CD147 shRNA真核表达质粒的构建及鉴定

目的针对CD147基因的不同部位,构建不同CD147 shRNA表达质粒载体,在转录后水平抑制CD147的表达,对其克隆进行鉴定并挑选出抑制效率最高的克隆.方法用DNA重组技术将针对人CD147基因的不同部位所设计的3对shRNA序列克隆到真核表达质粒pGE-1中,构建CD147 shRNA表达质粒载体pGE-1 CD147shRNA1、2、3.用阳离子脂质体介导转染人卵巢癌高转移细胞系HO-8910pm,经G418筛选后获得克隆进行鉴定.结果3个CD147 shRNA表达载体pGE-1CD147shRNA1、2、3经PCR、限制性酶切及部分序列分析证明基因插入正确.半定量RT-PCR、Western blotting均证实:转染细胞8910 pm/pGE-1 CD147shRNA,与亲本细胞相比,CD147的表达在mRNA和蛋白水平均明显下降.结论成功构建了CD147 shRNA表达载体pGE-1 CD147shRNA,并筛选出特异而高效地阻断CD147表达的克隆.此实验结果为进一步研究CD147蛋白分子的生物学功能及应用奠定了基础,并对shRNA设计靶序列的选择有一定指导意义.     

CD147反义RNA表达质粒的构建及克隆鉴定

目的　构建CD14 7反义RNA表达质粒载体。方法　用DNA重组技术将人CD14 7基因反向克隆到真核表达质粒PCD NA3.1中 ,构建成CD14 7反义RNA表达质粒PCDNAasCD14 7。用阳离子脂质体介导转染人卵巢癌细胞系 8910 ,经G4 18筛选后获得的克隆进行鉴定。结果　CD14 7反义RNA表达质粒载体PCDNAasCD14 7经限制性酶切及部分序列分析证明基因插入正确。流式细胞仪及免疫组化SP法染色均证实 :转染细胞 8910 /PCDNAasCD14 7,与亲本细胞相比 ,CD14 7的表达明显下降。结论　成功构建了CD14 7反义RNA表达质粒载体PCDNAasCD14 7,此实验结果为进一步研究CD14 7蛋白分子的生物学功能以及为卵巢癌的基因治疗研究奠定了基础。     

Silencing of IQGAP1 by shRNA inhibits the invasion of ovarian carcinoma HO-8910PM cells in vitro

Background

IQGAP1 is a scaffolding protein and overexpressed in many human tumors, including ovarian cancer. However, the contribution of IQGAP1 to invasive properties of ovarian cancer cells remains unknown. Here, we investigated the effect of IQGAP1-specific short hairpin RNA (shRNA) expressing plasmids on metastatic potential of ovarian cancer HO-8910PM cells.

Methods

We used RT-PCR and Western blot analysis to characterize expression of IQGAP1 in three human ovarian cancer-derived cell lines SK-OV-3, HO-8910 and HO-8910PM. We then determined whether expression of endogenous IQGAP1 correlated with invasive and migratory ability by using an in vitro Matrigel assay and cell migration assay. We further knocked down IQGAP1 using shRNA expressing plasmids controlled by U1 promoter in HO-8910PM cells and examined the proliferation activity, invasive and migration potential of IQGAP1 shRNA transfectants using MTT assay, in vitro Matrigel-coated invasion assay and migration assay.

Results

IQGAP1 expression level seemed to be closely associated with the enhanced invasion and migration in ovarian cancer cell lines. Levels of both IQGAP1 mRNA and protein were significantly reduced in HO-8910PM cells transfected with plasmid-based IQGAP1-specific shRNAs. RNAi-mediated knockdown of IQGAP1 expression in HO-8910PM cells resulted in a significant decrease in cell invasion and migration.

Conclusion

Our findings support the hypothesis that IQGAP1 promotes tumor progression and identify IQGAP1 as a potential therapeutic strategy for ovarian cancer and some other tumors with over-expression of the IQGAP1 gene.     

