| Bladder cancer (BC) is the first most common cancer in urinary system in China, which is a serious threat to human health. With respect to the pathological type, most of BC belongs to bladder urothelial carcinoma (BUC). The recurrence and invasion of BC is closely related to the pathological grade which is very important to evaluate the prognosis of BC. According to the degree of pathological grade, BC can be divided into papillary urothelial neoplasms of low malignant potential (PUNLMP), low grade urothelial carcinoma and high grade urothelial carcinoma. Pathological stage of BC refers to infiltration depth of the tumor and the metastatic status, which is not only the most valuable parameter judging the prognosis of BC but also the important basis for the BC treatment. According to the seventh edition of TNM staging principle made by union for international cancer control (UICC), BC can be divided into nonmuscle-invasive bladder cancer (NMIBC) including Tis, Ta and T1stage of BC and muscle-invasive bladder cancer (MIBC) including equal to or greater than the T2stage of BC.To date, transurethral resection of bladder tumor (TURBT) and postoperative adjuvant intravesical chemotherapy is still the main treatment for NMIBC, whereas the main treatment for NMIBC is radical cystectomy and pelvic lymphadencetomy with urinary diversion. However, because bladder cancer cell population present complex heterogeneity with various biological behavior, approximately80%of patients with NMIBC suffer from recurrence within1to2years and about8%will progress to MIBC although most of BC are characterized by NMIBC. For patients with MIBC, despite radical cystectomy and systemic therapy,50%of patients will die from metastasis. Therefore, to further explore the mechanism of the occurrence and development of BC and develop new effective treatments is still an important subject in the future.RNA interference (RNAi) is a phenomenon which is induced by small double-stranded RNA which can combine with mRNA with complementary sequence and degrade the mRNA. RNAi can silence specific gene with high efficiency, which is a kind of self-protective mechanism intrinsic to the cells against the invasion of exogenous gene. Using RNAi technology, the siRNA imported into tumor cell can lead to mRNA with homologous sequence to degrade, block specific gene expression with high efficiency, make cell present phenotype of specific gene deletion, and then achieve the goal of treatment for some diseases. So, RNAi may be widely used in gene therapy of malignant tumor. Using RNAi technology to block expression of genes or factors closely related to the occurrence and development of BC will change the present status of treatment for BC with more efficient and specific characteristics.Homeobox (HOX) genes are highly conserved clusters of DNA in evolution. The proteins coded by HOX genes are acted as transcriptional regulation factors in process of animal embryo development. Following maternal genes, fertilized egg genes and segmentation genes, these proteins present as expression before various specific types of cells in each parasegment differentiate and regulate the gene expression of the constituted structures in anterior-and posterior-axis of the body, thus forming expression pattern with strict space-time specificity, which play a key role in the development of embryonic somite and organs and the position of their corresponding location. In addition to regulate the differentiation and proliferation of embryonic cells, HOX genes play an important role in the regulation of the differentiation and development of tissues in adult. Recent studies indicated that the HOX genes may play an important role in occurrence and progression of some cancers. The changed expression of HOX genes makes the development of the individual and the formation of tissues and organs during the embryonal stages abnormal and induces malignant proliferation of cells and carcinogenesis in adult.Engrailed (EN) is one of HOX genes and widely exists in various phyla including annelids, mollusca, insects, echinoderm, chordate and vertebrata. Due to different functions in vertebrata, EN is divided into EN1and EN2which are functionally complementary in development.EN2is a highly conserved HOX gene and widely exists in various kinds of animals, which plays a key role in early embryonic development, particularly in nervous system development process. Additionally, EN2gene may be involved in establishment and maintenance of space integrity in the specific region of developing tissues and organs. Recent studies suggested that EN2play an important role in occurrence and progression of some cancers, such as breast cancer, prostate cancer, lung cancer and ovarian cancer, et al, which indicate that it may have the function of the proto-oncogene.However, to date, there