Integrin alpha9 (ITGA9) expression and epigenetic silencing in human breast tumors

Abstract

Integrin alpha9 (ITGA9) is one of the less studied integrin subunits that facilitates accelerated cell migration and regulates diverse biological functions such as angiogenesis, lymphangiogenesis, cancer cell proliferation and migration. In this work, integrin alpha9 expression and its epigenetic regulation in normal human breast tissue, primary breast tumors and breast cancer cell line MCF7 were studied. It was shown that integrin alpha9 is expressed in normal human breast tissue. In breast cancer, ITGA9 expression was downregulated or lost in 44% of tumors while another 45% of tumors showed normal or increased ITGA9 expression level (possible aberrations in the ITGA9 mRNA structure were supposed in 11% of tumors). Methylation of ITGA9 CpG-island located in the first intron of the gene was shown in 90% of the breast tumors with the decreased ITGA9 expression while no methylation at 5'-untranslated region of ITGA9 was observed. 5-aza-dC treatment restored integrin alpha9 expression in ITGA9-negative MCF7 breast carcinoma cells, Trichostatin A treatment did not influenced it but a combined treatment of the cells with 5-aza-dC/Trichostatin A doubled the ITGA9 activation. The obtained results suggest CpG methylation as a major mechanism of integrin alpha9 inactivation in breast cancer with a possible involvement of other yet unidentified molecular pathways.

Figure 1

ITGA9 expression in normal and tumor human breast tissue. (A) Multiplex RT-PCR. Intensity of the amplified ITGA9 fragments normalized to that of GAPDH (TotalLab Programme). Representative gel from multiplex RT-PCR is shown (inset). (B) Quantitative Real-Time RTPCR. ITGA9 expression normalized to 1000 b-actin molecules. The graph shows the mean expression levels from triplicate experiments (± SD) (OriginPro 8.1). 1 and 2, normal breast tissue; 84–32, breast tumors.

Figure 2

Diagram of subdivision of patients for different groups in dependency of ITGA9 expression change in breast tumors.

Figure 3

Multiplex RT-PCR analysis of ITGA9 expression with different primer pairs. (A) Scheme of the primers used in the study. (B) Representative gel from multiplex RT-PCR. DNA fragments amplified with two different primers pairs are shown. GAPDH expression was used as an internal standard. (C) ITGA9 expression levels normalized to that of GAPDH (TotalLab Programme). The graph shows the mean expression levels from triplicate experiments (± SD) (OriginPro 8.1). 1 and 2, normal breast tissue samples; 109, 110, 125 and 126, breast tumors; C and T, control and tumor breast tissue (match pair for each patient), respectively.

Figure 4

Methylation of ITGA9 CpG-island in primary human breast tumors. (A) ITGA9 expression in breast tumors; a representative gel from multiplex RT-PCR. (B) Methyl-specific PCR on ITGA9 CpG-island. (C) Bisulfite sequencing. 301–328, breast tumors; C and T, control and tumor breast tissue (match pair for each patient); +, positive PCR control; −, negative PCR control; M, DNA marker; Meth and Unmeth, primers for methylated or un-methylated DNA sequence, respectively.

Figure 5

Activation of ITGA9 expression in MCF7 breast carcinoma cells by 5-deoxyazacytidine treatment. (A) Representative gel showing ITGA9 amplified by multiplex RT-PCR. (B) Intensity of the amplified ITGA9 DNA fragments normalized to that of GAPDH (TotalLab Programme). Bars represent the mean ± SD from triplicate experiments (OriginPro 8.1). The lane order on the gel corresponds to the bars on the histogram. 5-aza-dC, 5-azadeoxycytidine; TSA, Trichostatin A.