靶向RhoA shRNA对卵巢癌HO8910细胞体外侵袭能力影响的研究

目的:观察RNA干扰(RNAi)技术对卵巢上皮癌HO8910细胞株RhoA基因表达的抑制作用,探讨其对HO8910细胞侵袭、迁移的影响。方法:构建RhoA基因特异性短发夹状RNA(shRNA)真核表达载体,以脂质体介导转染卵巢癌细胞HO8910,实时荧光定量PCR和蛋白质印迹法检测RNAi后HO8910中RhoA的mRNA及蛋白表达水平变化,Transwell小室体外侵袭和划痕试验检测细胞侵袭和迁移能力。结果:转染RhoA shRNA(质粒1、2和3)组的RhoA mRNA的表达明显弱于未转染任何质粒的空白组和对照组,P<0.05,且质粒2组表达最低(27.9%),抑制效率最高(72.1%),具有序列特异性。转染序列特异性RhoA shRNA的细胞(实验组)与对照组比较,RhoA蛋白的表达分别为(6.08±2.24)%和(59.25±17.96)%,P<0.05;细胞平均侵袭能力测值为18.82±2.61和35.25±5.12,P<0.05;愈合百分比为(43.80±6.21)%和(69.35±4.42)%,P<0.05。结论:靶向RhoA的shRNA可抑制卵巢癌HO8910细胞RhoA基因的表达,能降低细胞体外侵袭和迁移能力。     

Knockdown of UHRF1 by Lentivirus-mediated shRNA Inhibits Ovarian Cancer Cell Growth

Human UHRF1 (ubiquitin-like PHD and RING finger domain-containing 1) has been reported to beover-expressed in many cancers, but its role in ovarian cancer remains elusive. Here, we determined whetherknockdown of UHRF1 by lentivirus-mediated shRNA could inhibit ovarian cancer cell growth. Lentivirusmediatedshort hairpin RNAs (lv-shRNAs-UHRF1) were designed to trigger the gene silencing RNA interference(RNAi) pathway. The efficiency of lentivirus-mediated shRNA infection into HO-8910 and HO-8910 PM cells wasdetermined using fluorescence microscopy to observe lentivirus-mediated GFP expressionand was confirmed to beover 80 percent. UHRF1 expression in infected HO-8910 and HO-8910 PM was evaluated by real-time PCR andWestern blot analysis. The Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability; flow cytometryand Hoechst 33342 assay was applied to measure cell cycle arrest and apoptosis. Cell invasion was assessedusing transwell chambers. Our results demonstrated that the loss of UHRF1 promoted HO-8910 and HO-8910PM cell apoptosis, while inhibiting cell proliferation. In addition, UHRF1 knockdown significantly inhibitedthe invasion of human ovarian cancer cells. In the present study, we also showed that depleting HO-8910 cellsof UHRF1 caused activation of the DNA damage response pathway, with the cell cycle arrested in G2/M-phase.The DNA damage response in cells depleted of UHRF1 was illustrated by phosphorylation of CHK (checkpointkinase) 2 on Thr68, phosphorylation of CDC25 (cell division control 25) on Ser 216 and phosphorylation ofCDK1 (cyclin-dependent kinase 1) on Tyr 15.     

Effect of inhibition of the adrenomedullin gene on the growth and chemosensitivity of ovarian cancer cells

Adrenomedullin (AM), a potent vasodilator peptide, is present in various types of tumors. Here, we constructed short hairpin RNA (shRNA) in order to target the AM gene in?vitro using RNA interference (RNAi) technology. HO8910 ovarian cancer cells were transfected, and the effects of AM on proliferation and chemosensitivity of the cells were examined. RT-PCR, real-time PCR and western blot analysis were performed to detect the AM gene and protein expression. The MTT assay was used to observe the effect of AM on proliferation and chemosensitivity of the cells. Also, the protein levels of bcl-2 and the extracellular regulated protein kinase (ERK) were evaluated by western blot analysis. We found that silencing of the AM gene inhibited the proliferation and increased the chemosensitivity of HO8910 cells, reduced the expression of AM mRNA and protein as well as downregulated bcl-2 and p-ERK expression. We, therefore, conclude that silencing of the AM gene in HO8910 ovarian cancer cells inhibited the proliferation and increased the chemosensitivity of the cells through downregulation of ERK and bcl-2 expression. Thus, anti-AM treatment together with suppression of ERK and bcl-2 expression provides a novel research approach for ovarian cancer.     

RNAi-mediated silencing of CD147 inhibits tumor cell proliferation,invasion and increases chemosensitivity to cisplatin in SGC7901 cells in vitro

Background

CD147 is a widely distributed cell surface glycoprotein that belongs to the Ig superfamily. CD147 has been implicated in numerous physiological and pathological activities. Enriched on the surface of many tumor cells, CD147 promotes tumor growth, invasion, metastasis and angiogenesis and confers resistance to some chemotherapeutic drugs. In this study, we investigated the possible role of CD147 in the progression of gastric cancer.

