Expression Of Survivin And Its Splice Variants, Survivin-2B And Survivin-â–³Ex3 In Bladder Urothelial Carcinoma And Their Clinical Significance

Background and ObjectiveCancer is a multi-step, multi-stage and multiple genes involved disease. As with other types of tumors, the occurrence and development of bladder cancer is controlled through the gene level and the apparent genetic effect on cell proliferation and programmed cell death (apoptosis).The abnormal expression of anti apoptotic genes will lead to prolonged cell survival, the tumor began to start procedures to expedite the process or transfer. With the completion of human genome project, genetic screening of all genetic research has become a hot spot, which has great guiding significance in exploration of tumor progression and metastasis genes, determination of the prognosis, treatment selection and development of new therapeutic measures and programs. There are two of the most important apoptosis regulatory protein family:Bcl-2 protein family and the inhibitor of apoptosis proteins (inhibitor of apoptosis family of protein, IAPs).IAPs include 1 to 3 baculovirus IAP repeat domain (BIR), each BIR domain include 60～70 amino acids, repeat domain (BIR) is considered to play a major role in anti-apoptosis. Ambrosini form Yale University selected an anti apoptosis gene survivin by using of cDNA clone of effector cell protease receptor-1 in a human gene library screening, which is the most powerful inhibitor of apoptosis protein, involved in inhibiting apoptosis, regulation of cell cycle to decide the process of tumor development and progression. It is commonly expressed in human malignant tumors, but rarely expressed in normal differentiated tissues. At present, domestic and international study found that survivin gene has an important role in the development of bladder cancer, pointing out that the high sensitivity and specificity of survivin can be used as bladder cancer diagnosis and prognosis markers. With its in-depth study, scientists found that there are multiple splice variants, in which survivin-2B and survivin-ΔEx3 role gained attention because of its unique features and function. Survivin-2B increased 69bp exon 2B on the basis of survivin, encodes a 165 amino acid insertion of exon 2B interfere BIR domain, which made its anti-apoptotic activity significantly decreased. Survivin-ΔEx3 was chosen to cut off exon 3, causing regional coding sequence changes in exon 4, moving the stop codon to 3 UTR, encoding 137 amino acids, resulting in enhanced anti-apoptotic activity.At present, some studies have shown that survivin-2B and survivin-ΔEx3 play different role in tumor development. Survivin and its splice variants survivin-2B and survivin-ΔEx3 can be detected In soft tissue sarcoma and gastric cancer.In the brain tumor, survivin-ΔEx3 was higher than benign group, and highly expressed in malignant brain, while survivin-2B is highly expressed in benign tissue. But so far, the study on the expression of survivin splice variants in bladder urothelial carcinoma is rare.Bladder cancer is the most common urinary tract cancer in our country. Main treatment of superficial bladder cancer is transurethral resection of the tumor and adjuvant intravesical chemotherapy._After treatment, the recurrence rate of up to 61%, most will appear grade and stage of progress or metastasis, thus losing the opportunity to radical surgery. Currently factors thought to lead to higher recurrence rate of bladder cancer patients include:1.Pathological types of bladder cancer is the main factor of its recurrence, in particular the risk of pelvic recurrence is directly related to the pathological type of tumor,2.Local recurrence of bladder cancer is closely related to stage and grade of primary tumor, according to the order of increasing.3. Resection is not deep enough because of limited operation scopes, causing cancer recurrence in residual cutting edge 4.Postoperative bladder irrigation can not be formal and regular follow-up. Current bladder cancer diagnosis and follow-up study mainly depends on cystoscopy and biopsy pathology, the number of tumors, tumor size, shape and location can be clearly found by cystoscopy and suspicious of the tumor will use biopsy pathological examination to confirm the diagnosis.However, these methods are invasive which will lead to patients pain, missed diagnosis of early lesions, complications and other disadvantages. So look for sensitivity, high specificity of biological markers of bladder cancer has great significant guidance meaning in prognosis of the disease will relapse or not and also can be used as a new target for targeted cancer therapy. Our study focuses on expression of survivin, and its splice variants survivin-2B and survivin-ΔEx3 in the bladder urothelial carcinoma and analyzes the relevance of its expression and clinical pathological data.Materials and Methods(1) materials55 cases bladder cancer specimens of surgical resection (including initial and recurrent) in our hospital during December 2007 to June 2009, confirmed by pathology, without preoperative chemotherapy radiotherapy.45 cases of males and 10 females, Aged from 30 to 87 years, median age was 62.9 years. According to WHO 2004 histological grade:25 cases of low grade,30 cases of high-level; TNM clinical staging system groups:36 cases of Ta～T1,19 cases of T2～T4.10 cases of normal adjacent tissues,10 cases of normal bladder tissue taken from patients with benign prostatic hyperplasia over the same period for the control group._Washed organizations by sterile normal saline, cut into the size of about 0.2cm3 immediately and placed in EP tub in -80℃refrigerator, the rest sent for pathological examination. Survivin rabbit anti-human monoclonal antibody (Qi Kang Bio), SP immunohistochemistry kit (Boster), Trizol Reagent, RT-PCR kit, PCR primers and probes (Daan company), ABI 3900 high-desktop DNA synthesizer, HC-3018R High-speed refrigerated centrifuge(Tech Invo), ABI 9700 PCR instrument, ABI 7500 automated fluorescent quantitative PCR instrument.