Expression And Clinical Significance Of SATB1 In Bladder Urothelial Carcinoma

The process of tumor formation is a multi-stage, complex process and involves a variety of genetic and epigenetic changes, including two mechanism, they are genetic mechanism and the epigenetic mechanism. Genetic mechanism including gene mutation, chromosome loss and rearrangement, resulting in structural abnormality of the gene product; and when the tumor can transfer, it will get even more new capabilities, such as activity and invasiveness and viability in the cycle; This is also a multi-stage and complex process and also involves a variety of genetic and epigenetic changes. In recent years, several studies have shown that SATB1 gene has an important role in tumor progression and metastasis. SATB1 gene changes may cause changes in more than 1000 genes, thereby affect the complex process of tumor metastasis, and the incremental adjustment of SATB1 gene may be the switch gene cluster to tumor cell metastasis. With the completion of the human genome project, a variety of genetic screening has become hotspot, exploring tumor progression and metastasis-related genes on the prognosis of the tumor have important guiding significance to guide treatment measures and to development new therapeutic measures.BackgroundBladder urothelial carcinoma is the most common bladder tumor, the diagnosis of bladder urothelial carcinoma is mainly dependent on invasive cistoscopy and biopsy of the bladder.cystoscopy Can found whether is bladder tumor, and the number of tumor, the size of the tumor, the shape and location of the tumor. And can made biopsy to confirm the diagnosis. Bladder cancer is usually multifocal. Carcinoma in situ may be similar to the inflammation, dysplasia and other diseases, showing light red velvet-like mucosal change, or it even can be normal. On a single papilloma, if other parts of the bladder mucosa showed urine cytology negative, do not advocate routinely random biopsy, because it was discovered the possibility of carcinoma in situ is very low. Easy to relapse is a feature of the resection that retain the bladder, even if the bladder regularly supplemented by intravesical instillation of chemotherapy, there are still 10%-40% relapse rate, most of which also appeared grade and stage of progress or metastasis. Currently higher postoperative recurrence rate of factors are:(1) The pathological types of bladder cancer is the main factor affecting local recurrence, particularly the risk of pelvic recurrence and the pathological type of direct correlation; (2) primary tumor stage and grade is also closely related to the local recurrence of bladder cancer; (3) postoperative local recurrence of bladder tumor related to tumor location and the resection scope, tumor located at the top, anterior and lateral wall revealed good, have high rate of surgical excision and the local recurrence rate is low. Triangle area, the bottom of the ureteral openings due to the low lesion narrow and deep operative field, revealing the difficulties caused by operation of limited resection does not extend to depth, which leads to cutting edge cancer tissue residues lead to cancer recurrence; (4) after surgery can not have a regular bladder perfusion and regular follow-up. Therefore, the search for a specific, high sensitivity marker, which can predict the biological behavior of urothelial carcinoma of bladder tumor, as a index to predict the tumor progression and metastasis, in order to guide the choice of clinical treatment programs are particularly important. SATB1 was significantly expressed mainly in the thymus cells, SATB1 is special AT rich sequence binding protein, it contains two CUT motif, can form a structure similar to the PDZ dimer, and have very high affinity with the nuclear matrix attachment regions (MAR) combined thus affecting the DNA sequence of construction of rings involved, it participate in chromosome remodeling and histone acetylation, methylation and other processes, and has a close relevance with DNaseⅠhypersensitive sites. Studies have shown that the absence of SATB1 in mice, affecting at least 2% of the genome-wide gene expression abnormalities. SATBl involved in gene transcriptional repression, SATB1 inhibited the expression of heavy chainμ-chain enhancer MAR linked to heterologous reporter gene under the premise of integration in a stable and immunoglobulin. Study found that SATB1 binding in the mouse mammary tumor virus long terminal repeat (LTR) within the negative regulatory element on the inhibition of virus gene transcription. SATB1 also have an impact on the expression of the myc oncogene. Comparison before and after removing SATB1 several metastatic cell type in the changes in gene expression profiling results showed that SATB1 controls 1/4 to 1/3 "poor prognosis genes", including the control of adhesion, cell cycle, extracellular matrix, a number of signaling pathway, intercellular connections and apoptosis genes. Previous studies have shown that, SATB1 in specific chromosomal location made DNA folded into a dense ring of chromatin modifications affect protein and the role of transcription factor binding; subsequently in those parts of gene expression and synergy of multiple changes of acquired modifications. Thus, defects in cell and genetic variation of cells, SATB1 may be an incremental adjustment about progression and metastasis of tumor cells to switch gene cluster.OBJECTIVEStudies have shown that, SATB1 in several kinds of tumors is up-regulation, and significantly elevated in the tumors that potential malignant, easy to progression and metastasis, its change in the tumors can be detected in the early days. SATB1 gene and breast cancer metastasis was the earliest and most in-depth study, Han's study showed that, SATB1 played a key role in breast cancer progression, in highly metastatic breast cancer cell lines detected SATB1 mRNA and protein, in a high degree of malignancy of primary breast cancer samples expressed SATB1 protein; in the 1318 cases breast cancer samples from patients with histological analysis showed that high expression of SATB1 and short