腺病毒介导的RNAi沉默CD147基因对T-24膀胱癌细胞生物学行为的影响
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摘要
研究背景
膀胱癌是我国泌尿系统最常见的恶性肿瘤。90%以上源自于移行上皮,初发病例中75%为表浅性肿瘤(pTis,pTa,pT1),25%在初诊时为侵润性肿瘤或者已发生远处转移。经尿道膀胱肿瘤电切(TURBT)辅以术后膀胱灌注化疗是现阶段治疗表浅性膀胱肿瘤的主要手段,但术后复发率可高达85%,一旦复发,其生物学行为也随之改变,往往向更高的病理分级及临床分期发展,超过20%的复发性肿瘤最终进展为侵润性癌。相对于表浅性肿瘤来说,侵润性病变治疗棘手,预后也明显不同。尽管膀胱癌根治术被认为是治疗侵润性膀胱癌(pT2-pT4)的金标准,但有50%的患者在术后2年内发生远处转移,而且大大降低了患者的生活质量,患者5年生存率不到50%。因此,寻求新的干预策略对提高膀胱癌的疗效具有特殊的迫切性和重要性。
造成膀胱癌患者死亡的主要原因是膀胱癌的侵袭性。其侵袭过程中的一个重要因素是细胞外基质的降解,其中,起重要作用的是一类Zn2+依赖的基质金属蛋白酶MMPs (Matrix Metalloproteinases)。MMPs能够破坏局部组织结构和基底膜屏障,促进肿瘤的生长和转移,并且可促进肿瘤新生血管的形成。CD147亦称EMMPRIN,是单次跨膜糖蛋白,属于免疫球蛋白超家族(IgSF)成员。CD147在多种恶性肿瘤细胞中高表达,其表达水平和肿瘤的侵袭和转移等密切相关。CD147主要刺激肿瘤细胞和邻近的纤维母细胞分泌大量的MMPs,通过影响肿瘤的生长、侵袭、转移和血管形成等促进恶性肿瘤的侵袭和转移。CD147分子还可以通过旁分泌途径促进MMPs产生；在肿瘤细胞表面与MMP-1形成复合物,使肿瘤细胞表面局部MMPs聚集,有利于其发挥降解胶原作用,从而促进肿瘤细胞的侵袭。
然而,CD147是否参与调节膀胱癌的侵润及转移过程尚未见报道。为了研究CD147和膀胱癌之间的关系,我们首先应用免疫组化检测CD147基因在膀胱癌组织中的表达情况,分析CD147与膀胱癌分期、分级等临床病理参数的关系,然后针对CD147构建了RNA干扰载体,观察其对离体膀胱癌T-24细胞侵袭的抑制作用,阐明膀胱癌进展的分子机制,为研究膀胱癌更有效的治疗手段提供理论和实验依据。
研究方法
1、免疫组化检测CD147在膀胱癌组织中的表达情况,分析CD147与膀胱癌分期、分级等临床病理参数的关系。
2、CD147基因siRNA重组腺病毒载体(Ad-CD 147 siRNA)的构建及效果鉴定。
(1)RT-PCR检测Ad-CD 147 siRNA对人膀胱癌T-24细胞CD147基因mRNA表达的影响。
(2) Western Blot检测Ad-CD147 siRNA对人膀胱癌T-24细胞CD147基因蛋白表达的影响。
3、Ad-CD147 siRNA对膀胱癌T-24细胞增殖、迁移、侵袭能力的影响
(1)MTT检测Ad-CD147 siRNA对T-24细胞增殖能力的影响。
(2)划痕实验检测Ad-CD147 siRNA对T-24细胞迁移能力的影响。
(3) Transwell检测Ad-CD147 siRNA对T-24细胞侵袭能力的影响。
(4)明胶酶谱实验检测T-24细胞感染Ad-CD147 siRNA后MMP-2、MMP-9的分泌情况。
(5) RT-PCR检测T-24细胞感染Ad-CD147 siRNA后VEGF mRNA表达水平。
(6) ELISA检测T-24细胞感染Ad-CD147 siRNA后VEGF分泌水平。
