Original Scientific Article
Suppression effects of excessively expressed gene bcl-2 in cell lines of prostate cancer
Igor Esmerov * ,
Branko Atanasov ,
Aleksandra Angeleska ,
Radmila Crceva ,
Ljupco Mickov ,
Ljupco Angelovski ,
Slavica Josifovska ,
Nikolaj Markov ,
Nikola Adamov

Mac Vet Rev 2022; 45 (2): 187 - 199

10.2478/macvetrev-2022-0028

Received: 01 July 2022

Received in revised form: 14 September 2022

Accepted: 19 September 2022

Available Online First: 28 September 2022

Published on: 15 October 2022

Correspondence: Igor Esmerov, iesmerov@gmail.com
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Abstract

The aim of this study was to construct two plasmid-specific shRNA transcripts of the bcl-2 gene in order to prepare for reverse of cell apoptosis. The plasmid was designed according to a previously published sequence of interfering RNA following an appropriate reference, using appropriate software. By annulling complementary oligonucleotides, doublestranded inserts were formed. Recombinant shRNA-encoding plasmids were constructed by digestion of psiRNAx7SKGFPzeo plasmid (psiRNA-x7SKGFPzeo, with restrictive endonuclease BbsI electrophoresis in ultra-pure agarose with low melting point (LMP-Agarose). For each of the constructs, a suitable double-stranded insert downstream of x7SK (strong RNA III promoter) with T4 DNA ligase was cloned. The control plasmid psiRNAScr was used directly for transformation. The PC-3 cell lines were transfected with 2 plasmids, psiRNA-Bcl-2 and psiRNAScr to suppress the bcl-2 gene construct. The results have shown that the lowest level of bcl-2 genes was 48 h, and even lower 72 h after the transfer, and the mRNA levels returned to normal in 120 h. An increase in the percentage of cells with spontaneous apoptosis has been observed with successful inhibition of the bcl-2 gene. The induction of apoptosis in transfected cells increased the percentage of necrotic cells proportionally. The percentage of apoptotic cells transfected with psiRNA-bcl-2 plasmid increased proportionally to the increase of hydrogen peroxide concentration. The transfection of the PC-3 cell line from prostate cancer with constructed shRNA plasmid has induced suppression of bcl-2 gene expression versus control Scr plasmid. Suppression of bcl-2 gene expression significantly increased cell sensitivity to apoptosis induction.

Keywords: shRNA plasmid, Bcl-2, apoptosis, cytotoxicity, prostate cancer

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Copyright

© 2022 Esmerov I. This is an open-access article published under the terms of the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Conflict of Interest Statement

The authors have declared that no competing interests exist.

Citation Information

Macedonian Veterinary Review. Volume 45, Issue 2, Pages 187-199, e-ISSN 1857-7415, p-ISSN 1409-7621, DOI: 10.2478/macvetrev-2022-0028, 2022

The all content of the Macedonian Veterinary Review, except where otherwise noted, is licensed under a Creative Commons Attribution License.