RNA干扰Krüppel样转录因子8对肾癌786-0细胞体内外生长的影响
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摘要
肾细胞癌(renal cell carcinoma，RCC)是泌尿系统的常见肿瘤，占全部成人肿瘤发病率的2％。根治性肾癌切除术(radical nephrectomy，RN)是治疗局限性肾细胞癌最有效的方法，由于缺乏早期诊断及对晚期患者的有效治疗，此类患者仍保持着较高的死亡率。25％的肾细胞癌患者确诊时已处于局部进展期或发生转移。放、化疗对晚期肾细胞癌治疗效果不佳，5年生存率仅为5％-10％。白介素(interleukin)和干扰素(interferon)单一或联合免疫治疗由于毒性大，临床使用上受到限制，而且免疫治疗总缓解率不高。因此了解肾细胞癌转移和进展的分子机制对于提高早期诊断和患者生存率的治疗十分重要。
Krüppel样转录因子8(Krüppel-like factor 8，KLF8)是Krüppel样转录因子家族成员之一。KLF8在恶性肿瘤中呈高表达，正常组织中低表达。相关研究表明KLF8通过激活细胞周期蛋白D1(cyclin D1)的启动子，在粘着斑激酶(focal adhesionkinase，FAK)调控细胞周期、细胞致癌性转化、侵袭力、上皮间质转型(epithelial tomesenchymal transition，EMT)方面发挥重要作用。但KLF8在肾细胞癌的发生、发展、侵袭方面所起的作用尚不清楚。
本课题首先应用逆转录-聚合酶链反应(reverse transcription-polymerasechain reaction，RT-PCR)和免疫组织化学方法检测42例散发性肾细胞癌患者的KLF8基因和蛋白质的表达情况，初步探讨了KLF8基因与肾细胞癌发生、发展的可能相关性。接下来将构建的经化学修饰的小干扰RNA(small interference RNA，siRNA)转染到表达KLF8基因的肾癌786-0细胞中。明确KLF8-siRNA在基因和蛋白水平能够有效抑制KLF8的表达。并研究干扰KLF8基因后对细胞生长、细胞侵袭力、细胞凋亡和细胞周期，对FAK、cyclinD1、钙粘蛋白(E-cadherin)基因和蛋白表达的影响。然后接种肾癌786-0细胞形成裸鼠移植瘤模型后，探讨RNA干扰KLF8基因后对裸鼠致瘤性及肾移植瘤内KLF8基因和蛋白表达的影响。本课题采用RNA干扰方法研究KLF8基因对肾癌786-0细胞增殖和肿瘤生长的影响，为肾癌的基因治疗提供了一定的实验依据。
第一部分
KLF8在肾癌组织中的表达及临床意义
目的：探讨Krüppel样转录因子8(KLF8)在肾透明细胞癌的表达及其临床意义。
方法：应用RT-PCR和免疫组织化学方法从基因和蛋白水平检测了42例原发性散发肾透明细胞癌患者和癌旁正常肾组织中KLF8 mRNA和蛋白的表达情况。
结果：肾癌组织中的KLF8mRNA相对表达量为(0．869±0．049)，癌旁正常肾组织的KLF8mRNA相对表达量为(0．416±0．018)，组间比较差异有统计学意义(P＜0．001)。42例肾癌组织中有36例表达KLF8蛋白，表达率为85．7％(36／42)；42例癌旁正常肾组织有16例表达KLF8蛋白，表达率为38．1％(16／42)，肾癌组织KLF8蛋白表达水平显著高于癌旁正常肾组织，组间比较差异有统计学意义(P＜0．001)。KLF8 mRNA和蛋白表达与肿瘤大小(P＜0．001)和临床分期(P＜0．001)相关，与年龄、性别、细胞分化程度无关(P＞0．05)。
结论：肾癌组织KLF8mRNA和蛋白表达增高，可能与肾癌的发生、进展相关，有可能成为肾癌基因治疗的靶点。
第二部分
RNA干扰KLF8基因有效序列的构建和筛选
目的：探讨RNA干扰KLF8基因有效序列的构建和筛选。
方法：设计和合成可抑制KLF8表达的经过化学修饰的siRNA，并转染进入肾癌786-0细胞中。利用RT PCR和Western blot方法检测KLF8mRNA和蛋白表达情况。筛选出一条抑制效率最高的KLF8-siRNA进入下一步研究。
结果：与空白组(0．977±0．003)和阴性对照组(0．917±0．014)相比，各干扰组KLF8 mRNA相对表达量分别siRNA1(0．276±0．017)，siRNA2(0．084±0．005)，siRNA3(0．316±0．014)，组间差异有统计学意义(P＜0．001)。与空白组(0．822±0．016)和阴性对照组(0．807±0．019)相比，各干扰组KLF8蛋白相对表达量分别siRNA1(0．491±0．012)，siRNA2(0．264±0．029)，siRNA3(0．401±0．013)，组间差异有统计学意义(P＜0．001)。空白组与阴性对照组KLF8 mRNA和蛋白表达相比，组间比较差异无统计学意义(P＞0．05)。与RT-PCR结果一致，siRNA2抑制KLF8蛋白效率最高。
结论：构建成功的KLF8-siRNA序列可有效抑制肾癌786 0细胞中的KLF8基因和蛋白表达，KLF8 siRNA2序列抑制效率最高，选择它进入下一步研究。
第三部分
RNA干扰KLF8基因对肾癌786-0细胞体外生长的影响目的：探讨RNA干扰KLF8基因后对肾癌786-0细胞体外生长的影响。方法：将KLF8-siRNA2序列转染进入肾癌786-0细胞中。利用RT-PCR和Westernblot方法检测cyclinD1、FAK、E-cadherin mRNA和蛋白表达情况；MTT法检测肾癌细胞生长情况；Matrigel Invasion Assay方法检测肾癌细胞侵袭能力；流式细胞术检测肾癌细胞周期及细胞凋亡情况。
结果：与空白组(0．693±0．008)、(0．487±0．020)和阴性对照组(0．715±0．010)、(0．490±0．021)相比，KLF8-siRNA2组cyclinD1 mRNA(0．217±0．018)、FAK mRNA(0．1 13±0．012)表达均受到抑制(P＜0．001)；与空白组(0．780±0．017)、(0．810±0．021)和阴性对照组(0．607±0．024)、(0．593±0．008)相比，KLF8-siRNA2组cyclinD1蛋白(0．323±0．061 1)、FAK蛋白(0．380±0．021)表达均受到抑制(P＜0．001)；而空白组与阴性对照组cyclinD1、FAK mRNA和蛋白表达相比，组间差异无统计学意义(P＞0．05)。E-cadherin mRNA和蛋白表达上调。与空白组和阴性对照组相比，MTT显示KLF8-siRNA2组肾癌细胞生长减慢(P＜0．05)；Matrigel Invasion Assay法显示KLF8-siRNA2组细胞侵袭能力下降(P＜0．001)；流式细胞术分析表明与空白组和对照组相比，KLF8-siRNA2组G0／G1期细胞比例增加(P＜0．001)，S期比例减少(P＜0．001)，凋亡细胞比例增加(P＜0．001)。
