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Linc-ROR has a Potential ceRNA Activity for OCT4A by Sequestering miR-335-5p in the HEK293T Cell Line

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Abstract

Linc-ROR has a regulatory role in reprogramming, and the core stem cell transcription factors, OCT4, SOX2, and NANOG, regulate its expression. MicroRNAs (miRNAs) are also a critical constituent of pivotal posttranscriptional regulatory pathways. One of such interactions is a competing endogenous RNA interaction that connects small and long non-coding RNAs with coding transcripts. Here, we aimed to investigate the existence of such associations between OCT4A, Linc-ROR, hsa-miR-335-5p, and hsa-miR-544. Bioinformatic analysis was performed to evaluate the expression status of OCT4A, Linc-ROR, miR-335, and miR-544 throughout differentiation as well as in various differentiated cells. The complete lengths of OCT4A and Linc-ROR, and OCT4A 3′-UTR were cloned in the luciferase reporter vector, and the precursors of miR-335 and miR-544 were cloned in expression vectors. Following the overexpression of miR-335 and miR-544 in the 5637 cell line, the endogenous expression of OCT4A and Linc-ROR was evaluated. Afterward, the expression vectors of miRNAs and the reporter vectors of OCT4A/Linc-ROR were co-transfected in the HEK293T cell line. Via the Dual-Luciferase assay, the effect of the overexpression of miRNAs on their two possible targets (Linc-ROR and OCT4A) was investigated. The bioinformatic analysis demonstrated a relatively similar expression pattern for OCT4A and Linc-ROR, while miR-335 showed a different expression status. Both miR-335 and miR-544 inhibited the endogenous expression of OCT4A. The Dual-Luciferase assay likewise confirmed the inhibitory effect of miR-335 and miR-544 on OCT4A expression. In contrast, the miR-335 inhibitory effect was reversed in the presence of Linc-ROR, resulting in the upregulation of OCT4A. Such evidence suggests that Linc-ROR may compete with OCT4A to interact with miR-335.

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Acknowledgements

We thank Dr. Saman Hosseinkhani (Tarbiat Modares University) for providing a luminometer. This work was supported in part by a research grant awarded to Dr. Mahshid Malakootian from the Iranian Council of Stem Cell Research and Technology (Grant No. 186) and the Cardiogenetic Research Center, Rajaie Cardiovascular Medical and Research Center, Iran University of Medical Sciences, Tehran, Iran (Grant No. 94109).

Funding

This work was supported in part by a research grant awarded to Dr. Mahshid Malakootian from the Iranian Council of Stem Cell Research and Technology (Grant No. 186) and the Cardiogenetic Research Center, Rajaie Cardiovascular Medical and Research Center, Iran University of Medical Sciences, Tehran, Iran (Grant No. 94109).

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Authors and Affiliations

  1. Molecular Genetics Department, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

    Elham Taheri Bajgan & Seyed Javad Mowla

  2. Department of Biology, Islamic Azad University Tehran Science and Research Branch, Tehran, Iran

    Akram Gholipour

  3. Center for Therapeutic Innovation and Department of Psychiatry and Behavioral Sciences, University of Miami, Miami, FL, 33136, USA

    Mohammadali Faghihi

  4. Cardiogenetic Research Center, Rajaie Cardiovascular Medical and Research Center, Iran University of Medical Sciences, Tehran, Iran

    Mahshid Malakootian

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  1. Elham Taheri Bajgan
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  2. Akram Gholipour
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  3. Mohammadali Faghihi
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  4. Seyed Javad Mowla
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  5. Mahshid Malakootian
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Contributions

ETB: experiment design, lab work, data production, data interpretation, and writing first draft of manuscript; AG: bioinformatic data analysis and data interpretation; MF: project design and data interpretation; SJM: project design, data interpretation, and manuscript edit; MM: project design, experiment design, data interpretation, and manuscript final edit.

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Correspondence to Mahshid Malakootian.

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Nucleotide sequence data reported are available in GenBank databases under the accession numbers OK304860, OK304861, OK304862, OK304863, and OK304864.

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Taheri Bajgan, E., Gholipour, A., Faghihi, M. et al. Linc-ROR has a Potential ceRNA Activity for OCT4A by Sequestering miR-335-5p in the HEK293T Cell Line. Biochem Genet 60, 1007–1024 (2022). https://doi.org/10.1007/s10528-021-10140-0

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