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In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice
Published on: August 23, 2019
Epigenetic regulation of thyroid hormone receptor beta in renal cancer
Anna Wojcicka1, Agnieszka Piekielko-Witkowska2, Hanna Kedzierska2
1Department of Biochemistry and Molecular Biology, Centre of Postgraduate Medical Education, Warsaw, Poland; Genomic Medicine, Department of General, Transplant and Liver Surgery, Medical University of Warsaw, Warsaw, Poland.
Abstract:
Thyroid hormone receptor beta (THRB) gene is commonly deregulated in cancers and, as strengthened by animal models, postulated to play a tumor-suppressive role. Our previous studies revealed downregulation of THRB in clear cell renal cell carcinoma (ccRCC), but the culpable mechanisms have not been fully elucidated. Since epigenetic regulation is a common mechanism influencing the expression of tumor suppressors, we hypothesized that downregulation of THRB in renal cancer results from epigenetic aberrances, including CpG methylation and microRNA-dependent silencing. Our study revealed that ccRCC tumors exhibited a 56% decrease in THRB and a 37% increase in DNA methyltransferase 1 (DNMT1) expression when compared with paired non-neoplastic control samples. However, THRB CpG methylation analysis performed using BSP, SNaPshot and MSP-PCR consistently revealed no changes in methylation patterns between matched tumor and control samples. In silico analysis resulted in identification of four microRNAs (miR-155, miR-425, miR-592, and miR-599) as potentially targeting THRB transcript. Luciferase assay showed direct binding of miR-155 and miR-425 to 3'UTR of THRB, and subsequent in vivo analyses revealed that transfection of UOK171 cell line with synthetic miR-155 or miR-425 resulted in decreased expression of endogenous TRHB by 22% and 64%, respectively. Finally, real-time PCR analysis showed significant upregulation of miR-155 (354%) and miR-425 (162%) in ccRCC when compared with matched controls. Moreover, microRNA levels were negatively correlated with the amount of THRB transcript in tissue samples. We conclude that CpG methylation is not the major mechanism contributing to decreased THRB expression in ccRCC. In contrast, THRB is targeted by microRNAs miR-155 and miR-425, whose increased expression may be responsible for downregulation of THRB in ccRCC tumors.
Insights
Thyroid hormone receptor beta (THRB) is downregulated in kidney cancer. MicroRNAs miR-155 and miR-425, not DNA methylation, appear to be the primary cause of this decrease.
Area of Science:
- Molecular Biology
- Epigenetics
- Oncology
Background:
- Thyroid hormone receptor beta (THRB) is a tumor suppressor gene often deregulated in cancers.
- Previous studies indicated THRB downregulation in clear cell renal cell carcinoma (ccRCC), but the underlying mechanisms remained unclear.
- Epigenetic modifications, such as DNA methylation and microRNA silencing, are common drivers of tumor suppressor gene downregulation.
Purpose of the Study:
- To investigate the epigenetic mechanisms, specifically CpG methylation and microRNA-dependent silencing, responsible for THRB downregulation in ccRCC.
- To determine the role of specific microRNAs in regulating THRB expression in ccRCC.
Main Methods:
- Quantitative real-time PCR and Western blot to assess THRB and DNMT1 expression.
- Bisulfite sequencing (BSP), SNaPshot, and methylation-specific PCR (MSP-PCR) to analyze THRB CpG methylation.
- In silico analysis and luciferase assays to identify and validate microRNA targets of THRB.
- In vivo transfection experiments to assess the effect of microRNAs on THRB expression.
- Real-time PCR to quantify microRNA levels in ccRCC tissues.
Main Results:
- ccRCC tumors showed a significant decrease in THRB expression (56%) and an increase in DNMT1 expression (37%) compared to non-neoplastic controls.
- No significant changes in THRB CpG methylation patterns were observed between ccRCC and control samples.
- In silico and luciferase assays confirmed that miR-155 and miR-425 directly target the THRB 3'UTR.
- Transfection with miR-155 or miR-425 led to decreased endogenous THRB expression in ccRCC cells.
- miR-155 and miR-425 were significantly upregulated in ccRCC tissues and negatively correlated with THRB transcript levels.
Conclusions:
- CpG methylation is not the primary mechanism driving THRB downregulation in ccRCC.
- Increased expression of miR-155 and miR-425 is responsible for the reduced THRB levels observed in ccRCC.
- Targeting miR-155 and miR-425 may represent a potential therapeutic strategy for ccRCC.
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