结论：RNA干扰KLF8基因后可下调cyclinD1、FAK mRNA和蛋白表达，下调KLF8同时可上调E-cadherin的表达，同时减慢肾癌细胞的生长；降低细胞侵袭力；影响细胞周期及增加凋亡。KLF8在促进肾癌细胞生长、调控细胞周期，抑制细胞凋亡、维持细胞侵袭力等方面可能具有重要作用。KLF8可能是FAK调控肾细胞周期的关键因子，两者之间存在双向调节。RNA干扰下调KLF8基因有可能逆转肾细胞癌中EMT。
第四部分
RNA干扰KLF8基因对肾癌786-0细胞体内生长的影响
目的：探讨干扰KLF8基因后对体内人肾癌细胞致瘤性的影响。
方法：18只裸鼠随机分成空白组和阴性对照组、KLF8-siRNA2组，每组6只。采用肾癌786-0细胞悬液皮下接种3．5×10~6／0．2ml(RP1640和PBS液各为0．1ml)的方法制作裸鼠移植瘤模型。肾移植瘤成功后，分别给予等量PBS液、50μgscramble RNA液及50μgKLF8-siRNA2液进行肿瘤注射治疗，3天1次，连续6周。于治疗6周后处死裸鼠，计算肿瘤瘤重及肿瘤体积及绘制生长曲线；并应用RT-PCR及Western blot方法检测KLF8在mRNA和蛋白质水平的表达情况。
结果：治疗6周后KLF8-siRNA2组裸鼠肿瘤的肿瘤重量(2．073±0．067)g小于空白组(3．317±0．144)g和阴性对照组(3．333±0．185)g，组间差异有统计学意义(P＜0．001)，而空白组和阴性对照组组间差异无统计学意义(P＞0．05)。与空白组(0．488±0．021)和阴性对照组(0．483±0．017)相比，KLF8-siRNA2组KLF8mRNA(0．269±0．028)的表达显著降低，组间差异有统计学意义(P＜0．001)；与空白组(0．785±0．016)和阴性对照组(0．731±0．023)相比，KLF8-siRNA2组KLF8蛋白表达下降(0．278±0．044)，组间差异有统计学意义(P＜0．001)。而空白组和阴性对照组KLF8mRNA和蛋白相比，组间差异无统计学意义(P＞0．05)。
结论：转染KLF8-siRNA2后可有效抑制裸鼠模型肿瘤的生长并降低了瘤体内KLF8基因和蛋白的表达，提示KLF8基因在肾癌的发生、发展过程中可能发挥着重要的作用。本研究也为以KLF8为靶点的肿瘤基因沉默疗法提供了可靠的理论和实验基础。
Renal cell carcinoma (RCC) is the common genitourinary tract tumor andaccounts for approximately 2% of adult cancer.Radical nephrectomy is the mosteffective treatment for localized renal cell carcinoma.RCC remains the highmortality due to the lack of early detection methods and effective treatmentsfor late-stage cancers.Twenty-five percent of patients were diagnosed withlocally invasive or metastatic RCC.The lack of sensitive efficacy ofchemotherapy and radiation therapy in late-stage RCC has led to a 5-year survivalranging from 5% to i0%.The use of recombinant human interleukin-2 (IL-2) andrecombinant human interferonα(IFN-α), either alone or in combination,however, is limited by their toxicity and generally poor overall responserates.Understanding the molecular mechanisms underlying RCC progression andmetastasis is important for developing new strategies for early diagnosis andtherapies required for improvement of patient survival.
Kruppel like factor 8 ( KLF8 ) was one of Kruppel like transcription factorfamily.Over-expressed KLF8 has recently been found in some of human malignanttumors,while it is low expressed in the normal tissues.KLF8 has been thoughtas a key transcription factor downstream of FAK in the regulation of the cellcycle by activating cyclin D1 gene promoter and also plays a critical role inoncogenic transformation、cell invasion and epithelial to mesenchymaltransition (EMT). Especially,whether and how KLF8 might play a role in RCCtumorigenesis、progression and invasion is little known.
