reporter vector containing 3′ UTR of EIF4E, respectively along with miR-497 mimics suppressed luciferase activity. (F) MALAT1 knockdown reduced levels of EIF4E mRNA expression by ~63%. Bioinformatics tools including Miranda, TargetScan and Diana Tools were utilized to determine putative miRNA targets. Total RNA was collected from H295R cells following day 3 post transfection. mRNA expression of the targets was measured by RT-qPCR. Error bars show s.d., n = 3. *P < 0.05, ***P < 0.001.

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    Schematic of miR-497-MALAT1/EIF4E interplay feedback axis. LOH at 17p13.1 as shown in Libe et al. (2007) leads to the underexpression of miR-497. This affects the feedback loop between miR-497 and MALAT1 and their downstream targets, EIF4E and SFPQ, which regulates cellular proliferation and cell cycle arrest. A full colour version of this figure is available at https://doi.org/10.1530/ERC-19-0036.

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