were rare studies about the expression of EN2and its significance in BC. In this study, we used immunohistochemistry (IHC), Western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time polymerase chain reaction (qRT-PCR) to investigate the expression of EN2in BUC tissues and cells. Moreover, RNAi technology was used to inhibit the expression of EN2gene, thus investigating the effect of EN2on cell proliferation, apoptosis and invasive ability of BUC in order to explore the role of EN2in occurrence and progression of BC and provide the treatment for bladder cancer with a new target.Part1Expression of EN2in bladder urothelial cancer tissuesObjects:EN2is a member of HOX genes family, which is found to be over-expressed in a number of cancers and may have the function of the proto-oncogene. Nevertheless, there were rare studies about the expression of EN2and its significance in BC. This study aims to investigate the expression of EN2protein and gene in BUC tissues and normal bladder mucous membrane tissues in order to preliminarily identify its function in occurrence and progression of BUC.Mthods:The tumor samples as experimental group (n=60) were collected from hospitalized patients with BUC between January2012and December2012while normal bladder mucous membrane tissues as control group (n=10) came from patients with chronic bladder mucosal inflammation. The harvested tissues were divided into two parts:one for IHC and another for western blot and RT-PCR.Results:EN2protein was found to be present in both cytoplasm and nucleus, but mainly in cytoplasm by IHC. Compared with normal bladder urothelial tissues, the expression intensity of EN2protein in BUC tissues significantly increased (P<0.05). Moreover, there existed in positive correlation between the expression intensity of EN2protein and the pathological grade and stage of BUC. However, there was no correlation between patients’gender and the expression intensity of EN2protein in BUC tissues with different pathological grade and stage (P>0.05). Interestingly, there existed in positive correlation between the expression intensity of EN2protein in BUC tissues with T2stage and patients’age by Spearman rank correlation analysis, that is, the older patients’age was, the higher the expression intensity of EN2protein in BUC tissues with T2stage was (P<0.05). For patients with Ta and T1stage of BC, there was no correlation between patients’age and the expression intensity of EN2protein in BUC tissues (P>0.05). Additionally, the expression of EN2protein and mRNA detected by Western blot and RT-PCR were consistent with the expression of EN2protein detected by IHC (P<0.05).Conclusions:EN2protein was mainly present in cytoplasm of urothelial cell with few in nucleus. Compared with normal bladder urothelial tissues, the expression intensity of EN2protein and gene in BUC tissues significantly increased. Furthermore, there existed in positive correlation between the expression intensity of EN2protein and the pathological grade and stage of BC without significant correlation with patients’gender and age with Ta and T1stage.Part2Expression of EN2in various human bladder urothelial cancer cell linesObjects:Based on preliminarily clarifying the expression of EN2in BUC tissues, the expression of EN2protein and gene in three kinds of human BUC cell lines and one kind of human normal bladder urothelial cell line was investigated and the cell line with the highest expression level of EN2was selected.Methods:Three kinds of human BUC cell lines (EJ, T24and RT4, respectively) and one kind of human normal bladder urothelial cell line (SV-HUC-1) were cultured in vitro. The expression of EN2protien and gene in various cell lines was determined with ELISA, Western blot and qRT-PCR technology, respectively.Results:Compared with normal bladder urothelial cell line, the expression of EN2protein in BUC cell lines significantly increased with ELISA and Western blot (P<0.05). In three kinds of human BUC cell lines, the expression of EN2protein in EJ cell line is the highest, followed by T24and RT4cell line whereas there was a little expression of EN2protein in normal bladder urothelial cell line (P<0.05). Additionally, the expression of EN2protein was also detected in the supernatant of culture medium for three kinds of BUC cell lines, the level of which presented the same trend as that in cell lines. Similarly, there was also a little expression of EN2protein in normal bladder urothelial cell line (P<0.05). The expression of EN2mRNA determined by RT-PCR was consistent with the expression of EN2protein detected by ELISA and Western blot.Conclusions:Compared with normal bladder urothelial cell line, the expression of EN2protein and gene in various BUC cell lines significantly increased. However, the expression of EN2protein and gene was significantly different