In the present study,we firstly investigated the expression of KLF8 mRNAand protein in 42 sporadic RCC samples by reverse transcription polymerase chainreaction (RT-PCR) and immunohistochemistry(IHC),explored the correlation ofKLF8 and the carcinogenesis with development of human renal cell carcinoma.Subsequently we constructed small interference RNA (siRNA) sequences targetingKLF8, transfected it into the human RCC cell line 786-0, identified thatKLF8-siRNA can inhibited expression of KLF8 mRNA and protein leveleffectively.Further,we explored changes in cell growth, invasiveness、cellcycle and cell apoptosis,studied the influence on the cyclin Dl and FAK、E-cadherin mRNA and protein when KLF8-siRNA transfected 786-0 cells.Inoculating the renal carcinoma cells and interference renal carcinomacells to the nude mice,the influence of KLF8 genes on the oncogenicity wasinvestigated.In this project,using the KLF8-siRNAmethod to study the influenceon the proliferation of renal carcinoma 786-0 cells and the development of tumor,providing the experimental evidences to the gene therapy of renal cellcarcinoma.
PartⅠ
The expression KLF8 in renal cell carcinomaand clinical significances
Objectives: To investigate the expression of KLF8inrenal cell carcinomaand clinical significances.
Methods:We firstly investigated the expression of KLF8 mRNA and proteinin 42 sporadic RCC samples and adjacent non-tumorous renal tissues by RT-PCRand IHC.
Results:RT-PCR results revealed that the relative expression of KLF8 mRNAin RCC and adjacent non-tumorous renal tissues was (0.869±0.321) and (0.416±0.117) respectively and statistically significant difference exists betweenthem (P＜0.001).KLF8 protein expression was positive in 36 cases of RCC tissues(36/42,85.7%),higher than that in adjacent non-tumorous tissues (16/42,38.1%,P＜0.001).The high levels of KLF8 mRNA and protein expression were relatedwith the larger tumor size (P＜0.001) and high clinical stage(P＜0.001),but notrelated with sex,age and cell differentiate (P＞0.05).
Conclusions:The KLF8 mRNA and protein expression were elevated in thehuman RCC.KLF8 was involved in the development and progression of RCC,whichmay be used as a target of renal cell carcinoma gene therapy.
PartⅡ
The construction and identification of RNA interferencingKLF8 sequences
Objectives:To investigate the construction and identification of RNAinterferencing KLF8 sequences.
Methods:Designing and synthesizing the siRNA which can inhibit the KLF8expression and transfecting with renal cancer cell 786-0. Checking theexpression level of KLF8 mRNA and protein by RT-PCR and Western blot.We wantedto select a most efficient siRNA sequences for further study.
Results:Compared with blank control (0.977±0.003) and scrambled siRNA(0.917±0.014),KLF8-siRNA significantly inhibited the relative expression ofKLF8 mRNA(P＜0.001): siRNA1 (0.276±0.017), siRNA2 (0.084±0.005), siRNA3(0.316±0.014). SiRNA2 was identified as the most potent sequence whichsuppressed KLF8 mRNA expression.
Western blot analysis also demonstrated a significant reduction in KLF8protein level(P＜0.001):siRNA1 (0.491±0.012),siRNA2 (0.264±0.029), siRNA3(0.401±0.013).when compared with that in blank control (0.822±0.016) and thatin scrambled siRNA (0.807±0.019).There was no statistically significancesbetween the expression of KLF8 mRNA and protein in blank control group and thatin scrambled siRNA group (P＞0.05).
In agreement with RT-PCR results, the expression of KLF8 protein wasdown-regulated effectively by siRNA2.
Conclusions: Constructing KLF8-siRNA can effectively inhibit theexpression KLF8 mRNA and protein in renal cancer cell 786-0.Subsequentexperiments focused on the siRNA2 because it was the most effective at inhibitingKLF8 mRNA and protein expression.
PartⅢ
RNA interferencing KLF8 inhibits the renal carcinoma786-0 cells growthin vitro
Objectives:To investigate RNA interferencing KLF8 inhibits the renalcarcinoma 786-0 cells growth in vitro.
Methods:KLF8-siRNA2 were transfected with renal carcinoma cell 786-0.Checking the expression level of cyclinD1 and FAK、E-cadherin mRNA and protein by RT-PCR and Western blot.The effects of siRNA targeting KLF8 on growth,invasiveness,cell cycle and cell apoptosis of 786-0 ceils were evaluated byMTT Assay,Matrigel Invasion Assay and flow cytometry in vitro.
Results: Compared with blank control (0.693±0.009)、(0.487±0.020)and scrambled siRNA group(0.715±0.018)、(0.490±0.012), the relative mRNAexpression of cyclin D1 (0.217±0.018) and FAK (0.113±0.012) wassignificantly in KLF8-siRNA2 group(P＜0.001).
The relative protein expression of cyclin D1 (0.217±0.018) and FAK(0.113±0.012) was significantly in KLF8-siRNA2 group (P＜0.001),when comparedwith blank control (0.780±0.017)、(0.810±0.021) and scrambled siRNAgroup(0.607±0.024)、(0.593±0.008).There was no statisticallysignificancesbetween that the expression ofcyclin D1、FAK mRNA and protein in blank controlgroup and that in scrambled siRNA group (P＞0.05).
