Deregulated expression of hnRNP A/B proteins in human non-small cell lung cancer: parallel assessment of protein and mRNA levels in paired tumour/non-tumour tissues

Abstract

Background: Heterogeneous nuclear ribonucleoproteins (hnRNPs) of the A/B type (hnRNP A1, A2/B1, A3) are highly related multifunctional proteins participating in alternative splicing by antagonising other splicing factors, notably ASF/SF2. The altered expression pattern of hnRNP A2/B1 and/or splicing variant B1 alone in human lung cancer and their potential to serve as molecular markers for early diagnosis remain issues of intense investigation. The main objective of the present study was to use paired tumour/non-tumour biopsies from patients with non-small cell lung cancer (NSCLC) to investigate the expression profiles of hnRNP A1, A2/B1 and A3 in conjunction with ASF/SF2.

Methods: We combined western blotting of tissue homogenates with immunohistochemical examination of fixed tissue sections and quantification of mRNA expression levels in tumour versus adjacent normal-looking areas of the lung in the same patient.

Results: Our study, in addition to clear evidence of mostly uncoupled deregulation of hnRNPs A/B, has revealed hnRNP A1 to be the most deregulated protein with a high frequency of over-expression (76%), followed by A3 (52%) and A2/B1 (43%). Moreover, direct comparison of protein/mRNA levels showed a lack of correlation in the case of hnRNP A1 (as well as of ASF/SF2), but not of A2/B1, suggesting that different mechanisms underlie their deregulation.

Conclusion: Our results provide strong evidence for the up-regulation of hnRNP A/B in NSCLC, and they support the existence of distinct mechanisms responsible for their deregulated expression.

Figure 1

A representative western blotting analysis of lung tissue homogenates. Total protein obtained from paired tumour (T) and non-tumour (N) tissues of NSCLC patients was resolved by SDS-PAGE (10% gel) followed by transfer to nitrocellulose membrane and western blotting. (A) Immunodetection within the same material of the hnRNP proteins (hnRNP A1, A2/B1, A3 and K/J) and (B) of the ASF/SF2 splicing factor was done by incubating the membrane with the appropriate antibodies. The parallel detection of β-actin served as an internal loading control for estimating relative protein expression levels.

Figure 2

Comparison of the relative expression levels of the hnRNP A/B proteins in paired tumour and normal biopsies. A histogram presenting the relative fold change of the tumour-to-normal (T/N) value of paired biopsies for hnRNP A1, A2/B1 and A3 protein levels individually for every patient (M1-M27) included in this analysis.

Figure 3

Estimates of hnRNP A1 and A2/B1 expression levels by immunohistochemistry. A representative picture showing staining with anti-hnRNP A1 or anti-hnRNP A2/B1 monoclonal antibodies of fixed lung sections of either non-involved (normal; a and b) or lung cancer tissues of the adenocarcinoma (c and d) or squamous cell carcinoma (e and f) subtype. Original magnification: all x200. A representative tissue area corresponding to panels a - f was selected and is shown in a magnified view as an insert picture.

Figure 4

Direct comparison of hnRNP A/B and ASF/SF2 protein and mRNA levels in paired tumour and normal biopsies. Histograms presenting the relative fold change in the tumour to normal (T/N) value of paired biopsies for hnRNP A1 (upper panel), A2/B1 (middle panel) and ASF/SF2 (lower panel) protein and mRNA levels in the indicated patients.