<abstract xmlns="http://www.w3.org/1999/xhtml">

<p>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, double-stranded inserts were formed. Recombinant shRNA-encoding plasmids were constructed by digestion of psiRNA-x7SKGFPzeo 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.</p>
</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, double-stranded inserts were formed. Recombinant shRNA-encoding plasmids were constructed by digestion of psiRNA-x7SKGFPzeo 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.

Suppression Effects of Excessively Expressed Gene BCL-2 in Cell Lines of Prostate Cancer

Ss Cyril and Methodius University in Skopje

Institute of Biology, Faculty for Natural Sciences and Mathematics, Ss Cyril and Methodius University in Skopje

Macedonian Veterinary Review

This work is licensed under the Creative Commons Attribution 4.0 International License.

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...

Suppression Effects of Excessively Expressed Gene BCL-2 in Cell Lines of Prostate Cancer

Data publikacji: 23 lis 2022

Zakres stron: 187 - 199

Otrzymano: 01 lip 2022

Przyjęty: 19 wrz 2022

DOI: https://doi.org/10.2478/macvetrev-2022-0028

Słowa kluczowe
shRNA plasmid, Bcl-2, apoptosis, cytotoxicity, prostate cancer

© 2022 Igor Esmerov et al., published by Sciendo

This work is licensed under the Creative Commons Attribution 4.0 International License.

Suppression Effects of Excessively Expressed Gene BCL-2 in Cell Lines of Prostate Cancer

Data publikacji: 23 lis 2022

Zakres stron: 187 - 199

Otrzymano: 01 lip 2022

Przyjęty: 19 wrz 2022

DOI: https://doi.org/10.2478/macvetrev-2022-0028

Słowa kluczoweshRNA plasmid, Bcl-2, apoptosis, cytotoxicity, prostate cancer

© 2022 Igor Esmerov et al., published by Sciendo

This work is licensed under the Creative Commons Attribution 4.0 International License.

Słowa kluczowe
shRNA plasmid, Bcl-2, apoptosis, cytotoxicity, prostate cancer