The relative mRNA and protein expression of E-cadherin was alsosignificantly up-regulated in KLF8-siRNA2 group.MTT assay showed that thegrowth of 786-0 cell in KLF8-siRNA2 group are slower than that of blank controland scrambled siRNA group (P＜0.05).Migrating numbers of cells was significantlyinhibited in KLF8-siRNA2 group by Matrigel Invasion Assay.KLF8-siRNA2 groupinduced an increase in G0/G1 phase cells(P＜0.001),decreased in Sphase(P＜0.001),the percentage of apoptotic cells in KLF8-siRNA2 group wereincreased(P＜0.001).
Conclusions:The siRNAi targeting KLF8 can significantly decrease theexpression mRNA and protein of cyclinD1 and FAK.Down-regulation KLF8 expressioncan induce up-regulation the expression mRNA and protein of E-cadherin.
It can decrease cell grow,suppressing cell invasion,affecting the cellcycle,increasing the percentage of cell apoptosis.KLF8 may play a important rolein promoting cell grow, maintainting cell invasion,mediating cell cycle,suppressing cell apoptosis.KLF8 may be involved in the carcinogenesis andprogression.It may be a key factor of FAK mediating in the regulation of cellcycle and dual regulation may be existed between KLF8 and FAK.Blocking KLF8expression by RNAi might inverse EMT in the renal cancer cells.
RNA interferencing KLF8 inhibits the renal carcinoma 786-0 cellsgrowth in vivo
Objective:Exploring the influence of RNA interferencing KLF8 inhibitthe renal carcinoma 786-0 cells oncogenicity in vivo.
Methods:18 Male Balb/c male nude mice were randomly divided into blankcontrol group、scrambled siRNA group、KLF8-siRNA2 group(n=6).Tumor wasimplanted by subcutaneous injection of 3.5×10~6 cells/mouse in 200μl of a50/50 dilution of RPMI 1640 in PBS into the left flanks of the nude mice.Whentumor nodules were growed ,equal volume of PBS was used for intratumorinjection as blank control group.Scrambled siRNA group received intratumorinjections (50μg/ mouse ) of scrambled siRNA and KLF8-siRNA2 group receivedintratumor injection of 50μg KLF8-siRNA2 every 3 day.All mices were executedin 6 weeks after the inoculation,stripped and put out the tumor which to beweighed and then calculated the tumor volume according to the formula.RI-PCRand western blot were used to determine the expression of KLF8 mRNA and proteinin three group.
Results:Tumor weight in KLF8-siRNA2 group (2.073±0.067)g was alsosignificantly decreased (P＜0.001) than that in blank controlgroup(3.317±0.144)g and in scrambled siRNA group(3.333±0.185)g after sixweeks,while there was no statistically significance between blank controlgroup and scrambled siRNA group (P＞0.05).
Compared with blank control (0.488±0.021) and scrambled siRNA group(0.483±0.017), the relative mRNA expression of KLF8 (0.269±0.028)wassignificantly in KLF8-siRNA2 group(P＜0.001).The relative protein expressionof KLF8(0.278±0.044) was significantly in KLF8-siRNA2 group(P＜0.001),whencompared with blank control (0.785±0.016)and scrambled siRNAgroup(0.731±0.023).There was no statistically significances between that theexpression of KLF8 mRNA and protein in blank control group and that in scrambledsiRNA group (P＞0.05).
Conclusions:After transfectd with KLF8-siRNA2,the formation anddevelopment of renal tumor model can be obviously inhibited.The expressionof KLF8 mRNA and protein in KLF8-siRNA2 group was also decreased.It suggestedthat KLF8 may play an important role in the carcinogenesis and progression.
Krüppel样转录因子8(Krüppel-like factor 8，KLF8)是Krüppel样转录因子家族成员之一。KLF8在恶性肿瘤中呈高表达，正常组织中低表达。相关研究表明KLF8通过激活细胞周期蛋白D1(cyclin D1)的启动子，在粘着斑激酶(focal adhesionkinase，FAK)调控细胞周期、细胞致癌性转化、侵袭力、上皮间质转型(epithelial tomesenchymal transition，EMT)方面发挥重要作用。但KLF8在肾细胞癌的发生、发展、侵袭方面所起的作用尚不清楚。
