SMARCB1 expression in epithelioid sarcoma is regulated by miR‐206, miR‐381, and miR‐671‐5p on Both mRNA and protein levels

Abstract

Proximal type epithelioid sarcoma shares similarities with malignant rhabdoid tumor, including the lack of nuclear immunoreactivity of SMARCB1. Biallelic mutation of SMARCB1 has been convincingly established as the cause of loss of protein expression in rhabdoid tumor, but the cause in epithelioid sarcoma remains unknown. In our previous work, we demonstrated that DNA hypermethylation and post-translational modification mechanisms were not involved. In this current work, we explored the hypothesis that miRNAs regulate SMARCB1 gene expression in epithelioid sarcomas. In silico target prediction analysis revealed eight candidate miRNAs, and quantitative PCR-in 32 formalin-fixed, paraffin-embedded tumor samples comprising 30 epithelioid sarcomas and two malignant rhabdoid tumors-demonstrated significant (P < 0.001) overexpression of four miRNAs in epithelioid sarcomas: miR-206, miR-381, miR-671-5p, and miR-765. Two human tumors (fibrosarcoma and colon adenocarcinoma) and a normal cell line (human dermal fibroblast) with retained SMARCB1 expression were cultured for miRNA transient transfection (electroporation) experiments. SMARCB1 mRNA expression was analyzed by quantitative real-time PCR and immunostaining of SMARCB1 was performed to examine the effect of miRNAs transfections on both RNA and protein levels. Only three of the overexpressed miRNAs (miR-206, miR-381, and miR-671-5p) could silence the SMARCB1 mRNA expression in cell cultures; most effectively miR-206. Transfection of miR-206, miR-381, miR-671-5p, and some combination of them also eliminated SMARCB1 nuclear staining, demonstrating a strong effect on not only mRNA but also protein levels. Our results suggest loss of SMARCB1 protein expression in epithelioid sarcoma is due to the epigenetic mechanism of gene silencing by oncomiRs.

Epigenetic regulation of SMARCB1 By miR‐206, ‐381 and ‐671‐5p is evident in a variety of SMARCB1 immunonegative soft tissue sarcomas, while miR‐765 appears specific for epithelioid sarcoma. A miRNA study of 223 soft tissue sarcomas

“…In this regard, ES is unique because it seems that in this tumor, both genetic and epigenetic regulation may play a role. In our recently published works, we on the one hand proved that loss of SMARCB1 expression in ES is caused neither by DNA hypermethylation nor by post‐translational modifications (Papp et al, ), and on the other hand, we also proved that the SMARCB1 gene is regulated by oncomiR‐206, oncomiR‐381 and oncomiR‐671‐5p (functionally active miRNAs) at both the mRNA and protein levels (Papp et al, ). We also found that miR‐765 was significantly overexpressed in ES, but miR‐765 had no functional activity to silence SMARCB1 mRNA (Papp et al, ).…”

“…In our recently published works, we on the one hand proved that loss of SMARCB1 expression in ES is caused neither by DNA hypermethylation nor by post‐translational modifications (Papp et al, ), and on the other hand, we also proved that the SMARCB1 gene is regulated by oncomiR‐206, oncomiR‐381 and oncomiR‐671‐5p (functionally active miRNAs) at both the mRNA and protein levels (Papp et al, ). We also found that miR‐765 was significantly overexpressed in ES, but miR‐765 had no functional activity to silence SMARCB1 mRNA (Papp et al, ). In the present study, we wanted to examine whether overexpression of functionally active miRNAs in silencing SMARCB1 mRNA is characteristic or not for the other types of SMARCB1 immunonegative soft tissue sarcomas and whether overexpression of miR‐765 is specific for ES or not.…”

Primary proximal epithelioid sarcoma of the lung successfully treated with pneumonectomy and adjuvant chemotherapy

“…It is interesting to note, with respect to our patient's history of HIV infection, that the INI1 gene produces a host factor for the HIV virus and its integration. Studies suggest these silencing events may occur through action of OncomiRs, or microRNAs that interfere with the coding of this protein 7. OncomiRs have been implicated in virus-induced tumourigenesis by Epstein-Barr virus.…”

MicroRNAs in Soft Tissue Sarcomas: Overview of the Accumulating Evidence and Importance as Novel Biomarkers

“…Papp et al hypothesized that miRNAs regulate SMARCB1 expression and analyzed eight candidate miRNAs selected from in silico analysis. RT-PCR using tumor samples identified the overexpression of miR-206, -381, -671-5p, and -765 in epithelioid sarcomas [69]. Examination of the effect of miRNA transfections revealed that three of the overexpressed miRNAs (miR-206, miR-381, and miR- 671-5p) could silence SMARCB1 mRNA expression in cell cultures.…”

“…Examination of the effect of miRNA transfections revealed that three of the overexpressed miRNAs (miR-206, miR-381, and miR- 671-5p) could silence SMARCB1 mRNA expression in cell cultures. They concluded that the epigenetic mechanism of gene silencing by miRNAs caused the loss of SMARCB1 expression in epithelioid sarcoma [69]. …”