Gene Silence Of CD147 By Adenovirus-mediated RNAi Influence The Biological Behavior Of Bladder Cancer Cell Line T-24

BackgroundBladder carcinoma is the most common urogenital malignancy in our country. More than 90% of them are transitional cell carcinomas (BTCC). Approximately 75% of of patients with primary bladder carcinoma present with superficial (pTis, pTa, pT1) disease, the remaining 25% present with muscle-invasive or metastatic disease. Patients with superficial bladder carcinoma can be treated by transurethral resection or intravesical immunotherapy and chemotherapy. Unfortunately, recurrence rates are high (30-85%), and approximately 20-30% of cases will progress to muscle-invasive disease, which has a poorer prognosis. Although radical cystectomy with urinary diversion is currently the standard treatment for patients with muscle-invasive bladder cancer (pT2-pT4), nearly half of such patients develop metastases within 2 years after cystectomy and subsequently die of the disease, the 5-year survival rate is 50% or less. Therefore, more effective strategies for the treatment of bladder cancer are clearly needed.High invasion is the principle reason to the mortality of bladder cancer. In the invasive and metastatic process of malignant tumor, an important step is the degradation of extracellular matrix (ECM). Molecules existing in ECM and receptors or ligands existing on the surfaces of tumor cells play critical roles. CD147, also known as extracelluar matrix metalloproteinase inducer (EMMPRIN), is a highly transmembrane glycosylated member of the immunoglobulin superfamliy molecules expressed on the cell surface of most tumor cells, and its expression was reported to correlate with tumor progression and invasion. CD147 contributes to tumor invasion and metastasis by stimulating fibroblasts nearby to secrete an increased amount of matrix metallproteinases (MMPs), which play key roles in several aspects of tumor progression, including growth, invasion, metastasis, angiogenesis. Other sutdies have shown that CD147 has paracrine effect on MMPs porduction by endothelial cells and forms a complex with MMP-1 at the tumor cell surface. It is important in modifying the tumor cell pericellular matrix concentrating and localizing this collagen degrading enzyme at the tumor cell surface to promote tumor cell invasion.However, very few attempts have been made to clarify an involvement of CD 147 in progression of bladder cancer. To study the relationship between CD 147 and bladder cancerm, we constructed a vector of RNA interference of CD147 and investigated its inhibitory effects on invasion of T-24 cells in vitro. Therefore, an understanding of the molecular mechanisms involved in bladder cancer progression should be helpful in developing more effective treatments for bladder cancer.Methods1. We examined the clinical significance of CD147 expression in human bladder cancer using tissue microarray (TMA) technology and immunohistochemistry.2. Constuction of Recombinant CD147 siRNA Adenovirus.(1) RT-PCR was used to testify the mRNA level of CD147 in Ad-CD147 siRNA group,Ad-siControl group and non-infection group.(2) Western Blot was used to testify the protein level of CD147 in Ad-CD147 siRNA group,Ad-siControl group and non-infection group.3.Effects of CD147 downregulation on the proliferation,magriation,and invasionness of T-24 bladder cancer cell line.(1) The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine cell proliferation in Ad-CD147 siRNA group, Ad-siControl group and non-infection group.(2) Cell migration was measured by the scratch wound healing assay in Ad-CD 147 siRNA group, Ad-siControl group and non-infection group.(3) The invasive capacity was determined by a Matrigel invasion assay in Ad-CD147 siRNA group, Ad-siControl group and non-infection group.(4) Gelatin zymography was done to determine the effect on reducing secretions of MMP-2 and MMP-9 in Ad-CD147 siRNA group, Ad-siControl group and non-infection group.(5) RT-PCR was used to determine the mRNA level of VEGF in Ad-CD147 siRNA group, Ad-siControl group and non-infection group.