研究结果
1、CD147在正常膀胱癌组织中无表达或表达非常微弱,相反,在膀胱癌组织中阳性表达率为73.6%(59／80),CD147表达于肿瘤细胞膜和细胞质中,此研究结果说明与正常膀胱组织相比,膀胱癌组织中CD147存在过度表达。不同分期、分级膀胱癌细胞着色结果分析发现,CD147表达与膀胱癌分期(P=0.033)、分级(P=0.028)相关,而与年龄(P=0.276)、性别(P=0.196)无关,说明膀胱癌细胞不仅表达CD147,并且其表达程度与膀胱癌恶性程度成正比。
2、成功构建了针对CD147基因的siRNA重组腺病毒表达载体(Ad-CD 147 siRNA),RT-PCR和Western Blot结果显示,Ad-CD147 siRNA高效而特异地沉默了膀胱癌T-24细胞CD147基因的表达。MOI=100时,干扰效应达90%以上。
3、MTT实验结果表明,Ad-CD147 siRNA感染T-24细胞3、4和5天后,细胞增殖水平显著下降,和未感染组(Mock)相比,其下降率分别为38%,41%和43%(P<0.01)。细胞划痕实验中,Ad-CD147 siRNA感染T-24细胞后,细胞迁移能力显著下降,Ad-CD147 siRNA感染组,阴性对照组(Ad-siControl)和未感染组(Mock)的细胞迁移距离分别为0.85mm、1.73 mm和1.8mm(P<0.01)。Transwell体外侵袭实验结果显示,Ad-CD 147 siRNA感染后,T-24细胞侵袭能力显著下降,与未感染组(Mock)和阴性对照组(Ad-siControl)相比,其侵袭能力分别分别下降了75%和70%(P<0.01)。明胶酶谱实验结果发现,与未感染组(Mock)相比,Ad-CD147 siRNA感染组MMP-2、MMP-9的分泌量分别下降了68.5%和37.1%(P<0.01). VEGF RT-PCR和ELISA结果发现,与未感染组(Mock)相比,Ad-CD 147 siRNA感染组显著下降了VEGF mRNA和蛋白表达水平,其下降率分别为57.2%和56.5%(P<0.01)
结论
1、CD147基因在膀胱癌组织中存在过度表达,其表达强度与膀胱癌的分期、分级相关,可能作为判断膀胱癌是否具备高转移潜能的指标。
2、构建了CD147基因siRNA重组腺病毒载体。
(1)感染T-24细胞,经RT-PCR和Western-Blot检测,CD147分子在mRNA和蛋白水平被阻断。
(2)功能实验结果显示,RNAi较好地抑制了膀胱癌细胞的生物学行为。主要表现在：
①细胞的增殖和迁移能力明显下降。
②RNAi封闭的膀胱癌细胞自身分泌MMPs和VEGF的能力的能力大为减弱。
③在MMPs和VEGF分泌量减少的情况下,膀胱癌细胞降解胶原和血管形成能力下降,侵袭力减弱。
3、用siRNA阻断CD147基因的表达,有可能提供膀胱癌基因治疗的新的靶点,是膀胱癌治疗的一个新的方向。
Background
Bladder 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.
Methods
1. 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.
Results
1.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).
Conclusions
1. 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.
