| IntroductionAge-adjusted standardized incidence of bladder cancer was0.38/million in male and0.14/million in female in2011in China. In recent years, the tumor incidence reports of some cities have showed that the incidence of bladder tumor is on the increase. According to pathological types, bladder cancer can be divided into urothelial (transitional) cell carcinoma, squamous cell carcinoma and gland carcinoma, followed by less common small cell carcinoma, hybrid carcinoma, carcinosarcoma and metastatic carcinoma. Among them, the bladder urothelial carcinoma (BUC) is the most common bladder cancer, accounting for more than90%, followed by squamous cell carcinoma, which is relatively rare and accounts for about3%～7%of bladder cancer. Bladder adenocarcinoma is even rarer, whose proportion is less than2%. The majority of patients with bladder cancer are diagnosed to be in good or medium differentiation of pathological staging, and the tumors do not invade into the muscular layer, And about10%of the patients among them eventually develop muscular invasive bladder cancer or distant metastatic bladder cancer. Clinical data showed that bladder cancer staging and grading are closely related to the disease progression. Bladder cancer staging refers to the infiltration depth and transfer situation of the tumor, which is the important parameter to estimate the prognosis of bladder tumor. The bladder cancer can be divided into the non-muscle-invasive bladder cancer (NMIBC, also know as superficial bladder cancer)(Tis, Ta, T1), and muscle-invasive bladder cancer (MIBC)(T2and above) in clinical. The progression risk of tumor with low grade and low stage is lower than that in tumor with high staging and high grade. Cystoscopy and intraoperative histopathologic biopsy are the most reliable methods for diagnosis of bladder cancer. By cystoscopy, the numbers, size, shape, position of the bladder tumors and the abnormalities of the surrounding bladder mucosa are clarified, at the same time the biopsies are performed to the tumor and suspicious lesions to make clear pathological diagnosis. Transurethral resection of bladder tumor (TUR-BT) is not only the main diagnosis method but also the main therapy for patients with superficial bladder carcinoma. The first pathological results obtained from the TUR-BT are required for the exact pathological grading and staging of bladder tumor. Radical cystectomy with urinary diversion is currently the standard treatment for patients with muscle-invasive bladder cancer, which is also the effective therapy for patients with invasive bladder cancer to improve the survival rate, reduce the local recurrence and distant metastasis.10%-15%of MIBC patients have accompanied with distant metastasis and (or) lymph node metastasis of bladder tumor cells when diagnosed. After TUR, up to50%of cases will occur metastasis with the5-year survival rate of36%-54%. For T3-T4and (or) high risk patients with lymph node metastasis and distant metastasis, the5-year survival rate is25%-35%. In recent years, with the deepening understanding of the tumor molecular mechanism, many tumor markers have been found that can be used for judging the prognosis of bladder cancer, both of which have a certain value in prognostic judgment of bladder cancer. But so far, there is still not an ideal marker can replace cystoscope and urine cytology for the diagnosis, treatment and postoperative follow-up and prognosis of bladder cancer to make enough judgment. Therefore, seeking a new diagnosis and treatment measures is of great significance to improve the early intervention and treatment of bladder cancer. This research aims at exploring the RNAi silencing CD147gene to change the molecular biology of bladder cancer cells in vitro, so as to provide new ideas for the treatment of bladder cancer.Bladder cancer staging depends on the invasive ability of bladder cancer cells. When invasion occurs, malignant tumor cell need to break through the basement membrane and the2layers of extracellular matrix (ECM) in interstitial space. The process is a multi-step process, including the ECM degradation, matrix permeability, tumor cells in and out of the blood vessels and invasion to the target tissue. Basement membrane components mediated by proteolytic enzyme and remodeling of large ECM molecules are both required for all these mentioned process. Study confirmed that molecules on the surface of the tumor cells can induce fibroblasts to secrete a kind of Zn2+dependent Matrix metalloproteinases (MMPs), which cannot only invade through the basement membrane barrier and realize the tumor cell metastasis but also can promote the body to generate new vessels feeding tumor, thus promoting the tumor growth. In vivo observations show that MMPs are mainly originated from mesenchymal cells in most malignant tumor, especially the fibroblasts. Fibroblasts produce very small amounts of MMPs in normal time, but cancer cells can secrete some stimulation factor, thus stimulating fibroblast cells to produce large amounts of MMPs. The stimulation factor, which has been confirmed in different Labs recently, is found a kind of adhesion molecules CD147on the surface of the tumor cells. It was first isolated from lung cancer cell line LX-1, which was named extracellular matrix metalloproteinases inducer originally. Later, homologous molecules of