| ObjectiveHeparanase (HPSE) as a kind of glucuronidase enzyme can uniquely degrade heparan sulfate proteoglycans in extracellular matrix (HPSG) in vivo, and promotes the invasion and metastasis of tumor. Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 participate in many pathophysiological processes, including tumor cells induced chemotaxis, whereas chemotaxis of tumor cells was closely related with the invasion and metastasis of tumor in vivo. Extracellular matrix degradation and tumor cell directional movement are two important the processes of invasion and metastasis of tumor. Studies show that high expression of heparanase in invasive breast cancer was related to metastasis. Activation of SDF-1/CXCR4 biological axis also play a key role in the process of breast cancer cells transfering to specific tissues and organs, but the expression of heparanase and SDF-1/CXCR4 biological axis in the pathogenesis of breast cancer and their synergy in breast cancer are not explored. In this study, immunohistochemistry and RT-PCR were applied to detect the expressions of heparanase, SDF-1 and CXCR4 in invasive breast carcinoma, ductal carcinoma in situ and corresponding normal breast tissues to research their functions in breast cancer growth, invasion, and metastasis and at the same time explore the correlation between them.Methods1. MaterialsInvasive breast carcinoma, ductal carcinoma in situ and corresponding normal breast tissues were taken from patients who underwent surgery from September 2002 to December 2007 in Fengtian Affiliated Hospital of Shenyang Medical College. Specimens would be detected by immunohistochemistry were fixed in 10% neutral formalin solution and paraffin embedded. Fresh tissue specimens would be detected by RT-PCR were immediately placed in-80℃refrigerator after surgery.2. ImmunohistochemistryFour-micrometer-thick sections were cut from tissue array blocks for immunohistochemistry (SP method). After xylene dewaxing, gradient alcohol rehydration, antigen retrieval, dropping SP assay reagents (specific operations were carried out according to manual), heparanase, SDF-1 and CXCR4 antibody (1:100 in PBS/1% bovine serum albumin), PBS as negative control, positive slices as positive control,4℃overnight, staining with AEC for 5-10min, counterstaining with hematoxylin, mounting with AEC slide mounting medium and neutral balsam.3. RT-PCRTotal RNA extraction process was performed according to the mannul of RNAiso Plus reagent. Retrotranscription was carried out according to operating instructions. PCR conditions were 94℃denaturation for 3 min,94℃denaturation 30 s,53.5℃, 59.5℃,56.5℃,58.5℃annealing 30s,72℃extension 1min,35 cycles at 72℃after 5 min. The sizes of PCR reaction products were 288 bp (HPSE),366 bp (CXCR4),124 bp (SDF-1) and 224 bp (GAPDH), PCR products were stained with EB, and detected by 2% agarose gel electrophoresis, while experiment was repeated three times. Photos were taken and pictures were analyzed with Image J software.4. Statistical analysisThe SPSS 13.0 software was employed to analyze the data. P<0.05 was considered as statistical significance.Results1. ImmunohistochemistryThere were differential heparanase, SDF-1 and CXCR4 expressions in breast invasive carcinoma and ductal carcinoma in situ, ductal carcinoma in situ and corresponding normal breast tissues (P<0.05). The expressions of heparanase, SDF-1 and CXCR4 in breast invasive carcinoma were positively correlated (P<0.05), while the expressions of heparanase and CXCR4 correlated with lymph node metastasis and staging (P<0.05).2. RT-PCRThere were differential heparanase mRNA expressions in invasive breast cancer and ductal carcinoma in situ, invasive breast cancer and corresponding normal breast tissues (P<0.05). The heparanase mRNA expression of ductal carcinoma in situ and corresponding normal breast tissues was not significantly different (P> 0.05). SDF-1 and CXCR4 mRNA in breast invasive carcinoma, ductal carcinoma in situ and corresponding normal breast tissues were all differernt (P<0.05), the heparanase, SDF-1 and CXCR4 in invasive breast carcinoma was positively correlated (P<0.05).ConclusionHeparanase and SDF-1/CXCR4 biological axis played an important role in promoting breast cancer formation and the invasion and metastasis of breast cancer, and they might play a synergistic effect.
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