Expression and methylation status of LAMA2 are associated with the invasiveness of nonfunctioning PitNET

Abstract

The laminin subunit alpha 2 (LAMA2) gene encodes an alpha 2 chain, which constitutes one of the subunits of laminin 2 (merosin) and laminin 4 (s-merosin). In the current study, we investigated the relationship between LAMA2 promoter methylation status and the invasiveness of clinically nonfunctioning pituitary adenomas (PitNETs). Specimens from patients with nonfunctioning PitNET were classified into three groups according to preoperative computed tomography (CT)/magnetic resonance imaging findings: a normal group (n = 6), non-invasive group (n = 11) and invasive group (n = 6). LAMA2 expression was assessed using quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting, and the methylation status of the LAMA2 promoter region was observed using sodium bisulfite sequencing. Furthermore, 5-aza-2-deoxycytidine was used to explore the relationship between decreased LAMA expression and methylation in PitNET cells. According to the RT-qPCR and western blotting results, LAMA2 expression was downregulated in invasive PitNET, while the methylation of the LAMA2 promoter was increased. Methylation of the LAMA2 promoter decreased the expression of LAMA2. Thus, changes in LAMA2 expression due to promoter methylation were inversely correlated with the invasiveness of PitNET and the protein functions as a tumor suppressor. In addition, overexpression and demethylation of LAMA2 suppressed the invasion of PitNET cells, partially by exerting effects on the PTEN-PI3K/AKT signaling pathway and matrix metalloproteinase-9 (MMP-9). Furthermore, a xenograft model was also generated, and LAMA2 overexpression significantly suppressed tumor growth in vivo. Thus, LAMA2 expression and methylation patterns might be used as biomarkers to predict the prognosis of patients with PitNET.

Keywords: LAMA2; invasion; methylation; nonfunctioning pituitary adenoma.

Figure 1.

Differential expression of LAMA2 in the normal pituitary and nonaggressive and aggressive pituitary adenomas. (a) RT-qPCR analysis of LAMA2 mRNA expression in the normal pituitary and nonaggressive and aggressive pituitary adenomas; (b) western blotting analysis of the LAMA2 protein in the normal pituitary and nonaggressive and aggressive pituitary adenomas; the GAPDH protein was used as an internal control for western blotting; (c) quantitative analyses of western blotting results. ^*p < 0.05; ^**p < 0.01; and ^***p < 0.001. CT, ; GAPDH, ; LAMA2, laminin subunit alpha 2; mRNA, messenger ribonucleic acid; RT-qPCR, quantitative real-time polymerase chain reaction.

Figure 2.

Methylation status of LAMA2 in the normal pituitary and nonaggressive and aggressive pituitary adenomas. (a) Description of the methylated region of the LAMA2 promoter; (b) a representative methylation pattern of the CpGs in tumor tissues with different levels of invasiveness after bisulfite treatment; each line represents one PCR product, and the PCR products are shown for each sample. ●: methylated CpG; ○: unmethylated CpG; (c) percent methylation of the LAMA2 promoter region. All data are presented as means ± SEM; ^***p < 0.001. CpG, cytosine–phosphate–guanosine islands; GC, ; LAMA2, laminin subunit alpha 2; PCR, polymerase chain reaction; SEM, standard error of the mean.

Figure 3.

Demethylation of LAMA2 affects LAMA2 expression and the proliferation of pituitary adenoma cells. (a) Relative optical density (OD) of GH3 pituitary adenoma cells at 24, 48 and 72 h after treatment with the indicated concentrations of 5-aza-2-deoxycytidine. OD values are reported relative to the control (the concentration of DAC: 0 nmol/l) and were determined using an MTT assay; (b) after 72 h of treatment, the viability of passage two GH3 cells was significantly decreased with increasing concentrations of 5-aza-2-deoxycytidine [passage 8 (black) versus passage 10 (gray)], p < 0.01); (c) RT-qPCR analysis of LAMA2 mRNA expression 72 h after treatment with the indicated concentrations of 5-aza-2-deoxycytidine; (d) levels of the LAMA2 and GAPDH proteins measured in GH3 pituitary adenoma cells at 72 h after treatment with the indicated concentrations of 5-aza-2-deoxycytidine; quantitative analyses of western blotting results in GH3 pituitary adenoma cells are shown. All data are presented as means ± SEM, ^*p < 0.05 and ^**p < 0.01. PCR. DAC, ; GAPDH, ; GH3, ; LAMA2, laminin subunit alpha 2; mRNA, messenger ribonucleic acid; MTT, ; RT-qPCR, quantitative real-time polymerase chain reaction; SEM, standard error of the mean.

Figure 4.

Increased LAMA2 expression suppresses the invasion of GH3 cels. (a) Western blotting showing the LAMA2 levels in the control group, Lenti-LAMA2 group and 5-aza-2-deoxycytidine treatment group; (b) According to the results of the transwell assay, the number of invaded cells in the LAMA2 overexpression group was less than 40% of the number in the control group; (c, d) increased protein levels of epithelial markers (E-cadherin) was observed using western blotting (C), and decreased expression of genes related to migration and invasion (N-cadherin, β-catenin, MMP-9, and Snail) was observed using RT-qPCR (d) in cells overexpressing LAMA2 or treated with 5-aza-2-deoxycytidine; GAPDH was used as a positive control. n = 3; ^*p < 0.05 and ^**p < 0.01. GH3, ; GAPDH, ; LAMA2, laminin subunit alpha 2; MMP-9, metalloproteinase-9; RT-qPCR, quantitative real-time polymerase chain reaction.

Figure 5.

LAMA2 affected the PTEN-PI3K/AKT signaling pathway and MMP-9 expression. (a) Western blots showing protein expression; (b) quantitative analyses of western blotting results; (c) schematic depicting the mechanism by which LAMA may exert an effect on the PI3K/AKT signaling pathway and MMP-9 expression via PTEN. n = 3; ^*p < 0.05. AKT, ; LAMA2, laminin subunit alpha 2; MMP-9, metalloproteinase-9; mRNA, messenger ribonucleic acid; PTEN, ; PI3K, ; PIP, .

Figure 6.

LAMA2 regulated the growth of GH3 cells in vivo. (a) GH3 cells that had been transfected with the empty vector or LAMA2 vector were subcutaneously injected into nude mice, and the tumors that were excised from different groups are shown; (b) growth curve showing the changes in mouse tumor volumes after the administration of vehicle or Lenti-LAMA2; (c) the weights of the excised tumors in each group. n = 5; ^**p < 0.01. GH3, ; LAMA2, laminin subunit alpha 2.