本课题首先应用逆转录-聚合酶链反应(reverse transcription-polymerasechain reaction，RT-PCR)和免疫组织化学方法检测42例散发性肾细胞癌患者的KLF8基因和蛋白质的表达情况，初步探讨了KLF8基因与肾细胞癌发生、发展的可能相关性。接下来将构建的经化学修饰的小干扰RNA(small interference RNA，siRNA)转染到表达KLF8基因的肾癌786-0细胞中。明确KLF8-siRNA在基因和蛋白水平能够有效抑制KLF8的表达。并研究干扰KLF8基因后对细胞生长、细胞侵袭力、细胞凋亡和细胞周期，对FAK、cyclinD1、钙粘蛋白(E-cadherin)基因和蛋白表达的影响。然后接种肾癌786-0细胞形成裸鼠移植瘤模型后，探讨RNA干扰KLF8基因后对裸鼠致瘤性及肾移植瘤内KLF8基因和蛋白表达的影响。本课题采用RNA干扰方法研究KLF8基因对肾癌786-0细胞增殖和肿瘤生长的影响，为肾癌的基因治疗提供了一定的实验依据。
第一部分
KLF8在肾癌组织中的表达及临床意义
目的：探讨Krüppel样转录因子8(KLF8)在肾透明细胞癌的表达及其临床意义。
方法：应用RT-PCR和免疫组织化学方法从基因和蛋白水平检测了42例原发性散发肾透明细胞癌患者和癌旁正常肾组织中KLF8 mRNA和蛋白的表达情况。
结果：肾癌组织中的KLF8mRNA相对表达量为(0．869±0．049)，癌旁正常肾组织的KLF8mRNA相对表达量为(0．416±0．018)，组间比较差异有统计学意义(P＜0．001)。42例肾癌组织中有36例表达KLF8蛋白，表达率为85．7％(36／42)；42例癌旁正常肾组织有16例表达KLF8蛋白，表达率为38．1％(16／42)，肾癌组织KLF8蛋白表达水平显著高于癌旁正常肾组织，组间比较差异有统计学意义(P＜0．001)。KLF8 mRNA和蛋白表达与肿瘤大小(P＜0．001)和临床分期(P＜0．001)相关，与年龄、性别、细胞分化程度无关(P＞0．05)。
结论：肾癌组织KLF8mRNA和蛋白表达增高，可能与肾癌的发生、进展相关，有可能成为肾癌基因治疗的靶点。
第二部分
RNA干扰KLF8基因有效序列的构建和筛选
目的：探讨RNA干扰KLF8基因有效序列的构建和筛选。
方法：设计和合成可抑制KLF8表达的经过化学修饰的siRNA，并转染进入肾癌786-0细胞中。利用RT PCR和Western blot方法检测KLF8mRNA和蛋白表达情况。筛选出一条抑制效率最高的KLF8-siRNA进入下一步研究。
结果：与空白组(0．977±0．003)和阴性对照组(0．917±0．014)相比，各干扰组KLF8 mRNA相对表达量分别siRNA1(0．276±0．017)，siRNA2(0．084±0．005)，siRNA3(0．316±0．014)，组间差异有统计学意义(P＜0．001)。与空白组(0．822±0．016)和阴性对照组(0．807±0．019)相比，各干扰组KLF8蛋白相对表达量分别siRNA1(0．491±0．012)，siRNA2(0．264±0．029)，siRNA3(0．401±0．013)，组间差异有统计学意义(P＜0．001)。空白组与阴性对照组KLF8 mRNA和蛋白表达相比，组间比较差异无统计学意义(P＞0．05)。与RT-PCR结果一致，siRNA2抑制KLF8蛋白效率最高。
结论：构建成功的KLF8-siRNA序列可有效抑制肾癌786 0细胞中的KLF8基因和蛋白表达，KLF8 siRNA2序列抑制效率最高，选择它进入下一步研究。
第三部分
RNA干扰KLF8基因对肾癌786-0细胞体外生长的影响目的：探讨RNA干扰KLF8基因后对肾癌786-0细胞体外生长的影响。方法：将KLF8-siRNA2序列转染进入肾癌786-0细胞中。利用RT-PCR和Westernblot方法检测cyclinD1、FAK、E-cadherin mRNA和蛋白表达情况；MTT法检测肾癌细胞生长情况；Matrigel Invasion Assay方法检测肾癌细胞侵袭能力；流式细胞术检测肾癌细胞周期及细胞凋亡情况。
结果：与空白组(0．693±0．008)、(0．487±0．020)和阴性对照组(0．715±0．010)、(0．490±0．021)相比，KLF8-siRNA2组cyclinD1 mRNA(0．217±0．018)、FAK mRNA(0．1 13±0．012)表达均受到抑制(P＜0．001)；与空白组(0．780±0．017)、(0．810±0．021)和阴性对照组(0．607±0．024)、(0．593±0．008)相比，KLF8-siRNA2组cyclinD1蛋白(0．323±0．061 1)、FAK蛋白(0．380±0．021)表达均受到抑制(P＜0．001)；而空白组与阴性对照组cyclinD1、FAK mRNA和蛋白表达相比，组间差异无统计学意义(P＞0．05)。E-cadherin mRNA和蛋白表达上调。与空白组和阴性对照组相比，MTT显示KLF8-siRNA2组肾癌细胞生长减慢(P＜0．05)；Matrigel Invasion Assay法显示KLF8-siRNA2组细胞侵袭能力下降(P＜0．001)；流式细胞术分析表明与空白组和对照组相比，KLF8-siRNA2组G0／G1期细胞比例增加(P＜0．001)，S期比例减少(P＜0．001)，凋亡细胞比例增加(P＜0．001)。
结论：RNA干扰KLF8基因后可下调cyclinD1、FAK mRNA和蛋白表达，下调KLF8同时可上调E-cadherin的表达，同时减慢肾癌细胞的生长；降低细胞侵袭力；影响细胞周期及增加凋亡。KLF8在促进肾癌细胞生长、调控细胞周期，抑制细胞凋亡、维持细胞侵袭力等方面可能具有重要作用。KLF8可能是FAK调控肾细胞周期的关键因子，两者之间存在双向调节。RNA干扰下调KLF8基因有可能逆转肾细胞癌中EMT。
第四部分
RNA干扰KLF8基因对肾癌786-0细胞体内生长的影响
目的：探讨干扰KLF8基因后对体内人肾癌细胞致瘤性的影响。
方法：18只裸鼠随机分成空白组和阴性对照组、KLF8-siRNA2组，每组6只。采用肾癌786-0细胞悬液皮下接种3．5×10~6／0．2ml(RP1640和PBS液各为0．1ml)的方法制作裸鼠移植瘤模型。肾移植瘤成功后，分别给予等量PBS液、50μgscramble RNA液及50μgKLF8-siRNA2液进行肿瘤注射治疗，3天1次，连续6周。于治疗6周后处死裸鼠，计算肿瘤瘤重及肿瘤体积及绘制生长曲线；并应用RT-PCR及Western blot方法检测KLF8在mRNA和蛋白质水平的表达情况。
结果：治疗6周后KLF8-siRNA2组裸鼠肿瘤的肿瘤重量(2．073±0．067)g小于空白组(3．317±0．144)g和阴性对照组(3．333±0．185)g，组间差异有统计学意义(P＜0．001)，而空白组和阴性对照组组间差异无统计学意义(P＞0．05)。与空白组(0．488±0．021)和阴性对照组(0．483±0．017)相比，KLF8-siRNA2组KLF8mRNA(0．269±0．028)的表达显著降低，组间差异有统计学意义(P＜0．001)；与空白组(0．785±0．016)和阴性对照组(0．731±0．023)相比，KLF8-siRNA2组KLF8蛋白表达下降(0．278±0．044)，组间差异有统计学意义(P＜0．001)。而空白组和阴性对照组KLF8mRNA和蛋白相比，组间差异无统计学意义(P＞0．05)。
结论：转染KLF8-siRNA2后可有效抑制裸鼠模型肿瘤的生长并降低了瘤体内KLF8基因和蛋白的表达，提示KLF8基因在肾癌的发生、发展过程中可能发挥着重要的作用。本研究也为以KLF8为靶点的肿瘤基因沉默疗法提供了可靠的理论和实验基础。
Renal cell carcinoma (RCC) is the common genitourinary tract tumor andaccounts for approximately 2% of adult cancer.Radical nephrectomy is the mosteffective treatment for localized renal cell carcinoma.RCC remains the highmortality due to the lack of early detection methods and effective treatmentsfor late-stage cancers.Twenty-five percent of patients were diagnosed withlocally invasive or metastatic RCC.The lack of sensitive efficacy ofchemotherapy and radiation therapy in late-stage RCC has led to a 5-year survivalranging from 5% to i0%.The use of recombinant human interleukin-2 (IL-2) andrecombinant human interferonα(IFN-α), either alone or in combination,however, is limited by their toxicity and generally poor overall responserates.Understanding the molecular mechanisms underlying RCC progression andmetastasis is important for developing new strategies for early diagnosis andtherapies required for improvement of patient survival.