膀胱癌是我国泌尿系统最常见的恶性肿瘤。90%以上源自于移行上皮,初发病例中75%为表浅性肿瘤(pTis,pTa,pT1),25%在初诊时为侵润性肿瘤或者已发生远处转移。经尿道膀胱肿瘤电切(TURBT)辅以术后膀胱灌注化疗是现阶段治疗表浅性膀胱肿瘤的主要手段,但术后复发率可高达85%,一旦复发,其生物学行为也随之改变,往往向更高的病理分级及临床分期发展,超过20%的复发性肿瘤最终进展为侵润性癌。相对于表浅性肿瘤来说,侵润性病变治疗棘手,预后也明显不同。尽管膀胱癌根治术被认为是治疗侵润性膀胱癌(pT2-pT4)的金标准,但有50%的患者在术后2年内发生远处转移,而且大大降低了患者的生活质量,患者5年生存率不到50%。因此,寻求新的干预策略对提高膀胱癌的疗效具有特殊的迫切性和重要性。
造成膀胱癌患者死亡的主要原因是膀胱癌的侵袭性。其侵袭过程中的一个重要因素是细胞外基质的降解,其中,起重要作用的是一类Zn2+依赖的基质金属蛋白酶MMPs (Matrix Metalloproteinases)。MMPs能够破坏局部组织结构和基底膜屏障,促进肿瘤的生长和转移,并且可促进肿瘤新生血管的形成。CD147亦称EMMPRIN,是单次跨膜糖蛋白,属于免疫球蛋白超家族(IgSF)成员。CD147在多种恶性肿瘤细胞中高表达,其表达水平和肿瘤的侵袭和转移等密切相关。CD147主要刺激肿瘤细胞和邻近的纤维母细胞分泌大量的MMPs,通过影响肿瘤的生长、侵袭、转移和血管形成等促进恶性肿瘤的侵袭和转移。CD147分子还可以通过旁分泌途径促进MMPs产生；在肿瘤细胞表面与MMP-1形成复合物,使肿瘤细胞表面局部MMPs聚集,有利于其发挥降解胶原作用,从而促进肿瘤细胞的侵袭。
然而,CD147是否参与调节膀胱癌的侵润及转移过程尚未见报道。为了研究CD147和膀胱癌之间的关系,我们首先应用免疫组化检测CD147基因在膀胱癌组织中的表达情况,分析CD147与膀胱癌分期、分级等临床病理参数的关系,然后针对CD147构建了RNA干扰载体,观察其对离体膀胱癌T-24细胞侵袭的抑制作用,阐明膀胱癌进展的分子机制,为研究膀胱癌更有效的治疗手段提供理论和实验依据。
研究方法
1、免疫组化检测CD147在膀胱癌组织中的表达情况,分析CD147与膀胱癌分期、分级等临床病理参数的关系。
2、CD147基因siRNA重组腺病毒载体(Ad-CD 147 siRNA)的构建及效果鉴定。
(1)RT-PCR检测Ad-CD 147 siRNA对人膀胱癌T-24细胞CD147基因mRNA表达的影响。
(2) Western Blot检测Ad-CD147 siRNA对人膀胱癌T-24细胞CD147基因蛋白表达的影响。
3、Ad-CD147 siRNA对膀胱癌T-24细胞增殖、迁移、侵袭能力的影响
(1)MTT检测Ad-CD147 siRNA对T-24细胞增殖能力的影响。
(2)划痕实验检测Ad-CD147 siRNA对T-24细胞迁移能力的影响。
(3) Transwell检测Ad-CD147 siRNA对T-24细胞侵袭能力的影响。
(4)明胶酶谱实验检测T-24细胞感染Ad-CD147 siRNA后MMP-2、MMP-9的分泌情况。
(5) RT-PCR检测T-24细胞感染Ad-CD147 siRNA后VEGF mRNA表达水平。
(6) ELISA检测T-24细胞感染Ad-CD147 siRNA后VEGF分泌水平。
研究结果
1、CD147在正常膀胱癌组织中无表达或表达非常微弱,相反,在膀胱癌组织中阳性表达率为73.6%(59／80),CD147表达于肿瘤细胞膜和细胞质中,此研究结果说明与正常膀胱组织相比,膀胱癌组织中CD147存在过度表达。不同分期、分级膀胱癌细胞着色结果分析发现,CD147表达与膀胱癌分期(P=0.033)、分级(P=0.028)相关,而与年龄(P=0.276)、性别(P=0.196)无关,说明膀胱癌细胞不仅表达CD147,并且其表达程度与膀胱癌恶性程度成正比。
2、成功构建了针对CD147基因的siRNA重组腺病毒表达载体(Ad-CD 147 siRNA),RT-PCR和Western Blot结果显示,Ad-CD147 siRNA高效而特异地沉默了膀胱癌T-24细胞CD147基因的表达。MOI=100时,干扰效应达90%以上。
3、MTT实验结果表明,Ad-CD147 siRNA感染T-24细胞3、4和5天后,细胞增殖水平显著下降,和未感染组(Mock)相比,其下降率分别为38%,41%和43%(P<0.01)。细胞划痕实验中,Ad-CD147 siRNA感染T-24细胞后,细胞迁移能力显著下降,Ad-CD147 siRNA感染组,阴性对照组(Ad-siControl)和未感染组(Mock)的细胞迁移距离分别为0.85mm、1.73 mm和1.8mm(P<0.01)。Transwell体外侵袭实验结果显示,Ad-CD 147 siRNA感染后,T-24细胞侵袭能力显著下降,与未感染组(Mock)和阴性对照组(Ad-siControl)相比,其侵袭能力分别分别下降了75%和70%(P<0.01)。明胶酶谱实验结果发现,与未感染组(Mock)相比,Ad-CD147 siRNA感染组MMP-2、MMP-9的分泌量分别下降了68.5%和37.1%(P<0.01). VEGF RT-PCR和ELISA结果发现,与未感染组(Mock)相比,Ad-CD 147 siRNA感染组显著下降了VEGF mRNA和蛋白表达水平,其下降率分别为57.2%和56.5%(P<0.01)
结论
1、CD147基因在膀胱癌组织中存在过度表达,其表达强度与膀胱癌的分期、分级相关,可能作为判断膀胱癌是否具备高转移潜能的指标。
2、构建了CD147基因siRNA重组腺病毒载体。
(1)感染T-24细胞,经RT-PCR和Western-Blot检测,CD147分子在mRNA和蛋白水平被阻断。