between BUC cell lines with different malignant degrees, that is, compared with BUC cell line with lower malignant degree the expression of EN2protein and gene in BUC cell line with higher malignant degree more significantly increased. Moreover, EN2protein may enter into the extracellular environment with secretion, leakage or other ways, thus further into the urine.Part3Effects of interference of EN2gene expression by RNAi on biological behaviors of human BUC cellObjects:Based on clarifying the expression of EN2proten and gene in BUC cell lines, the EN2gene was repressed by chemosynthetic siRNA against EN2with RNAi technology, and then the proliferation, invasion and apoptosis of human BUC cell were detected and the function of EN2in occurrence and progression of BUC was further investigated.Methods:The BUC cell line (EJ cell line) with the highest expression level of EN2identified by part2experiment was selected. The siRNA against EN2with high specificity was chemically synthesized and then successfully transfected into EJ cell. Transfection efficiency was observed under the inverted flurescence microscopy to identify the best transfection concentration. The expression of EN2mRNA in EJ cell with successfully transfected-siRNAs was detected by qRT-PCR to identify the siRNA with the best inhibition efficiency. The expression of EN2protein was observed with the confocal laser scanning microscopy after the siRNA was transfected. The expression of EN2protein and gene was determined with Western blot and qRT-PCR after the siRNA was transfected, respectively. The apoptosis of EJ was detected with flow cytometry while the proliferation and invasion of EJ cell was determined MTS method and matrigel invasion assay, respectively.Results:At the48th hour after transfection, the EN2protein was mainly present in the cytoplasm, rarely in the nuclear, under the confocal laser scanning microscopy, indicating that the cytoplasm and/or nuclear in the positive cells was dyed green whereas the cells in the blank group were not dyed. Furthermore, compared with the non-interfered group, the fluorescence intensity in the interfered group was significantly lower, indicationg that the expression of EN2protein was significantly downreulated with the inhibition rate of50.99±0.01%. At the24th and48th hour after the siRNA was successfully transfected into EJ cell, compared with the blank and non-interfered group, the expression of EN2protein and gene in EJ cells in the interfered group significantly decreased with qRT-PCR and Western blot, respectively (P<0.05). Compared with non-interference group and negative control group, the OD value in EJ cell in the transfected group had no significant difference (P>0.05) at the24th h after the transfection of siRNA, which indicated that the proliferation of EJ cell in the transfected group was not significantly affected at this moment (P>0.05). At the48th h after the transfection of siRNA, the OD value in EJ cell in the transfected group significantly decreased in comparison with non-interference group and negative control group, indicating that the inhibition rate rapidly increased to27.85%, and further increasing to43.02%at the72nd hour, which suggested that the proliferation of EJ cell in the transfected group was significantly inhibited (P<0.05). Matrigel invasion assay indicated that the invasive cell number of EJ cell had no difference between non-interference group and negative control group (29.27±4.08and29.47±3.18, respectively)(P>0.05) whereas the invasive cell number of EJ cell in the transfected group significantly decreased in comparison with non-interference group and negative control group (only5.13±1.68)(P<0.05), which suggested that invasive ability to EJ cell in transfected group was significantly inhibited. Flow cytometry analysis indicated that the apoptosis rate of EJ cell in transfected group significantly increased in comparison with non-interference group and negative control group at the48th h after the transfection of siRNA (P<0.05). For EJ cell in transfected group, the early, late and total apoptosis rate were9.72±0.58%,8.46±0.62%and18.19±1.21%, respectively. Compared with non-interference group and negative control group, the proportion of apoptotic cells in the transfected group significantly increased (P<0.05) whereas the proportion of living cells in the transfected group significantly decreased (P<0.05).Conclusions:The siRNA against EN2with high specificity may significantly downregulate the expression of EN2in BUC cell, thus significantly inhibit the proliferation and invasion of BUC cell while promoting the apoptosis of BUC cell. Therefore, EN2is expected to become the new target for treatment of BC, particularly providing a new pathway with gene therapy of BC in the future. |
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