Ma et al. Journal of Experimental & Clinical Cancer Research 2010, 29:85 http://www.jeccr.com/content/29/1/85 Table 1: Relative content of mRNA of BMP-2 and its receptors in ovarian tissue Ovarian cancer Benign ovarian tumor Normal ovarian tissue P value BMP-2 0.875 ± 0.136 1.409 ± 0.089 1.598 ± 0.082 0.001 aP = 0.548, comparison between benign ovarian tumor and normal ovarian tissue. BMPRIA 1.525 ± 0.158 1.569 ± 0.198 1.455 ± 0.176 0.680 BMPRIB 0.808 ± 0.137 1.173 ± 0.143 1.234 ± 0.162 0.001a BMPRII 0.834 ± 0.138 1.016 ± 0.119 1.273 ± 0.179 0.001 Page 3 of 6 70°C for later use. With a prestained marker serving as an index, the necessary gels were selected after polyacrylam- ide gel electrophoresis was performed, and a nitrocellu- lose filter was used for the transfer print. The primary antibody concentration was 1:100 and the secondary antibody was 1:2,000. By using alkaline phosphatase col- oration, the protein hybridization band was scanned with a GIS-2020 digital image analysis system and the absor- bance value was assayed. The ratios of BMP-2, BMPRIA, BMPRIB, BMPRII, and β-actin were calculated for the semiquantitative analysis. Immunohistochemistry Paraffin slices were treated according to the SABC immu- nohistochemical kit, and results were analyzed using a double-blind method. Five high-power fields (×400) were selected at random, and two pathologists evaluated scores independently. PBS, instead of the primary anti- body, was used as negative control, and specimens were scored according to the intensity of the dye color and the number of positive cells. The intensity of the dye color was graded as 0 (no color), 1 (light yellow), 2 (light brown), or 3 (brown), and the number of positive cells was graded as 0 (<5%), 1 (5-25%), 2 (25-50%), 3 (51-75%), or 4 (>75%). The two grades were added together and specimens were assigned to one of 4 levels: 0-1 score (-), 2 Figure 1 The mRNA expression of BMP-2 and its receptors detect- ed by RT-PCR 1: Ovarian cancer tissue; 2: Benign ovarian tumor tissue; 3: Normal ovarian tissue; M: Marker. scores (+), 3-4 scores (++), more than 5 scores (+++). The positive expression rate was expressed as the percent of the addition of (++) and (+++) to the total number. Statistical analysis Statistical analysis was performed with SPSS version 11.0 software, and P < 0.05 was considered to be statistically significant. Statistical tests used included the chi square test and analysis of variance. Results RT-PCR The mRNA expression levels of BMP-2, BMPRIB, and BMPRII in ovarian cancer tissues was significantly lower than those in benign ovarian tumors or normal ovarian tissue. No significant differences in BMPRIA mRNA expression level were observed among the three kinds of tissue (Table 1 and Figure 1). The relative content of the proteins was expressed as mean ± standard deviation (SD). Western blot The relative content of the proteins BMP-2, BMPRIB, and BMPRII in ovarian cancer tissue was significantly lower than those in benign ovarian tumors or normal ovarian tissue. No significant differences in BMPRIA protein expression level were observed among the three kinds of tissue (Table 2 and Figure 2). The relative content was expressed as mean ± standard deviation (SD). Immunohistochemistry Positively stained BMP-2 and its receptors BMPRIA, BMPRIB, and BMPRII were mainly located in the cyto- plasm of ovarian cancer cells and appeared as light brown and brown particles (Figure 3). Retrospective analysis of follow-up visits of patients showed that the total five-year survival rate of 100 patients was 32% with a mean survival time of 32.42 ± 22.62 months. The five-year survival rate after surgery of ovarian cancer patients with positive expression of BMP- 2, BMPRIB, and BMPRII was remarkably higher than that of patients with negative expression of BMP-2, BMPRIB, and BMPRII. BMPRIA expression was not associated