CD147have been found in different species (chicken, mouse), which are given different names.CD147, also known as extracelluar matrix metalloproteinase inducer (EMMPRIN), is a highly transmembrane glycosylated member of the immunoglobulin superfamily (IgSF) expressed on the cell surface of most tissue cells. It is not only highly expressed in various tumor cells and tissue but also has a correlation with inflammatory responses, such as rheumatic arthritis, cardiovascular disease and sperm differentiation. Immunohistochemical results showed that there is a close relationship between the MMPs expression and CD147expression in a wide variety of tumor stromal fibroblasts. EMNPRIN plays a role in interaction between tumor and fibroblasts, as well as tumor and endothelial cells, and it also can stimulate the tumor cells’autocrine of MMPs, especially the increased secretion of MMPs-2. Moreover, it can also regulate endothelial cells to generate MMPs through paracrine, thus the feeding vessels of the tumor cells are induced so as to promote the growth of tumor tissue. EMNPRIN mainly achieves the goal by inducing peripheral expression of invasive malignant tumor. Furthermore, EMNPRIN molecules also bind MMPs on the surface of the tumor cell surface to form complexes, which is favorable for the biodegradation of matrix around the tumor cells, thus making tumor cells easily to local invasion and distant metastasis. The compound function may be related with the adhesion between tumor cells and extracellular matrix as well as between tumor cells and mesenchymal cells. CD147can be combined into a variety of cells including endothelial cells and fibroblasts in the soluble recombinant form. With the soluble form of recombined CD147adenovirus vector to stimulate fibroblast, high expression of MMPs can be induced. Experimental study show that it is ineffective to use MMPs inhibitor alone to treat tumors, and in vitro researches on melanoma and oral squamous cancer cells testified that CD147antibody can significantly inhibit tumor invasion and metastasis, suggesting that inducer CD147with the MMPs as the target may be an effective way of tumor therapy.RNA interfering (RNAi) refers to that the dsRNA (double stranded RNA) can result in specific degradation with its homologous mRNA after introduction into the cells, leading to gene silencing. Since it occurs after the transcription, so it is also known as gene silencing. Large pieces of dsRNA can lead to extensive nonspecific gene silencing in mammalian cells. Elbashir et al. found that introduction of synthetic dsRNA with the size of21-23nt into cultured mammalian cells will not cause nonspecific interferon-like reaction. Instead, it can lead to gene silencing with specific sequence, which becomes a remark of RNAi application in mammals. dsRNA with the size of21-23nt is called small interfering RNA (siRNA). It has the comparable function to those whose gene is knocked out. The technology is fast, easy, effective and stable, which becomes a new approach for the study of the gene function in recent years.The methods to synthesize siRNA include chemical synthesis, in vitro transcription, in vivo vector expression, siRNA expressing framework and in vitro enzyme digestion. The former two methods can only lead to a short RNAi effect after transfection of siRNA into cells. Therefore, various studies over the interference are limited by time. In vivo vector expression method is the in vivo expression of siRNA mediated by plasmid or viral vector. With reduplication and amplification ability, they can maintain gene silencing for a longer time, which is applicable to long-term interference research. By designing CD147-siRNA, its appropriate target sequences and sources are selected and subsequently the CD147-siRNA recombinant adenovirus expression vector is successfully constructed. Through observation of the effect on the biological invasion of in vitro bladder cancer cells, the molecular mechanism of bladder cancer invasion is clarified to provide new ideas for effective treatments on bladder cancer.Materials and methodsMain experimental materials1.76cases of patients were enrolled in this study, who were undergone operation due to transitional urothelial cell carcinoma between September2011and February2014in uropoiesis surgical department of the first affiliated hospital of Gannan Medical College. Among them, normal paracarcinoma tissues were taken from16patients to form tissue microarray of the bladder cancer.62cases were male and14cases were female, with the median age of57years old (range:22-75years old), all of whom were not given chemotherapy and other adjuvant therapy before operation, and all the tumors are single. According to the classification criteria of urothelial carcinoma issued by the world health organization (WHO) in1999, the postoperative pathological histology classification was performed:12G1,25G2and39G3. Clinical pathological classification was based on the TNM criteria of Union for International Cancer Control (UICC):28Tl,22T2,20T3and6T4.2. Human bladder cancer cell lines are purchased from the Institute of Biochemistry and Cell Biology (Shanghai), Chinese Academy of Sciences.3. CD147siRNA recombinant adenovirus expression vector and the negative control adenovirus are synthesized with the assistance of Shanghai GenePharma Co., LTD.Main experimental