Identification of proteins with different abundance associated with cell migration and proliferation in leiomyoma interstitial fluid by proteomics

Abstract

Uterine leiomyoma is the most common female reproductive tract benign tumor. Little is known about protein composition and changes in the leiomyoma interstitial fluid (IF). The present study focused on changes in protein abundance in the IF of leiomyoma. Leiomyoma IFs and adjacent myometrial IFs were obtained and analyzed by two-dimensional electrophoresis (2-DE) coupled with mass spectrometry and western blotting for 2-DE data validation. A total of 25 unique proteins were observed to change significantly (P<0.05). Of these proteins with different abundance, 22 had not been previously identified in leiomyoma IF. In silico analysis predicted that three of these proteins were secreted via classical mechanisms, while 22 were secreted via non-classical mechanisms. Ingenuity Pathway Analysis identified 17 proteins associated with cellular migration and proliferation. Among these, phosphoglycerate mutase 1 had not been previously associated with leiomyoma. The abundance of seven proteins was further validated by western blotting. A comparative proteomic approach identified a number of proteins associated with cellular migration and proliferation, with changes in abundance in IF likely to be involved in tumor development. Further studies will be required to investigate the role of these proteins in leiomyoma IF and their possible association with tumor development and growth.

Keywords: 2-D electrophoresis; interstitial fluid; leiomyoma; mass spectrometry; myometrium; proteomics; secreted protein.

Figure 1.

Two-dimensional electrophoresis map of the (A) normal myometrium IF and (B) leiomyoma IF proteome. Immobilized pH gradient pH 3–10 non-linear strips were used for the first dimension and 12% polyacrylamide gels were used for the second dimension. IF, interstitial fluid. Circles indicate different proteins identified.

Figure 2.

Top-scored molecular networks involving proteins with different abundance in the leiomyoma interstitial fluid, according to the results of Ingenuity Pathway Analysis software. Proteins in the network are represented by their gene symbols.

Figure 3.

Western blot analysis of CRABP2, CALR, LUM, PRDX1, PGAM1, ANXA2 and ALDOA in paired MIF and LIF. The intensity of the immunostained bands was normalized against the total protein intensities measured from the same blot stained with Red Ponceau S. The bar graph shows the relative expression (band density) of proteins in the MIF and LIF. The results are shown as a histogram (mean) with error bars representing the standard deviation. All differences were observed to be significant (Wilcoxon signed-rank test for matched samples, P<0.05). MIF, myometrium IF; LIF, leiomyoma IF; IF, interstitial fluid; CRABP2, cellular retinoic acid binding protein 2; CALR, calreticulin; LUM, lumican; PRDX, peroxiredoxin; PGAM1, phosphoglycerate mutase 1; ANXA2; annexin A2; ALDOA, aldolase A.