Methods

Short hairpin RNA (shRNA) expressing vectors targeting CD147 were constructed and transfected into human gastric cancer cells SGC7901 and CD147 expression was monitored by quantitative realtime RT-PCR and Western blot. Cell proliferation, the activities of MMP-2 and MMP-9, the invasive potential and chemosensitivity to cisplatin of SGC7901 cells were determined by MTT, gelatin zymography, Transwell invasion assay and MTT, respectively.

Results

Down-regulation of CD147 by RNAi approach led to decreased cell proliferation, MMP-2 and MMP-9 activities and invasive potential of SGC7901 cells as well as increased chemosensitivity to cisplatin.

Conclusion

CD147 involves in proliferation, invasion and chemosensitivity of human gastric cancer cell line SGC7901, indicating that CD147 may be a promising therapeutic target for gastric cancer.     

Inhibition of CD147 gene expression via RNA interference reduces tumor cell invasion, tumorigenicity and increases chemosensitivity to cisplatin in laryngeal carcinoma Hep2 cells

CD147, also named extracelluar matrix metalloproteinase inducer (EMMPRIN), is a member of the immunoglobulin family and a glycoprotein enriched on the surface of tumor cells, which promotes invasion, metastasis, growth and survival of malignant cells, and is known to confer resistance to some chemotherapeutic drugs. To determine the possible role of CD147 in the invasive properties of laryngeal carcinoma, we used an RNA interference approach to silence CD147 expression in the Hep2 cell line at high levels of CD147 expression. Our results showed that CD147 expression was significantly impeded at both mRNA and protein levels, which resulted in a decrease of the Hep2 invasion activity in vitro and tumorigenicity in nude mice. The suppression of CD147 expression also sensitized cells to cisplatin. Our current results indicated that CD147 was a laryngeal carcinoma-related gene and CD147 might be a potential target for therapeutic anti-cancer drugs.     

巨噬细胞游走抑制因子在卵巢癌侵袭中的作用与意义

背景与目的:巨噬细胞游走抑制因子(maerophage migration inhibitory factor,MIF)与肿瘤的恶性进展密切相关.本研究探讨MIF基因对卵巢癌HO-8910和OVCAR-3细胞侵袭和增殖能力的影响及其在卵巢癌组织中表达的意义.方法:瞬时转染小干扰RNA(small interfering RNA,siRNA)靶向敲除MIF基因,RT-PCR和Western blot检测MIF在mRNA和蛋白水平的表达,通过体外迁移、侵袭实验和噻唑蓝比色法(MTT)分析MIF对HO-8910和OVCAR-3细胞迁移、侵袭和增殖能力的影响;免疫组织化学检测MIF蛋白在卵巢癌组织中的表达情况.结果:与阴性对照组细胞比较,瞬时转染MIF siRNA的HO-8910和OVCAR-3细胞中MIF基因表达水平明显降低.MTT结果显示,转染MIF siRNA的两株卵巢癌细胞增殖率显著低于阴性对照组(P＜0.05).转染MIF siRNA的HO-8910细胞穿膜细胞数(MIF-si1:48.0±7.3;MIF-si2:38.0±3.6)与阴性对照组(78.0±8.5)比较差异有统计学意义(P＜0.05),其穿过铺了Matrigel膜的细胞数(MIF-sil:35.0±5.0:MIF-si2:30.0±5.6)也显著少于阴性对照组(P＜0.05).同样,转染了MIF siRNA的OVCAR-3细胞穿膜细胞数(MIF-si1:40.0±4.5;MIF-si2:42.0±3.0)与阴性对照组(65±2.1)比较,差异也有统计学意义(P＜0.05),其穿过铺了Matrigel膜的细胞数(MIF-sil:25.0±3.0:MIF-si2:27.0±3.4)也明显低于阴性对照组(P＜0.05).53.6%(37/69)卵巢癌组织中有MIF蛋白表达.MIF蛋白表达与肿瘤临床分期呈显著正相关(P＜0.01).结论:MIF基因可能通过促进肿瘤细胞的增殖和侵袭能力在卵巢癌的发生发展中发挥重要作用,有可能成为分子靶向治疗卵巢癌的潜在靶点.     