survival; in vitro experiments, down the expression of SATB 1 is not only reversed the invasiveness of the cells, and inhibit tumor growth, while the ectopic SATB1 expression in non-invasive cell obtained invasion force. The study also confirmed that SATB1 protein expression is not limited in the late phase of disease, in early stage of breast cancer, its existence can also be observed prior to lymph node metastasis. The latest study on the SATB1 gene and small cell lung cancer metastasis research suggests that SATB1mRNA in small cell lung cancer tissue than in normal tissue expression was up regulated, expression levels 13-fold than normal tissue, the difference was significant, suggested that SATB1 gene expression and small cell lung cancer may have some relevance, their relationship is unclear. SATB1mRNA in small cell lung cancer expression level do not related about sex, age, histological type, but have significantly related to the clinical stage, histological grade, lymph node metastasis, in which lymph node metastasis group and non-transfer group were 23.63-fold and 5.57-fold than normal tissues, they suggesting that SATB1 gene expression and small-cell lung cancer development and invasion may have certain relevance. Bladder urothelial carcinoma is the most common bladder tumor in our country, the current diagnosis is mainly dependent on their diagnosis of invasive bladder microscopic examination and biopsy. Easy to relapse is its feature, even if the bladder regularly supplemented by intravesical instillation of chemotherapy, there are still 10%-40% relapse rate, most of which also appeared grade and stage of progress or metastasis,while the loss of opportunities for radical surgery. Current bladder urothelial carcinoma of SATB1 protein expression is still poorly understood, and don't have the relationship about bladder tumor with the parameters of the relationship between biological behavior and clinical research. Our aim is to study the experimental study SATB1 in bladder urothelial carcinoma tissues and its relationship with tumor biological behavior.Method(1) Using two-step immunohistochemical to detected SATB1 expression in 68 cases of bladder urothelial carcinoma and 17 cases of normal bladder:Slice, dewaxing, hydration after antigen retrieval and clear peroxidase, slices with a 1:100 dilution of anti-SATB1,37℃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 lmin, 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 Bei Hang 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 SATB1 expression was positive. statistical methods:The SPSS13.0 Statistical Package for the right SATB1 protein in 68 cases of bladder cancer patients, respectively, on the age group, gender group, tumor grade and clinical stage grouping positive expression rate of packet data analysis and processing, usingχ2 test to P<0.05 for the difference statistically significant.(2) RT-PCR determination the expression of SATB1mRNATake 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 ACTB 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. 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 measured the SATB1 gene and amplification within the reference gene ACTB ratio of fragment density to calculate the relative content of SATB1 gene. Used SPSS13.0 Statistical Package for the right SATB1mRNA relative expression levels in 34 cases of bladder cancer patients, respectively, on the age group, gender group, tumor grade and clinical stage grouping analysis and processing in groups, using T test to P<0.05 for the difference was statistically significant.ResultsImmunohistochemistry test results show that, SATB1 expression in cancer positive rate of 57.4%,23.5% higher than the normal tissues positive rates, Chi-square test,χ2 value of 6.224, P value of 0.013 (P<0.05), SATB1 in bladder urothelial carcinoma of the positive rate of gender, age, tumor grade relationship is not clear, specific values for chi-square testχ2 values were 0.363,0.176,1.882, P values were 0.547,0.675,0.170 (P> 0.05), but have relationship with the clinical stage and lymph node metastasis, the specific values for chi-square testχ2 values were 5.537,6.918 P values were 0.019,0.009 (P<0.05), RT-PCR test results show that, SATB1mRNA expression in cancer tissues relative content is 0.105±0.064, and normal bladder tissue relative content is 0.017±0.007,SATB1mRNA expression level in cancer tissue significantly more than normal tissue, for two independent samples t test, t value is-7.864, P value is 0.000 (P<0.001) the difference is significant, SATB1mRNA in bladder carcinoma expression's relationship with age, tumor grade is not clear, specific values for two independent samples t test, t values were 0.372,-0.640, P values were 0.717,0.528 (P> 0.05), but with the clinical stage, gender, lymph node metastasis, the specific values for two independent samples t test, t values were-4.857,3.681,-6.685, P values were 0.000,0.001,0.000 (P<0.05), in which the clinical stage, metastasis is significantly correlated (P<0.001). Carried out at the same time, immunohistochemistry and PT-PCR of the specimens seen, SATB1 expression of positive samples relative expression of its SATB1mRNA content of 0.144±0.059 SATB1 protein expression was significantly higher than negative relative expression levels of specimens SATB1mRNA 0.055±0.023, specific values for two independent samples t test, t value is5.997, P value is 0.000 (P<0.001). The results suggest that SATB1 gene expression and the development of bladder urothelial carcinoma and invasion have certain relevance. Conclusion1. SATB1 positive rate in bladder urothelial carcinoma was significantly higher than normal bladder mucosa, SATB1mRNA in the expression level of cancer tissue were significantly increased compared with normal tissue, indicating that SATB1 in bladder urothelial carcinoma existed in the expression and have an important role in the occurrence and development of bladder urothelial carcinoma.2. SATB1 may play a catalytic role, in the occurrence and development of bladder urothelial carcinoma, and have correlation with clinical stage, lymph node metastasis.