Kruppel like factor 8 ( KLF8 ) was one of Kruppel like transcription factorfamily.Over-expressed KLF8 has recently been found in some of human malignanttumors,while it is low expressed in the normal tissues.KLF8 has been thoughtas a key transcription factor downstream of FAK in the regulation of the cellcycle by activating cyclin D1 gene promoter and also plays a critical role inoncogenic transformation、cell invasion and epithelial to mesenchymaltransition (EMT). Especially,whether and how KLF8 might play a role in RCCtumorigenesis、progression and invasion is little known.
In the present study,we firstly investigated the expression of KLF8 mRNAand protein in 42 sporadic RCC samples by reverse transcription polymerase chainreaction (RT-PCR) and immunohistochemistry(IHC),explored the correlation ofKLF8 and the carcinogenesis with development of human renal cell carcinoma.Subsequently we constructed small interference RNA (siRNA) sequences targetingKLF8, transfected it into the human RCC cell line 786-0, identified thatKLF8-siRNA can inhibited expression of KLF8 mRNA and protein leveleffectively.Further,we explored changes in cell growth, invasiveness、cellcycle and cell apoptosis,studied the influence on the cyclin Dl and FAK、E-cadherin mRNA and protein when KLF8-siRNA transfected 786-0 cells.Inoculating the renal carcinoma cells and interference renal carcinomacells to the nude mice,the influence of KLF8 genes on the oncogenicity wasinvestigated.In this project,using the KLF8-siRNAmethod to study the influenceon the proliferation of renal carcinoma 786-0 cells and the development of tumor,providing the experimental evidences to the gene therapy of renal cellcarcinoma.
PartⅠ
The expression KLF8 in renal cell carcinomaand clinical significances
Objectives: To investigate the expression of KLF8inrenal cell carcinomaand clinical significances.
Methods:We firstly investigated the expression of KLF8 mRNA and proteinin 42 sporadic RCC samples and adjacent non-tumorous renal tissues by RT-PCRand IHC.
Results:RT-PCR results revealed that the relative expression of KLF8 mRNAin RCC and adjacent non-tumorous renal tissues was (0.869±0.321) and (0.416±0.117) respectively and statistically significant difference exists betweenthem (P＜0.001).KLF8 protein expression was positive in 36 cases of RCC tissues(36/42,85.7%),higher than that in adjacent non-tumorous tissues (16/42,38.1%,P＜0.001).The high levels of KLF8 mRNA and protein expression were relatedwith the larger tumor size (P＜0.001) and high clinical stage(P＜0.001),but notrelated with sex,age and cell differentiate (P＞0.05).
Conclusions:The KLF8 mRNA and protein expression were elevated in thehuman RCC.KLF8 was involved in the development and progression of RCC,whichmay be used as a target of renal cell carcinoma gene therapy.
PartⅡ
The construction and identification of RNA interferencingKLF8 sequences
Objectives:To investigate the construction and identification of RNAinterferencing KLF8 sequences.
Methods:Designing and synthesizing the siRNA which can inhibit the KLF8expression and transfecting with renal cancer cell 786-0. Checking theexpression level of KLF8 mRNA and protein by RT-PCR and Western blot.We wantedto select a most efficient siRNA sequences for further study.
Results:Compared with blank control (0.977±0.003) and scrambled siRNA(0.917±0.014),KLF8-siRNA significantly inhibited the relative expression ofKLF8 mRNA(P＜0.001): siRNA1 (0.276±0.017), siRNA2 (0.084±0.005), siRNA3(0.316±0.014). SiRNA2 was identified as the most potent sequence whichsuppressed KLF8 mRNA expression.