Inhibition of basigin expression in glioblastoma cell line via antisense RNA reduces tumor cell invasion and angiogenesis

Basigin/CD147, also named extracelluar matrix metalloproteinase inducer (EMMPRIN), has been implicated in playing very important roles in several aspects of tumor progression. In this study, we examined the inhibitory effects of antisense RNA of CD147 on invasion and angiogenesis of human glioblastoma U251 cells in vitro. The U251 cell line was transfected by a plasmid containing antisense CD147 cDNA. Gelatin zymography was used to determine the effect on reducing secretions of MMP-2 and MMP-9 of the transfected cells. Boyden chamber was employed to test the invasion of U251 cells in vitro. We found that downregulation of CD147 resulted in reducing secretions of MMP-2, MMP-9, and VEGF. Moreover, the invasion of stable antisense transfectants was inhibited. Wound-induced migration assay also showed decreased migration in stable antisense transfectants compare to parental- and empty vector-transfected cells. Taken together, these results provide evidence that invasion of human glioblastoma cells can be inhibited by antisense RNA of CD147. Basigin/CD147 may be used as a potential target of drugs for anti-invasion and metastasis of human glioblastoma cells.     

Inhibition of CD147 expression reduces tumor cell invasion in human prostate cancer cell line via RNA interference

CD147, also named extracelluar matrix metalloproteinase inducer (EMMPRIN), has been proved to be involved in the invasion and metastasis processes of tumor cells in many types of cancers. To determine the role of CD147 in the invasiveness properties of prostate cancer, we successfully downregulated CD147 by RNA interference (RNAi) technology, in PC-3 cell line at high level of CD147 expression. PC-3 cells were transfected with a pSilencer 4.1-CMV neo Vector coding for an RNA composed of two identical 19-nucleotide sequence motifs in an inverted orientation, separated by a 9-bp spacer to form a hairpin dsRNA capable of mediating target CD147 inhibition. Gelatin zymography was employed to determine the effect on reducing secretions of MMP-2 and MMP-9 of the transfected cells. Matrigel invasion assay was performed to evaluate the invasion ability of PC-3 cells in vitro. Our results showed that CD147 expression was significantly inhibited by small interfering RNAs (siRNA) transfectants in PC-3 cells at mRNA and protein levels, which resulted in dramatic reduction of invasion ability in tumor cells. Moreover, downregulation of CD147 resulted in reducing secretions of MMP-2, MMP-9. Taken together, CD147 downregulation by RNAi technology decreases the invasive capability of prostate cancer cells, demonstrating that stable expression of siRNA CD147 could potentially be an experimental approach for prostate cancer gene therapy.     

Involvement of GRP78 in the Resistance of Ovarian Carcinoma Cells to Paclitaxel

Background: Glucose regulated protein 78 (GRP78) is a type of molecular chaperone. It is a possible candidateprotein that contributes to development of drug resistance. We first examined the involvement of GRP78 inchemotherapy-resistance in human ovarian cancer cell. Materials and Methods: The expression of GRP78mRNA and protein were examined by RT-PCR and western blotting, respectively, in human ovarian cancer cellsline (HO-8910). Sensitivity of HO-8910 to paclitaxel was determined with methyl thiazolyl tetrazolium (MTT).Suppression of GRP78 expression was performed using specific small-interfering RNA (siRNA) in HO-8910cells, and cell apoptosis was assessed by flow cytometry. Statistical analysis was performed using the SPSS 15.0statistical package. Results: HO-8910 cells, with high basal levels of GRP78, exhibited low sensitivity to paclitaxel.The mRNA and protein levels of GRP78 were dramatically decreased at 24h, 48h and 72h after transfection andthe sensitivity to paclitaxel was increased when the GRP78 gene was disturbed by specific siRNA transfection.Conclusions: The results suggested that high GRP78 expression might be one of the molecular mechanismscausing resistance to paclitaxel, and therefore siRNA of GRP78 may be useful in tumor-specific gene therapyfor ovarian cancer.     

