Expression And Significance Of Nodal Protein In Bladder Urothelial Carcinoma

Research background:Bladder tumor is a common malignant tumor of the urogenital system, which is agreat threat to the survival of the patients with the disease. In the worldwide, the incidence of bladder tumor in man ranks eighth in all kinds of tumor, while in women ranks25th. The ratio of men to women that develop bladder cancer is approximately3:1. According to statistics, there are69250cases of bladder tumor diagnosed for the first time around the world in2011, and it is the eighth leading cause of death from cancer. In our country, bladder tumors is the most common tumor of the urogenital system, and shows a continuous increasing tendency in recent years. Most of bladder tumor cases are bladder urothelial carcinoma in histology, account for about90%. According to the depth of cancer cells invading and the presence of transition, bladder cancer can be divided into the non-muscle layer invasive bladder cancer (stage Tis, Ta, T1)and muscle layer invasive bladder cancer (stage T2above). The non-muscle layer invasive bladder cancer is characterized with heterogeneous group, including form a heterogeneous group, spanning from completely benign, noninvasive papillary tumors that rarely progress, to papillary lamina propria-invasive high-grade tumors. The malignant degree of muscle-invasive bladder cancer is extremely high, which has the characteristic of strong invasiveness. It can develop partly invasive growth to surrounding tissues and organs, and even transfer further through lymphatic and blood. The current diagnosis of bladder tumor is mainly dependent on cystoscopy and Pathology biopsy. Unfortunately, bladder microscopic examination is traumatic, which can bring pain to patients. And pathology biopsy can sometimes misdiagnose because of failures in taking samples. High rate of relapse is one of the characteristics of bladder cancer. Clinically, among patients with incipient bladder cancer, about70%of tumor belongs to non-muscle invasive bladder cancer. After having bladder tumor resection (transurethral resection of bladder tumor), even if patients regularly do instillation therapy, there is still a10%-40%recurrence rate. Most of the tumors may develop or transfer by stages, which may lose the opportunity of radical surgery. About30%of tumor is muscle invasive bladder cancer, even though they received radical bladder full cut surgery, postoperative about50%will finally died of tumor distant metastasis. In other words, no matter(take) transurethral bladder tumor electricity cut method or Bladder full cut method, postoperative recurrence is high, tumor malignant degree is also rising with each recurrence, and tumor metastasis eventually occur. In addition to surgery, radiation and chemotherapy have certain effect, but the prognosis is generally poor, even it is easy to recurrence and multiple metastases such as bone transfer, Pulmonary metastasis and so on. As a result,to find a biomarker that has the potential to improve diagnosis, treatment and prognosis carries great practical significance. Nodal protein is an important embryonic morphogrnic factor belonging to TGF-beta superfamily, and its signaling pathway are essential for the induction of mesoderm and endoderm and the determination of the left-right axis. Recent investigation indictes that Nodal is highly expressed in tumor cell. The high expression relates with the tumor cell growing, invasion abihty and metastasis. However, The expression of Nodal protein in bladder cancer has not been fully elucidated, and few reports of study of Nodal protein relationship with bladder tumor biology behavior and clinical parametershave been seen. The research purpose of this experiment is to examines the expression of Nodal protein in bladder urothelial carcinoma (UCCB), and to investigate its clinical significance in the occurrence, development and prognosis of bladder urothelial carcinoma.Objective:To investigate the expression of embryonic morphogen Nodal in normal bladder specimen and bladder cancer and evaluate relationship of Nodal and bladder tumor occurrence and development.Methods:1、To collect17cases of bladder cancer specimen and6cases of adjacent normal bladder specimen, among the bladder cancer,5cases were grade I,7cases were grade Ⅱ,5cases were grade Ⅲ and7cases were Ta,6cases were T1,4cases were T2-T4. The sample taken from Jingzhou affiliate central Hospital of Tongji medical college in August2009to August2010. All patienls did not receive preoperative chemotherapy or radiotherapy.2、Detect the expression of Nodal protein in adjacent normal bladder specimen and bladder urothelial carcinoma by immunohistochemistrySlice, dewaxing, hydration after removal of endogenous peroxidase and antigen repair, slices with5%50ul sheep serum closed drops, room temperature incubated for10minutes, pour to serum, add appropriate