Downregulated expression of HSP27 in human low-grade glioma tissues discovered by a quantitative proteomic analysis

Abstract

Background: Heat shock proteins (HSPs), including mainly HSP110, HSP90, HSP70, HSP60 and small HSP families, are evolutionary conserved proteins involved in various cellular processes. Abnormal expression of HSPs has been detected in several tumor types, which indicates that specific HSPs have different prognostic significance for different tumors. In the current studies, the expression profiling of HSPs in human low-grade glioma tissues (HGTs) were investigated using a sensitive, accurate SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative proteomic strategy.

Results: The five HSP family members were detected and quantified in both HGTs and autologous para-cancerous brain tissues (PBTs) by the SILAC-based mass spectrometry (MS) simultaneously. HSP90 AB1, HSP A5(70 KDa), and especially HSP27 were significantly downregulated in HGTs, whereas the expression level of HSPA9 (70 KDa) was little higher in HGTs than that in PBTs. It was noted that the downregulation ratio of HSP27 was 0.48-fold in HGTs versus PBTs, which was further validated by results from RT-PCR, western blotting and immunohistochemistry. Furthermore, we detected HSP27 expression changes along with cell growth under heat shock treatment in glioma H4 cells.

Conclusion: The SILAC-MS technique is an applicable and efficient novel method, with a high-throughput manner, to quantitatively compare the relative expression level of HSPs in brain tumors. Different HSP family members have specific protein expression levels in human low-grade glioma discovered by SILAC-MS analysis. HSP27 expression was obviously downregulated in HGTs versus PBTs, and it exhibited temporal and spatial variation under heat shock treatment (43 degrees C/0-3 h) in vitro. HSP27's rapid upregulation was probably correlated with the temporary resistance to heat shock in order to maintain the survival of human glioma cells.

Figure 1

Incorporation of Leu-d₃ in β-actin at different passages of H4 cells. A-D represented samples respectively from 1, 3, 5 and 7 passages of labeled H4 cells. The represented pairs of peptides "SYELPDGQVITIGNER" from β-actin were analyzed at different time points by ESI-TOF/MS. The intensity ratio of pairs of isotope peaks (m/z, 897/895) was respectively about 65.78%, 94.92%, 97.23% and 97.67%. After seven passages, the incorporation of Leu-d₃ was exceed 97.5%, which indicated that the labeling was complete.

Figure 2

One pair of isotope peptides (m/z, 582/581) "EITALAPSTMK" of β-actin was chosen to be an internal standard to validate the mixture ratio. (A) showed the peak intensity of the isotope-labeling peptides from the protein mixture containing human glioma tissues (HGTs) and H4 cells. The SILAC ratio1 of HGTs versus labeled H4 cells was 1.03 ± 0.02. (B) represented the peak intensity of isotope-labeling peptides, which came from the protein mixture from para-cancerous brain tissues (PBTs) and labeled H4 cells. The SILAC ratio2 of PBTs versus labeled H4 cells was 0.98 ± 0.03. Thus, the change ratio of β-actin in HGTs versus PBTs was 1.05, near 1:1, which indicated the expression of β-actin was similar between the two tissues.

Figure 3

The representative pairs of isotope labeling peptides to quantify HSP expression in glioma tissues. (A)-(B) was the peak intensity of isotope labeling peptide "SQIHDIVLVGGSTR" of HSPA8(71 KD) respectively from HGTs/H4 (A) and PBTs/H4(B) samples. A: the SILAC ratio1 (HGTs/H4) of HSPA8 was 0.52 ± 0.9; B: the SILAC ratio2 (PBTs/H4) of HSPA8 was 0.55 ± 0.1. The change ratio (HGTs/PBTs) was 0.95 (0.52/0.55) indicated HSPA8 was no significant change between glioma tissues and para-cancerous brain tissues. Similarly, the pairs of isotope labeling peptide "LATQSNEITIPVTFESR" of HSP27(HSPB1) from HGTs/H4 (C) and PBTs/H4(D) samples for quantitation, and the change ratio of 0.48 (0.29/0.61) showed HSP27 was down-regulated in glioma tissues.

Figure 4

The expression validation of HSP27 in glioma tissues by RT-PCR (A) and Western blot (B). HGTs: human glioma tissues; PBTs: para-cancerous brain tissues. Marker: DNA Marker; The β-actin was taken as a loading control.

Figure 5

The expression and distribution of HSP27 in human glioma and para-cancerous brain tissues. (A) the HE staining of para-cancerous brain tissues, which have typical characterization of normal brain tissues; (B) HE staining of glioma tissues. HSP27 immunoreactivity in the representative para-cancerous brain tissue (C) and glioma tissue (D) (original magnification ×400). The positive staining of HSP27 in the neuronal somata and proximal processes was shown as yellow-brown granules indicated by arrows. The figure c₂-d₂ was the insert of its corresponding expanded figure c₁-d₁. The scale bar was 10 μm.

Figure 6

The expression variation of HSP27 protein under heat shock (A). The relative strength of band was determined based on the control by Quality-One software(Bio-Rad) (B). Compared with the control, HSP27 expression was increased to 3.83 and 5.36-fold at time point of 30 min and 1 h after heat shock, then gradually jumped back near to the background level at 2 h and 3 h, with 2.3 and 1.12-fold expression versus the control. CON: the control cells without heat shock.

Figure 7

The survival rate of H4 cells with heat shock. It was noticed that cell proliferation ability under heat shock at the time point of 30 min(OD_{595 nm}, 0.57 ± 0.03, n = 18) was almost similar with that at 1 h (OD_{595 nm}, 0.57 ± 0.04, n = 18), and a same status was observed at the time point of 2 h(OD_{595 nm}, 0.48 ± 0.03, n = 18) and 3 h (OD_{595 nm}, 0.47 ± 0.03, n = 18) treatment. However, at the time point of 0.5-1 h and 2-3 h treatment, compared with control (OD_{595 nm}, 0.63 ± 0.05, n = 18), cell growth had significantly decreased (p < 0.01) in these two groups. CON: the control cells without heat shock.