Expression And Correlation Of Mesothelin And CA125 In Patients With Epithelial Ovarian Tumors

Objective: Mesothelin is a tumor antigen that was originally identified by antibody CAK-1 on mesothelial cells, mesotheliomas and ovarian cancers. Using DNA microarrays find that mesothelin highly expresses in ovarian cells. Mesothelin is a 40 kDa glycosylphosphatidylinositol-linked glycoprotein. It is synthesized as a precursor of molecular mass 69kDa , which is then proteolytically processed into an N-terminal secreted form of 31kDa, and a membrane-bound form of 40 kDa, while 31kDa can be detected in serum. Recently investigations find that CA125 binds to mesothelin in a specific manner, which might be connected with their expression and function. The level of them in membrane of ovarian tumor cells and serum might be connected with clinic grade, size of tumor in body and the state of an illness. The reciprocity of them might lead ovarian tumor cells falling off, specific adheresion, planting with peritoneum mesothelial cells, lead to widely metastasia in abdominal cavity. Recently investigations find that epithelial ovarian cancer might come from epithelial ovarian malignant exchanges. To investigate the expression and correlation of mesothelin and CA125 in patients with normal ovarian and epithelial ovarian tumors at protein level; to investigate the expression of mesothslin in patients with normal ovarian and epithelial ovarian tumors at gene level. Methods: 107 fresh samples of epithelial ovarian cancer (45 serous, 16 mucinous, 46 endometrioid), 24benign ovarian tumor(9 serous, 15 mucinous), 18 normal epithelial ovarian were analyzed. All subjects were the admitted patients for operative treatment in our department. According to the classification of tumor stage suggested by WHO. Tissue specimens were snap-frozen in liquid nitrogen and transferred to a -80℃freezer for analysis or fixed in 4% formalin, embedded in paraffin. (1): Using RT-PCR methods to examine the expression of mesothelin gene: First total RNA was prepared by using Trizol reagent. After being washed in ethanol and resuspension in DEPC-treated water, RNA yield and purity were measured by spctrophotometric determination at 260nm and 280nm. And it was visualized on formaldehyde denaturing agarose gel. Second unique cDNA primers were designed for amplification of mesothelin from tissues by semiquantitative RT-PCR. They were as follows: mesothelin: 5'-AAC GGC TAC CTG GTC CTA G-3'(scene)and 5'-TTT ACT GAG CGC GAG TTC TC-3'(antiscene). β-actin 5'-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG-3'(scene)and 5'-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3'(antiscene). RT-PCR products are 226bp and 838bp respectively. Third, products of RT-PCR were electrophoresed in 1.5% agarose. Then mesothelin was evaluated by a Gel-analyzer software thatof corresponding β-actin mRNA was the expression parameter. Last statistical analysis was performed. Differences were considered to be statistically significant when the probability value was less than 0.05. (2): Using immunohistochemistry to detect the expression, orientation and with clinic pathology of mesothelin and CA125: Specimens were embedded in paraffin. Immunostaining with mesothelin or CA125 protein was performed by the labeled avidin-biotin. For staining, sections endogenous peroxidase activity was removed using 3%H202 in merhanol and nonspecific antigens were blocked with normal calf serum. The sections were then incubated for 60 minutes at 37℃with primary antibody in a humidity chamber, and a working dilution or 1:30 dilution was used. Staining was achieved using a biotinylated anti-mouse secondary antibody and antibody binding was amplified using biotin. The compkex was visualized using diaminovenzidine(DBA). An isotype IgGl, suitably diluted. Last statistical analysis was performed. Differences were considered to be statistically significant when the probability value was less than 0.05. Results: (1) Using immunohistochemistry to detect the expression, orientation and with clinic pathology of mesothelin and CA125: The expression of mesothelin and CA125 is different in normal,benign ovarian tumor and epithelial ovarian cancer at protein level. The expression of mesothelin and CA125 in epithelial ovarian cancer is significantly higher than that in benign ovarian tumor(P < 0.05), the expression ofpathology grade in G2,G3 is significantly higher than that in G1(P<0.05), the expression of histologic type in serous,endometrioid is significantly higher than that in mucinous (P<0.05), while in age,ascites and CA125 there is no difference( P>0.05). The expression of CA125 stage in Ⅲ,Ⅳis significantly higher than that inⅠ,Ⅱ(P<0.05). (2) Using RT-PCR methods to examine the expression of mesothelin gene: Expression of mesothelin mRNA is different in normal ovarian,benign ovarian tumor and epithelial ovarian cancer . The expression of mesothelin gene was not siginificantly higher in malignant than that in benign tumor(P>0.05).The expression level of MSLN mRNA in benign epithelial ovarian tumor and epithelial ovarian cancer is significantly higher than that in normal ovarian (P<0.05). In age,stage,histologic type and ascites the expression of them have no significant differences(P>0.05). Conclusion : The expression of mesothelin and CA125 is different in normal ovarian,benign ovarian tumor and epithelial ovarian cancer at protein level. There is significant correlation between mesothelin,CA125 expression and benign,ovarian cancer,grade and histologic type of epithelial ovarian cancer. Expression of mesothelin mRNA is different in normal ovarian,benign ovarian tumor and epithelial ovarian cancer . The expression level of MSLN mRNA in benign epithelial ovarian tumor and epithelial ovarian cancer is significantly higher than that in normal ovarian (P<0.05). In age,stage,histologic type...