(2)功能实验结果显示,RNAi较好地抑制了膀胱癌细胞的生物学行为。主要表现在：
①细胞的增殖和迁移能力明显下降。
②RNAi封闭的膀胱癌细胞自身分泌MMPs和VEGF的能力的能力大为减弱。
③在MMPs和VEGF分泌量减少的情况下,膀胱癌细胞降解胶原和血管形成能力下降,侵袭力减弱。
3、用siRNA阻断CD147基因的表达,有可能提供膀胱癌基因治疗的新的靶点,是膀胱癌治疗的一个新的方向。
Background
Bladder 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.
Methods
1. 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.
Results
1.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).
Conclusions
1. 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.
引文
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10 Sier CF, Zuidwijk K, Zijlmans HJ, et al.EMMPRIN-induced MMP-2 activaation cascade in human cervical squamous cell carcinoma. Int J Cancer.2006; 118:2991-2998.
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2 Dong Z, Bonfil RD, Chinni S, et al. matrix metalloproteinase activity and osteoclasts in experimental prostate cancer bone metastasis tissue. Am J Pathol.2005; 166:1173-1186.
3 John A, Tuszynski G. The role of matrix metalloproteinase in tumor angiogenesis and tumor metastasis. Pathol Oncol Res.2001; 7:14-23.
4 Reimers N, Zafrakas K, Assmann V, et al. Expression of extracellular matrix metalloproteinase inducer on micrometastatic and primary mammary carcinoma cells. Clin Cancer Res.2004; 10:3422-3428.
5 Riethdorf S, Reimers N, Assmann V et al. High incidence of EMMPRIN expression in human tumors. Int J Cancer.2006; 119:1800-1810.
6 Michele C, Madigan A, Kingsley J, et al. The role of extracellular matrix metalloproteinase inducer protein in prostate cancer progression. Cancer Immunol Immunother.2008; 57: 1367-1379.
7 Wu W, Wang RB. Liu H, et al. Prediction of prognosis in gallbladder carcinoma by CD147 and MMP-2 immunohistochemistry. Med Oncl.2008; 6:359-367.
8 Li HG. Xie DR. Shen XM, et al. Clinicopathological significance of expressionon paxillin, syndecan-1 and EMMPRIN in hepatocellular carcinoma. World J Gastroenterol.2005; 11: 1145-1451.
9 Vigneswaran N, Becker S, et al. Increased EMMPRIN(CD147) expression during oral carcinogenesis. Exp Mol Pathol.2006; 180:147-159.
10 Sier CF, Zuidwijk K, Zijlmans HJ, et al.EMMPRIN-induced MMP-2 activaation cascade in human cervical squamous cell carcinoma. Int J Cancer.2006; 118:2991-2998.
11 Zheng HC, Takahashi H, Murai Y, et al. Upregulated EMMPRIN might contribute to growth and angiogenesis of gastric carcinoma:a good marker for local invasion and prognosis. Brith J Cancer.2006; 95:1371-1378.
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