Epigenetically regulated MicroRNA-193b targets CCND1 in prostate cancer
Kaukoniemi, Kirsi (2024)
2024
Lääketieteen, biotieteiden ja biolääketieteen tekniikan tohtoriohjelma - Doctoral Programme in Medicine, Biosciences and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2025-03-10
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202410079134
https://urn.fi/URN:NBN:fi:tuni-202410079134
Tiivistelmä
Prostate cancer (PC) is the most commonly diagnosed cancer and one of the leading causes of cancer-related deaths in Europe. While the majority of patients are cured, some cases progress to advanced stages for which no curative treatments are available. Although current diagnostic methods are effective in detecting PC, they struggle to efficiently distinguish indolent tumors from aggressive ones, often leading to overtreatment. Consequently, new biomarkers are needed for both diagnostic and therapeutic purposes.
Epigenetic modifications regulate gene expression without affecting the underlying DNA sequence. Among these, DNA hypermethylation of gene promoters is the most extensively studied epigenetic alteration, typically associated with transcriptional silencing.
MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression post-transcriptionally and are integral components of the epigenetic regulatory network. Like protein-coding genes, miRNA genes are subjected to regulation at both genetic and epigenetic levels. In PC and other cancers, many miRNAs are differentially expressed.
The aim of this study was to validate the previously observed hypermethylation and reduced expression of miR-193b in PC and investigate whether miR-193b targets CCND1 in prostate cancer. CCND1 encodes for cyclin D1 protein, a key regulator of G1/S transition in the cell cycle. The results revealed that miR-193b is hypermethylated in PC samples compared to benign prostatic hyperplasia (BPH), and its expression is lower in more advanced pT3-stage prostatectomy tumors compared to pT2-stage tumors.
The interaction between miR-193b and CCND1 was investigated using various methods. Expression studies in 22Rv1 cells, which exhibit low endogenous miR-193b and high CCND1 levels, showed that forced overexpression of miR-193b led to reduced CCND1 mRNA and cyclin D1 protein levels. Additionally, the phosphorylation of RB, a target of the cyclin D1-CDK4/6 complex, was reduced. Luciferase assays demonstrated that miR-193b binds to the CCND1 3'UTR and this result was validated through rescue assay.
Cyclin D1 expression in clinical samples was evaluated using immunohistochemistry (IHC) on tissue microarrays. IHC analysis showed that cyclin D1 expression is elevated in castration-resistant prostate tumors compared to hormone-naïve tumors. Furthermore, a CDK4/6 inhibitor was applied to PC cell lines with varying endogenous levels of miR-193b and CCND1 expression. Growth of 22Rv1 and VCaP cells, which exhibit low endogenous miR-193b and high CCND1 expression, was significantly reduced following inhibitor treatment. In contrast, the inhibitor had no effect on the growth of PC-3 and DU145 cells, which have high endogenous miR-193b and low CCND1 expression.
Altogether, the data presented in this thesis demonstrates that miR-193b targets cyclin D1 in prostate cancer and that MIR193B promoter is hypermethylated and its expression is reduced in PC tumors.
Epigenetic modifications regulate gene expression without affecting the underlying DNA sequence. Among these, DNA hypermethylation of gene promoters is the most extensively studied epigenetic alteration, typically associated with transcriptional silencing.
MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression post-transcriptionally and are integral components of the epigenetic regulatory network. Like protein-coding genes, miRNA genes are subjected to regulation at both genetic and epigenetic levels. In PC and other cancers, many miRNAs are differentially expressed.
The aim of this study was to validate the previously observed hypermethylation and reduced expression of miR-193b in PC and investigate whether miR-193b targets CCND1 in prostate cancer. CCND1 encodes for cyclin D1 protein, a key regulator of G1/S transition in the cell cycle. The results revealed that miR-193b is hypermethylated in PC samples compared to benign prostatic hyperplasia (BPH), and its expression is lower in more advanced pT3-stage prostatectomy tumors compared to pT2-stage tumors.
The interaction between miR-193b and CCND1 was investigated using various methods. Expression studies in 22Rv1 cells, which exhibit low endogenous miR-193b and high CCND1 levels, showed that forced overexpression of miR-193b led to reduced CCND1 mRNA and cyclin D1 protein levels. Additionally, the phosphorylation of RB, a target of the cyclin D1-CDK4/6 complex, was reduced. Luciferase assays demonstrated that miR-193b binds to the CCND1 3'UTR and this result was validated through rescue assay.
Cyclin D1 expression in clinical samples was evaluated using immunohistochemistry (IHC) on tissue microarrays. IHC analysis showed that cyclin D1 expression is elevated in castration-resistant prostate tumors compared to hormone-naïve tumors. Furthermore, a CDK4/6 inhibitor was applied to PC cell lines with varying endogenous levels of miR-193b and CCND1 expression. Growth of 22Rv1 and VCaP cells, which exhibit low endogenous miR-193b and high CCND1 expression, was significantly reduced following inhibitor treatment. In contrast, the inhibitor had no effect on the growth of PC-3 and DU145 cells, which have high endogenous miR-193b and low CCND1 expression.
Altogether, the data presented in this thesis demonstrates that miR-193b targets cyclin D1 in prostate cancer and that MIR193B promoter is hypermethylated and its expression is reduced in PC tumors.