amount of the working liquid,4℃refrigerator incubation overnight, PBS buffer (PH7.4slightly) washed3times x5min, removing the PBS solution, each section plus50ul biotin labeled tow anti-working fluid, incubation20min at room temperature, PBS washed3times x5min, dropping50ul Streptomyces biotin-resistance of horseradish peroxidase solution, room temperature incubated for40min, PBS wash5min x3times, DAB chromogenic3-5min, under the microscope control, water washed to termination the reaction, hematoxylin about3min, washed water for blue of10-15min, gradient alcohol to dehydration, xylene totransparent, neutral gum cover the slice. Judging the results by two blinded pathologist used film-reading manner, combined with Bei Hang image analysis software. The standard about results determine:appear brown granular cytoplasm of cells aspositive cells, every slice select ten high power field(X400times), Determine the results of the standard:to cytoplasm tan granule cells as positive cells, the results using semi-quantitative scoring method, according to cell dyeing intensity (A) and (B) shows the percentage of the product to decide.3> Detecte the expression of Nodal mRNA in adjacent normal bladder specimen and bladder urothelial carcinoma by RT-PCR, and analyze the expression of Nodal mRNA relationship with malignancy level and clinical stage of bladder urothelial carcinomaGenBank to find Nodal mRNA sequence, and used Primer design software Premier online Primer5.0and Oligo6design related primers. Primers were synthesized from Shanghai ying jun biological technology co., LTD. A total RNA extraction step in accordance with the instructions for Trizol reagent, take70℃5min, then37℃5min,42℃60min,70℃10min to do reverse transcription for synthesis cDNA, add4ul reverse transcription of DNA product in25ul PCR reaction system,94℃5min,94℃30s,55℃45s,72℃lmin, a total of36cycle. Choose beta-actin for PCR inside. PCR products analyzed by agarose gel electrophoresis and gel imaging system, the calculation for the ratio of the indicators and beta actin A value. Used SPSS13.0software to statistical analyze comparison relationship between the biological behavior of bladder urothelial carcinoma and expression of Nodal mRNA, P<0.05for the difference is statistically significant.4、Detecte the expression of Nodal protein in adjacent normal bladder specimen and bladder urothelial carcinoma by Western blot, and analyze the expression of Nodal protein relationship with malignancy level and clinical stage of bladder urothelial carcinomaExtracte total protein of Specimens and quantitate, transfer film after8%SDS-polyacrylamide gel electrophoresis (SDS-PAGE) separate protein,37℃5%skimmed milk closed1h, a1:1000dilution of the mice Nodal monoclonal antibody,4℃incubation overnight, horseradish peroxidase labele two resistance,37℃incubated for2h, ECL development after washing membrane, dark room exposure. All experimental repeat3-5times. Scan protein bands and the calculate ratio of every band and internal β-actin absorbance value (A value), it represent each Nodal protein expression content in the specimen. Used SPSS13.0software to statistical analyze comparison relationship between the biological behavior of bladder urothelial carcinoma and expression of Nodal protein, P<0.05for the difference is statistically significant.Result:1、The immunohistochemical results show:therewas no expression of Nodal protein in normal bladder specimen, while the expression in bladder cancer specimen was high, and tan positive granule can be seen in tumor cells cytoplasm cells, there were no obvious positive staining in cell membrane and the nucleus, And tumor tissue has lost the normal cells form with the increase of pathological grade, cell arranget is disorganized, granule dyeing is stronger within the cytoplasm, show expression of Nodal protein is strong at this time.2、Western Blot and RT-PCR method test results showed that there was no expression of Nodal protein and mRNA in normal bladder specimen, while the expression in17cases bladder cancer specimen was high.3、To analyze each band the absorbance value through the imaging system showed that the expression level of Nodal protein and mRNA increased in accordance with pathology level and clinical stage increased, Ⅲ grade> Ⅱ grade(P<0.05)>I grade(P<0.01), stage T2-T4>Tl>Ta(all P<0.05).Conclusion:1、There is no expression of Nodal protein and mRNA in adjacent normal bladder specimen, while the expression was very high in bladder cancer specimen.2、The expression of Nodal protein and mRNA was significantly positive correlated with malignancy level and clinical stage of bladder cancer. Nodal may participate in the occurrence and development process of bladder cancer.3、Nodal may plays an important role in the process of occurrence and development of bladder cancer, and it is expected to become an important target for early detection and treatment of bladder cancer.