HMGA1基因小分子RNAi对卵巢癌转移抑制的实验研究

目的：探讨高迁移率蛋白家族A1（high mobility group A1,HMGA1）基因小分子干扰RNA对卵巢癌转移抑制的机理。方法：RT—PCR一步法检测3种不同的卵巢癌细胞株中HMGA1、E钙黏蛋白mRNA的表达水平;设计并合成HMGA1 siRNA,转染H08910PM细胞：半定量RT—PCR分析法观察HMGA1 siRNA转染对E钙黏蛋白mRNA表达的逆转作用,及对卵巢癌细胞株体外侵袭、运动的抑制作用。结果：RT—PCR半定量分析结果显示：OVCAR-3细胞中HMGA1 mRNA表达量相对较低（0．64±0．13）,而E-钙黏蛋白mRNA仅在OVCAR-3细胞中有表达;H08910PM细胞稳定转染HMGA1 siRNA后,转染组、对照组HMGA1 mRNA表达水平分别为0．16±0．08、0．47±0．11（P〈0．01）,E钙黏蛋白mRNA表达水平分别为0．38±0．07、0．09±0.05（P〈0．01）;体外运动实验显示,跨膜细胞数转染组明显低于对照组（P〈0．05）;重组细胞基底膜侵袭实验显示,穿透基底膜细胞数转染组明显低于对照组（P〈0．01）。结论：HMGA1基因与卵巢癌转移密切相关,HMGA1基因siRNA可上调卵巢癌细胞中E钙黏蛋白的表达,抑制卵巢癌细胞的运动和侵袭,为卵巢癌转移的基因治疗提供新的靶点。     

高转移人卵巢癌细胞系及其母系多基因表达研究

为研究高转移人卵巢癌细胞系ＨＯ８９１０ＰＭ及其母系ＨＯ８９１０多基因表达情况及其彼此关系,采用ＳＰ免疫组化染色方法,观察了９种基因产物的表达。结果显示,除ｂａｘ外,其余８种基因产物在２株细胞均呈不同程度的阳性表达。在ＨＯ８９１０ＰＭ细胞系中,以ｐ５３、ＣｙｃｌｉｎＤ１、ＣＤ４４Ｖ６、ＥＧＦＲ的表达强度强于母系;而ｐ１６、ｎｍ２３表达强度则较母系弱。２株细胞ｃｅｒｂＢ２、ｂｃｌ２表达无明显不同。结果表明,ＨＯ８９１０ＰＭ是一株较母系ＨＯ８９１０更具侵袭和转移生长潜能的细胞株。同时也证实肿瘤的发生、浸润和转移是多基因参于、多阶段协同作用的结果     

shRNA against CD44 inhibits cell proliferation, invasion and migration, and promotes apoptosis of colon carcinoma cells

CD44 is a causal factor for tumor invasion, metastasis and acquisition of resistance to apoptosis. CD44 knockdown using inducible short hairpin RNA (shRNA) significantly reduces cell growth and invasion. Short hairpin RNA against CD44 and pGFP-V-RS-vector was used for knockdown of CD44 expression in SW620 colon cancer cells. Cell growth, invasion and migration assay, immunofluorescence for β-catenin expression and western blotting for Wnt signaling molecules were analyzed. Cell cycle analysis and western blot analysis for apoptotic molecules were evaluated. Short hairpin RNA against CD44 reduced the expression of CD44. Cell proliferation, migration and invasion were markedly inhibited and apoptosis was increased in shRNA CD44-transfected cells. Knockdown of CD44 decreased the phosphorylation of PDK1, Akt and GSK3β, and β-catenin levels. Decreased phosphorylated Akt led to an increase in phosphorylated FoxO1 and induced cell cycle arrest in the G0-G1 phase and a decrease in the S phase. The levels of Bcl-2 and Bcl-xL expression were down-regulated, while the levels of BAX expression and cleaved caspase-3, -8 and -9 were increased. CD44 knockdown by way of shRNA inhibited cell proliferation and induced cell apoptosis. This can be used as a therapeutic intervention with the anti-survival/pro-apoptotic machinery in human colon cancer.     

人反义VEGF基因表达载体的构建及其对卵巢癌细胞增殖的影响

目的：构建人反义VEGF基因真核表达载体,进行抗血管生成治疗卵巢癌实验。方法：RT－PCR法获得人VEGF基因,将VEGF基因反向克隆入真核表达载体pcDNA3中,酶切鉴定结果。用此真核表达载体转染人卵巢癌细胞HO－8910,G418筛选获得阳性克隆,RNA斑点杂交鉴定HO－8910细胞中VEGF表达。Western blot及间接免疫荧光法检测转染前后HO－8910 细胞中VEGF蛋白表达。检测转染前后瘤细胞的生物学性状及裸鼠体内致瘤性。结果：RT－PCR法得到VEGF基因,并获得反向构建的VEGF真核表达载体。VEGF反义RNA部分阻断了HO－8910细胞中VEGF表达,转染后单个细胞的克隆形成能力明显减弱;细胞周期中,G1期细胞增多,S期细胞减少,细胞增殖能力降低;形态学超微结构显示细胞器扩张和肿胀,核染色质边集,凝集成块,可见明显的凋亡改变。裸鼠体内致瘤性降低。结论：成功构建了反义VEGF基因真核表达载体,该载体可明显抑制卵巢癌细胞增殖,为卵巢癌的基因治疗提供了一定的实验依据。     