Western blot analysis also demonstrated a significant reduction in KLF8protein level(P＜0.001):siRNA1 (0.491±0.012),siRNA2 (0.264±0.029), siRNA3(0.401±0.013).when compared with that in blank control (0.822±0.016) and thatin scrambled siRNA (0.807±0.019).There was no statistically significancesbetween the expression of KLF8 mRNA and protein in blank control group and thatin scrambled siRNA group (P＞0.05).
In agreement with RT-PCR results, the expression of KLF8 protein wasdown-regulated effectively by siRNA2.
Conclusions: Constructing KLF8-siRNA can effectively inhibit theexpression KLF8 mRNA and protein in renal cancer cell 786-0.Subsequentexperiments focused on the siRNA2 because it was the most effective at inhibitingKLF8 mRNA and protein expression.
PartⅢ
RNA interferencing KLF8 inhibits the renal carcinoma786-0 cells growthin vitro
Objectives:To investigate RNA interferencing KLF8 inhibits the renalcarcinoma 786-0 cells growth in vitro.
Methods:KLF8-siRNA2 were transfected with renal carcinoma cell 786-0.Checking the expression level of cyclinD1 and FAK、E-cadherin mRNA and protein by RT-PCR and Western blot.The effects of siRNA targeting KLF8 on growth,invasiveness,cell cycle and cell apoptosis of 786-0 ceils were evaluated byMTT Assay,Matrigel Invasion Assay and flow cytometry in vitro.
Results: Compared with blank control (0.693±0.009)、(0.487±0.020)and scrambled siRNA group(0.715±0.018)、(0.490±0.012), the relative mRNAexpression of cyclin D1 (0.217±0.018) and FAK (0.113±0.012) wassignificantly in KLF8-siRNA2 group(P＜0.001).
The relative protein expression of cyclin D1 (0.217±0.018) and FAK(0.113±0.012) was significantly in KLF8-siRNA2 group (P＜0.001),when comparedwith blank control (0.780±0.017)、(0.810±0.021) and scrambled siRNAgroup(0.607±0.024)、(0.593±0.008).There was no statisticallysignificancesbetween that the expression ofcyclin D1、FAK mRNA and protein in blank controlgroup and that in scrambled siRNA group (P＞0.05).
The relative mRNA and protein expression of E-cadherin was alsosignificantly up-regulated in KLF8-siRNA2 group.MTT assay showed that thegrowth of 786-0 cell in KLF8-siRNA2 group are slower than that of blank controland scrambled siRNA group (P＜0.05).Migrating numbers of cells was significantlyinhibited in KLF8-siRNA2 group by Matrigel Invasion Assay.KLF8-siRNA2 groupinduced an increase in G0/G1 phase cells(P＜0.001),decreased in Sphase(P＜0.001),the percentage of apoptotic cells in KLF8-siRNA2 group wereincreased(P＜0.001).
Conclusions:The siRNAi targeting KLF8 can significantly decrease theexpression mRNA and protein of cyclinD1 and FAK.Down-regulation KLF8 expressioncan induce up-regulation the expression mRNA and protein of E-cadherin.
It can decrease cell grow,suppressing cell invasion,affecting the cellcycle,increasing the percentage of cell apoptosis.KLF8 may play a important rolein promoting cell grow, maintainting cell invasion,mediating cell cycle,suppressing cell apoptosis.KLF8 may be involved in the carcinogenesis andprogression.It may be a key factor of FAK mediating in the regulation of cellcycle and dual regulation may be existed between KLF8 and FAK.Blocking KLF8expression by RNAi might inverse EMT in the renal cancer cells.
RNA interferencing KLF8 inhibits the renal carcinoma 786-0 cellsgrowth in vivo
Objective:Exploring the influence of RNA interferencing KLF8 inhibitthe renal carcinoma 786-0 cells oncogenicity in vivo.
Methods:18 Male Balb/c male nude mice were randomly divided into blankcontrol group、scrambled siRNA group、KLF8-siRNA2 group(n=6).Tumor wasimplanted by subcutaneous injection of 3.5×10~6 cells/mouse in 200μl of a50/50 dilution of RPMI 1640 in PBS into the left flanks of the nude mice.Whentumor nodules were growed ,equal volume of PBS was used for intratumorinjection as blank control group.Scrambled siRNA group received intratumorinjections (50μg/ mouse ) of scrambled siRNA and KLF8-siRNA2 group receivedintratumor injection of 50μg KLF8-siRNA2 every 3 day.All mices were executedin 6 weeks after the inoculation,stripped and put out the tumor which to beweighed and then calculated the tumor volume according to the formula.RI-PCRand western blot were used to determine the expression of KLF8 mRNA and proteinin three group.
Results:Tumor weight in KLF8-siRNA2 group (2.073±0.067)g was alsosignificantly decreased (P＜0.001) than that in blank controlgroup(3.317±0.144)g and in scrambled siRNA group(3.333±0.185)g after sixweeks,while there was no statistically significance between blank controlgroup and scrambled siRNA group (P＞0.05).
Compared with blank control (0.488±0.021) and scrambled siRNA group(0.483±0.017), the relative mRNA expression of KLF8 (0.269±0.028)wassignificantly in KLF8-siRNA2 group(P＜0.001).The relative protein expressionof KLF8(0.278±0.044) was significantly in KLF8-siRNA2 group(P＜0.001),whencompared with blank control (0.785±0.016)and scrambled siRNAgroup(0.731±0.023).There was no statistically significances between that theexpression of KLF8 mRNA and protein in blank control group and that in scrambledsiRNA group (P＞0.05).