(2)RT-PCR determination the expression of Survivin, survivin-2B and survivin-AEx3mRNATake appropriate organization to the new 1.5mlEP tube, glass rod grinding, adding Trizol 1ml, full oscillation mixing, add chloroform 0.2ml, tightly cover, forced shake 15s,15-30℃incubated with 2-3min,4℃12000r/min centrifugal 15min, the supernatant to a new 1.5 mL EP tube, with a total RNA extraction step in accordance with the instructions for Trizol reagent. According to literature selected P-actin as an internal reference gene, GenBank to find the target gene mRNA sequences in the CDS area. Used Primer express 2.0 software design specific primers, synthetic primer desktop used ABI 3900 high-throughput DNA synthesizer. Primer:5'-TCC ACT GCC CCA CTG AGA AC-3'and 5'-CCT TCC AGC TCC TTG AAG CA-3'for Survivin(67bp);5'-GCGGATCACGAGAGAGGAAC-3'and5'-AGTTTCAAAAATT CACCAAGGGTTA-3'for surviving-2B(102bp); 5'-AGTTTCAAAAATTCACCAAGGGTTA-3'and5'-GAATTTGAGGAAACTGCG GAGA-3'for survivin-ΔEx3 (119bp); 5'-GCA TGG GTC AGA AGG ATT CCT-3'和5'-TCG TCC CAG TTG GTG ACG AT-3'for internal referenceβ-actin (106bp) Reverse transcription reaction:Used ABI 9700 instrument, take 4μl RNA template to do reverse transcription reaction, the standard curve of quantitative method statement prepared and reference to the positive standard and its gradient, and negative quality control standard using sterile double-distilled water. Sample under test and positive standard fluorescence quantitative PCR reaction conditions:93℃3 minutes, then 93℃30 seconds,55℃45 seconds,72℃45 seconds, a total of 40 cycles. By measuring the Survivin, survivin-2B and survivin-AEx3 gene and internal reference geneβ-actin ratio of the density of the amplified fragments, calculate the relative expression level.Used SPSS 13.0 Statistical Package for relative expression levels in 55 cases of bladder cancer patients, respectively, on the tumor grade and clinical stage grouping analysis and processing in groups, using T test to P<0.05 for the difference was statistically significant.(3) Immunohistochemical staining Slice, dewaxing, hydration after antigen retrieval and clear peroxidase, slices with a 1:100 dilution of anti-Survivin,37℃120min, PBS washed 2min×3 times, dropping two anti-working fluid A fluid,37℃incubated for 20 min, PBS washed 2min×3 times, dropping two working solution of anti-B solution,37℃incubated for 20 min, PBS washed 2min×3 times, DAB chromogenic 2-5min, under the microscope control, water washed to termination the reaction, hematoxylin about 1min, washed water for blue of 5min, gradient alcohol to dehydration, xylene to transparent, neutral gum cover the slice, using PBS instead of a blank control of anti-doing. Judging the results by two blinded pathologist used film-reading manner, combined with BeiHang image analysis software. The standard about results determine:appear brown granular cytoplasm of cells as positive cells, every slice select five high power field (×400 times), each field of vision were randomly counting 100 cells, and seeking the average positive rate of the mean percentage of positive cells≥25% for the Survivin expression was positive.ResultsSurvivin, survivin-2B and survivin-ΔEx3 mRNA in bladder cancer were expressed, expression was 0.560±0.286,0.050±0.025,0.467±0.229;The control group, the expression of survivin-2B expression level was 0.017±0.010,_Survivin, survivin-ΔEx3 only trace expressed in 5 cases and 6 cases respectively, expression was 0.003±0.006,0.003±0.003. The expression level of cancer tissue were significantly increased compared with normal tissue, the difference was significant (P <0.01). Considered the survivin and its splice variants have played a important role in tumor development.Expression of Survivin, survivin-2B and survivin-ΔEx3 mRNA has statistically significance in different pathological grade and clinical stage of bladder cancer, (P <0.01)._With the increased pathological grade, clinical stage, Survivin and survivin-ΔEx3 mRNA expression increased; and survivin-2B mRNA expression decreased. So we may predict that survivin-ΔEx3 has the similar function with surviving._On the other hand, confirmed that survivin and survivin-ΔEx3 have anti-apoptosis function, and survivin-2B weakened the anti-apoptotic effect. Survivin-2B may also antagonize survivin and survivin-ΔEx3 anti-apoptotic function. Of 55 cases of bladder urothelial carcinoma,46 cases are positive expression, and 9 cases are negative, Survivin positive expression rate was 83.6%; 20 cases of normal bladder mucosa, 2 cases are positive cases, and 18 cases are negative Survivin expression positive rate was 10%. The chi-square test,χ2 value is 34.517, P =0.000, therefore, positive rate of Survivin expression in bladder urothelial carcinoma is higher than that in normal bladder mucosa. (P<0.01)Conclusions1. Expression levels of survivin, survivin-2B and survivin-ΔEx3 mRNA in bladder urothelial carcinoma were significantly increased compared to normal tissue, indicating that Survivin and its splice variants in bladder carcinoma overexpressed, which has an important role in development and process of Bladder urothelial carcinoma.2.Survivin, survivin-2B and survivin-ΔEx3 have different roles in occurrence and development of bladder cancer, and have a certain relevance with the pathologic grade, clinical stage.