miRNA regulation of Sirtuin-1 expression in human astrocytoma

Abstract

Sirtuins are a family of 7 histone deacetylases largely involved in the regulation of cell proliferation, survival and death. The role of sirtuins in tumorigenesis and cancer progression has been previously studied in certain cancer types. Few studies have investigated sirtuin expression in gliomas, with controversial results. The aim of the present study was to investigate the expression of sirtuin-1 (Sirt-1) in diffuse astrocytoma [low grade astrocytoma (LGA)], anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM) and in primary glioma cell lines: PLGAC (primary LGA cells); PAAC (primary AA cells); and PGBMC (primary GBM cells). Tumor samples were obtained from patients who underwent craniotomy for microsurgical tumor resection at the Neurosurgery Unit of the University of Messina between 2011 and 2014. Sirt-1 expression was qualitatively analyzed in 30 human glial tumor samples and 5 non-neoplastic brain tissue (NBT) specimens using immunohistochemistry and western blotting techniques. Sirt-1 expression was quantitatively analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In addition, Sirt-1 expression in primary cell lines was investigated by immunoblotting and RT-qPCR. Sirt-1 expression was downregulated in gliomas compared to NBTs. Sirt-1 levels also varied among different tumor grades, with more evident downregulation in high-grade (P<0.001) than low-grade tumors (P<0.01). These data were confirmed in cell lines, with the exception of upregulation of protein level in the highest malignancy grade cell lines. The present results suggest a role for miRNA-34a, miRNA-132 and miRNA-217 in the epigenetic control of Sirt-1 during gliomagenesis and progression, and demonstrate the different implications of Sirt-1 in human tissues and cell lines. Furthermore, the present results reveal that Sirt-1 may be an intrinsic regulator of tumor progression and the regulation of Sirt-1 involves complex molecular pathways. However, the biological functions of Sirt-1 in gliomagenesis require additional investigation.

Keywords: Sirtuin-1; astrocytoma; glioblastoma multiforme; glioma cell lines; histone deacethylases.

Figure 1.

(A) Western blot analysis of Sirt-1 in NBT, LGA, AA and GBM tissues. Representative autoradiography shows Sirt-1 and β-actin expression. Quantitative data indicate the mean ± standard deviation (error bars). *P<0.01, LGA and AA vs. NBT; **P<0.001, GBM vs. NBT. (B) Western blot analysis of Sirt-1 on primary glioma cell cultures. Representative autoradiography shows Sirt-1 expression and β-actin in NHA, primary LGA cells (grade II), primary AA cells (grade III) and primary GBM cells (grade IV). Quantitative data indicate the mean ± standard deviation (error bars). *P<0.001 vs. NHA. Sirt-1, sirtuin-1; NBT, neoplastic brain tissue; LGA, low grade astrocytoma; AA, anaplastic astrocytoma; GBM, glioblastoma multiforme; NHA, normal human astrocyte.

Figure 2.

Expression of Sirt-1 mRNA in (A) tumor tissues and (B) primary glioma cell cultures. (A) Sirt-1 expression was quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis on tumor tissues and normalized vs. NBT. Data are represented as log10 (relative quantification). Error bars indicate the standard deviation. ***P<0.0001, AA vs. NBT; *P<0.01, GBM vs. NBT. (B) mRNA levels of Sirt-1 assessed by RT-qPCR in grade II (primary LGA cells), III (primary AA cells) and IV (primary GBM cells) glioma cell lines and normalized vs. NHA. Data are indicated as log10 (relative quantification). Error bars represent the standard deviation. ***P<0.0001 III cells vs. NHA. Sirt-1, sirtuin-1; LGA, low grade astrocytoma; AA, anaplastic astrocytoma; GBM, glioblastoma multiforme; NBT, neoplastic brain tissue; NHA, normal human astrocyte.

Figure 3.

Expression of miR-132, miR-34a and miR-217. miRNAs expression were quantified by reverse transcription-quantitative polymerase chain reacion in primary glioma cell lines (panels A, C and E) and in tumor tissues (panels B, D and F). The data are expressed as the log10 RQ relative to U6. On the right: Aligment details with 3′-UTR region of SIRT-1. Sirt-1 is direct target of miR-34a, miR-217 and miR-132. The predicted highly conserved miRNAs targeting sequence located at the 3′ UTR of Sirt-1 mRNA found on microRNA.org. Sirt-1, sirtuin-1; LGA, low grade astrocytoma; AA, anaplastic astrocytoma; GBM, glioblastoma multiforme; miR, microRNA; 3′-UTR, 3′-untranslated region.