methods1. Immunohistochemistry was used to examine and quantize CD147expressions in bladder cancer tissue as well as normal paracarcinoma tissues with different stages and grades. Then whether there were differences between the expressions of CD147and the correlation between the expressions with different stages and grades were analyzed.2. The target sequence of CD147-siRNA and its source were searched online and then designed to help build Ad-CD147siRNA and the negative control adenovirus. Human bladder cancer cells were subcultured. The multiplicated adenovirus was used to transfect human bladder cancer cells. And it was determined whether the transfection was successful. CD147and primers of internal reference GAPDH were designed and synthesized. RT-PCR was used to testify the mRNA levels of CD147in Ad-CD147siRNA group, Ad-siControl group and non-infection group, thus determining its influences on mRNA expression. Corresponding to the results of RT-PCR, Western Blot was used to determine the protein levels of CD147in Ad-CD147siRNA group, Ad-siControl group and non-infection group. Therefore, the obtained influence on protein expression was used to judge the interference of Ad-CD147siRNA, which laid a foundation for the further research. 3. Ad-CD147siRNA and negative Ad-siControl group were used to transfect human bladder cancer cells respectively. Influence of Ad-CD147siRNA on bladder cancer cell migration was measured by the scratch wound healing assay. The invasive capacities of the tumor cells were determined by a Transwell invasion assay,Gelatin zymography was performed to determine the secretions of MMP-2after transfection with Ad-CD147siRNA, while the secretion of VEGF was evaluated with ELIS A.Experimental results1. CD147showed no expression or very weak expression in normal bladder tissue.. Conversely, the immunoreactive patterns of CD147were predominantly positive in the cancer tissues with the positive expression rate of72.3%(55/76). CD147was mainly stained near the cell membrane. CD147overexpression was observed in the tumors. Through immunohistochemical staining on bladder cancer cells at different stages and grades, there showed an association between CD147protein expression with tumor stage (P=0.005) and histologic grade (P=0.047). But CD147protein expression was not related with age(P=0.463) or gender(P=0.880). This suggested that bladder cancer cells did not only express CD147, but the expression levels are correlated with the malignant degree and invasion levels of the bladder cancer.2. Recombinant CD147siRNA Adenovirus (Ad-CD147siRNA) was successfully constructed and the infection rate was determined. RT-PCR analysis and Western Blot test were performed on this basis, and the results showed that Ad-CD147siRNA can specific and effective silence the expression of CD147genes in bladder cancer cells T-24. The above researches laid a foundation for further research on the influence of CD147gene RNAi on human bladder cancer cells.3. In scratch wound healing assay, the migration of tumor cells was significantly suppressed after infected by Ad-CD147siRNA (P<0.01), with the migration distances of Ad-CD147siRNA and Ad-siControl infected cells as well as no-infection group of0.76,1.73, and1.83mm, respectively. Moreover, the Ad-CD147siRNA infected cells showed a lower invasion level (P<0.01) compared with cells infected with Ad-siControl and without infection by73.8%and70.9%, respectively. The secretion of MMP-2and MMP-9was reduced (P<0.01) in the Ad-CD147siRNA group by60.8%and41.3%compared with the non-infection group in Gelatin zymography. The protein expression level in was significantly reduced (P<0.01) in the Ad-CD147siRNA group by52.5%compared with the non-infection group in ELISA results.Experimental conclusion1. CD147has week or even no expression in normal bladder tissue while overexpressed in bladder cancer, whose expression level is correlated with tumor stage and grade. It could be a useful indicator for the assessment of potent invasion of bladder cancer.2. Recombinant adenovirus vector carrying CD147gene siRNA can successfully transfect human bladder cancer cells, adenovirus is selected as a vector, while siRNA can maintain a long time gene silencing. For human bladder cancer cells transfected by adenovirus, RT-PCR and Western Blot results showed that CD147expression was significantly inhibited at mRNA and protein levels. The results of subsequent functional experiments showed that RNA interference via blocking CD147could inhibit the biological behavior of bladder cancer cells. It could be presented in the following aspects:(1) the migration ability of bladder cancer cells was reduced significantly.(2) The ability of bladder cancer cells to secrete MMP-2and VEGF was greatly decreased. Since the secretion of MMPs and VEGF were reduced and the ability of proteolytic degradation and angiogenesis of bladder cancer cells were decreased, so the invasion of bladder cancer cells was weakened.In conclusion, through the expression of CD147in bladder cancer cells and the correlation research on its biological behavior, it suggested that siRNA gene technology can block the expression of EMNPRIN. Therefore, it could be a potential new target for bladder cancer gene therapy and is a new trend for the treatment of bladder cancer. |
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