(6) Quantitative evaluation of VEGF in cell culture supernatants was with ELISA in Ad-CD 147 siRNA group,Ad-siControl group and non-infection group.Results1.Normal bladder epithelia showed either no protein expression or very weak staining. Conversely, the immunoreactive patterns of CD 147 were predominantly positively identified in the cancer tissues. CD147 stained mainly in the cell membrane and cytoplasm. There was an association between CD 147 protein expression and both tumor stage (P=0.033) and histologic grade (P=0.028). CD147 overexpression in the tumors was shown to be correlated with tumor stage and histologic grade suggesting that its expression might be important for the acquirement of malignant potential in bladder cancer.2. RT-PCR analysis indicated that Ad-CD147 siRNA infection inhibited CD147 mRNA expression level in a dose-dependent manner compared with mock and scrambled vector controls. Western Blot analysis was carried out to determine whether decreased protein expression level, as observed, correlated with decreased transcription of CD147 mRNA. A similar trend was observed by Western Blot analysis as well. The inhibitory effect of the Ad-CD147 siRNA was shown to be specific because a control Ad-siControl had no effect on CD147 expression level.3. Given that Ad-CD 147 siRNA could effectively decrease CD 147 expression, its effects on the proliferation of T-24 cells were first determined using MTT assay. Compared with Ad-siControl or mock infection, the proliferation of Ad-CD147 infection was significantly inhibited at 3 days of infection and the proliferation inhibition rates at 3,4 and 5 days were 38%,41% and 43%(P<0.01), respectively. We tested the effects of CD147 downregulation on cancer cell migration and invasion. The migration distance of mock, Ad-siControl and Ad-CD147 siRNA infected cells was 1.8, 1.73, and 0.85 mm, respectively. These findings suggest that the migration of Ad-CD147 siRNA infected cells was significantly suppressed (P<0.01). Moreover, the Ad-CD147 siRNA infected cells showed a low level of penetration through the Matrigel-coated membrane compared with cells infected with mock and Ad-siControl. Invasion of T-24 cells was reduced to 75% of that of the mock infected cells by Ad-CD147 siRNA infection. (P<0.01). The effect of CD147 on VEGF gene expression was analyzed by RT-PCR and ELISA. The VEGF protein and mRNA level were reduced by 56.5%(P<0.01) and 57.2%(P<0.01) in Ad-CD147 siRNA infected cells compared with the mocked infected cells, respectively. Ad-CD147 siRNA infection significantly decreased MMP-2 and MMP-9 secretion, as determined by gelatin zymography when compared with mock-or Ad-SV-infected cells. Secretion of MMP-2 and MMP-9 in the Ad-CD147 siRNA-infected cells was reduced by 68.5% and 37.1%, respectively, as compared with the mock infected cells (P<0.01).Conclusions1. CD147 is overexpressed in bladder cancer, and its overexpression correlated with tumor stage and histologic grade. It could be a useful indicator for the assessment of potent invasion and metastasis of bladder cancer.2. Adenovirus-delivered small interfering RNA (siRNA) vector of CD 147 was constructed successfully.3. RT-PCR and Western Blot results showed thtat CD147expression was signficantly inhibited by Ad-CD 147 siRNA infection in T-24 cell line at mRNA and protein levels.4. The results of functional experiments showed that downregulation of CD147 via RNA interference could influence the biological behaviour of tumor cells. It could be persented in the following aspects:(1) The capacity of proliferation and magriation was reduced signficiantly(2) Down-regulation of CD 147 inhibited the secretions of MMP-2, MMP-9 and the expression of VEGF. It mainly focused on the decrease of MMPs and VEGF quantity.(3)The suppression of CD147 expression in human bladder cancer cells could the invasion potential of cells. The inhibition of MMPs secretion and VEGF expression weakened the ability of proteolytic degradation and angiogenesis of bladder cancer cells, inhibited the invasion of them through reconstituted basement membrane in vitro.5. CD147 could potentially be a new experimental approach for bladder cancer gene therapy. Alteration of the malignant behavior of tumor cells by RNAi technology is a trend for the treatment of bladder cancer.