MTA1在卵巢癌中的表达及其对卵巢癌细胞侵袭转移影响的研究

  目的  研究转移相关基因1(metastasis-associated-gene1, MTA1)表达与卵巢癌发生发展转移的关系, 研究MTA1对卵巢癌侵袭转移能力的影响, 并探讨抑制卵巢癌侵袭转移的潜在靶点。  方法  免疫组织化学法检测110例卵巢癌组织中MTA1的蛋白表达水平, 分析MTA1蛋白表达与卵巢癌分化程度、临床分期及与腹腔转移的关系。并通过脂质体介导方法, 将特异性siRNA表达载体psilenter2.0-MTA1-siRNA转染入人卵巢癌细胞系HO-8910PM, 采用RT-PCR以及Western blot检测特异性siRNA对MTA1mRNA及蛋白表达的抑制效果。应用划痕损伤实验及Transwell实验检测MTA1对卵巢癌细胞侵袭转移能力的影响。  结果  MTA1随卵巢癌组织学分化程度的升高而降低, 呈负相关, MTA1的表达随着FIGO分期期别的增加而增加, 呈正相关, MTA1的表达随卵巢癌腹腔转移而增加, 呈正相关。RT-PCR及Western blot结果显示, siRNA成功抑制卵巢癌细胞系HO-8910PM中MTA1的表达。划痕损伤实验显示转染后划痕损伤愈合明显减慢, 迁移率明显降低, Transwell体外侵袭实验结果显示, 转染后穿膜细胞百分率显著降低(P < 0.05)。  结论  MTA1表达水平的增高与卵巢癌的分化程度、临床分期及远处转移密切相关, 体外研究显示抑制MTA1在卵巢癌细胞中的表达, 使细胞生长、侵袭及转移能力均受到抑制, 提示MTA1在卵巢癌的远处侵袭转移过程中发挥重要作用, 可能成为卵巢癌基因治疗的潜在靶点。      

CD147 silencing via RNA interference reduces tumor cell invasion, metastasis and increases chemosensitivity in pancreatic cancer cells

CD147, which belongs to the immunoglobulin superfamily, is a multifunctional glycoprotein that has been shown to increase tumor invasion, metastasis and multidrug resistance. To define the role of CD147 in invasion and metastasis more precisely, we utilized gene silencing to inhibit the expression of CD147 in pancreatic cancer cells. We observed that CD147 expression was significantly impeded at both the mRNA and protein levels and resulted in a decrease of MMP-2 and MMP-9 activities. There was also a decrease of MCT1 expression in the invasion and metastasis potential of pancreatic cancer cells, as well as increased chemosensitivity to gemcitabine in Panc-1 cells. Overall, these results suggest that CD147 plays an important role in the invasion, metastasis and chemosensitivity of the human pancreatic cancer cell line Panc-1, indicating that CD147 may be a promising therapeutic target for pancreatic cancer.     

A small interfering CD147-targeting RNA inhibited the proliferation, invasiveness, and metastatic activity of malignant melanoma

CD147 plays a critical role in the invasive and metastatic activity of malignant melanoma cells by stimulating the surrounding fibroblasts to express matrix metalloproteinases and vascular endothelial growth factor. We developed a system that blocks CD147 in the human malignant melanoma cell line, A375, using RNA interference. By transfecting melanoma cells with the small interfering RNA (siRNA) that targets human CD147, we were able to establish two stable clones in which CD147 expression was significantly down-regulated. This resulted in the decreased proliferation and invasion of A375 cells in vitro. CD147 siRNA also down-regulated the expression of vascular endothelial growth factor in these cells and reduced the migration of vascular endothelial cells. The reduction in the CD147 level suppressed the size of s.c. tumors and the microvessel density in an A375 s.c. nude mouse xenograft model. In addition, the in vivo metastatic potential of A375 cells transfected with CD147 siRNA was suppressed in a nude mouse model of pulmonary metastasis.