Conclusions:After transfectd with KLF8-siRNA2,the formation anddevelopment of renal tumor model can be obviously inhibited.The expressionof KLF8 mRNA and protein in KLF8-siRNA2 group was also decreased.It suggestedthat KLF8 may play an important role in the carcinogenesis and progression.
引文
1.Jemal A,Siegel R,Ward E,et al.Cancer Statistics 2007[J].CA Cancer J Clin,2007;57(1):43-66.
2.Motzer RJ,Bander NH,Nanus DM.Renal-cell carcinoma[J].N Engl J Med 1996;335(12):865-875.
3.Flanigan RC,Campbell SC,Clark JI,et al.Metastatic renal cell carcinoma[J].Current treatment options Oncology,2003,4(5):385-390.
4.Martel CL,Lara PN.Renal cell carcinoma:current status and future directions[J].Crit Rev Oncol Hematol,2003,45(2):177-190.
5.Bukowski RM.Natural history and therapy of metastatic renal cell carcinoma:the role of interleukin[J].Cancer,1997,80(7):1198-1120.
6.Messing EM,Manola J,Wilding G,et al.Phase Ⅲ study of interferon alfa-NL as adjuvant treatment for resectable renal cell carcinoma:an Eastern Cooperative Oncology Group/Intergroup trial[J].J Clin Oncol,2003,21(7):1214-1222.
7.Clark JI,Atkins MB,Urba WJ,et al.Adjuvant high-dose bolus interleukin-2 for patients with high-risk renal cell carcinoma:a cytokine working group randomized trial[J].J Clin Oncol,2003,21(16):3133-3140.
8.Eisen T,Oudard S,Szczylik C,et al.Sorafenib for older patients with renal cell carcinoma:subset analysis from a randomized trial[J].J Natl Cancer Inst,2008,100(20):1454-1463.
9.Escudier B,Eisen T,Stadler WM,et al.Sorafenib in advanced clear-cell renal-cell carcinoma.N Engl J Med,2007,356(2):125-134.
10. Tschoep K, Kohlmann A, Schlemmer M, et al. Gene expression profiling in sarcomas[J]. Crit Rev Oncol Hematol, 2007, 63(2):111-124.
11. Masiero M, Nardo G, Indraccolo S, Favaro E. RNA interference: implications for cancer treatment[J]. Mol Aspects Med,2007,28(1):143-166.
12. Imyanitov EN, Moiseyenko VM. Molecular-based choice of cancer therapy:realities and expectations[J]. Clin Chim Acta, 2007, 379(1-2):1-13.
13. McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs[J]. Nat Rev Genet,2002,3(10):737-747.
14. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J]. Nature 2001,411 (6836):494-498.
15. Wang X,Zhao J. KLF8 transcription factor participates in oncogenic transformation[J]. Oncogene, 2007,26(6):456-461.
16. Wang X, Zheng M, Liu G, et al. Kruppel-Like Factor 8 Induces Epithelial to Mesenchymal Transition and Epithelial Cell Invasion[J]. Cancer Res, 2007, 67(15) :7184-7193.
17. Zhao J, Bian ZC, Yee K, et al. Identification of transcription factor KLF8 as a downstreamtarget of focal adhesion kinase in its regulation of cyclin Dl and cell cycle progression[J].Mol Cell, 2003, 11 (6):1503-1515.
18. Naw shad A, L agamba D, Po lad A , et al. Transforming growth factor-β signaling during epithelial-mesenchymal transforation: Implication for embryogenesis and tumor metastasis[J].Cells Tissues Organs,2005,179(1) : 11-23.
19. Thiery JP. Epithelial mesenchymal transitions intumor progression [J]. Nat Rev Cancer, 2002, 2(6):442-454.
20. McLean GW, Carragher NO, Avizienyte E, et al. The role of focal-adhesion kinase in cancer-a new therapeutic opportunity[J].Nat Rev Cancer,2005, 5 (7): 505-515.
21. Van Slambrouck S, Grijelmo C, De Wever 0, et al. Activation of the FAK-src molecular scaffolds and pl30Cas-JNK signaling cascades by alphal-integrins during colon cancer cell invasion[J].Int J Oncol,2007, 31(6):1501-1508.
22. Golubovskaya VM, Kweh FA, Cance WG. Focal adhesion kinase and cancer[J].Histol Histopathol, 2009, 24(4):503-510.
1.Wang X,Zhao J.KLF8 transcription factor participates in oncogenic transformation[J].Oncogene,2007,26(6):456-461.
2.Wang X,Zheng M,Liu G,et al.Kruppel-Like Factor 8 Induces Epithelial to Mesenchymal Transition and Epithelial Cell Invasion[J]. Cancer Res, 2007, 67(15): 7184-7193.
3. Storkel S, Eble JN, Adlakha K, et al. Classification of renal cell carcinoma[J]. Cancer, 1997, 80(5):987-989.
4. Tuna B, Yorukoglu K, Gurel D, et al. Significance of COX-2 expression in human renal cell carcinoma[J]. Urology, 2004, 64(6):1116-1120.
5. Pootrakul L, Datar RH, Shi SR, et al. Expression of stress response protein Grp78 is associated with the development of castration-resistant prostate cancer[J]. Clin Cancer Res. 2006,12(20Pt1):5987-5993.
6. Linehan WM, Vasselli J, Srinivasan R, et al. Genetic basis of cancer of thekidney: disease-specific approaches to therapy[J]. Clin Cancer Res,2004,10(18 Pt 2): 6282-6289.
7. CohenHT, McGovern FJ. Renal cell carcinoma [J]. New Eng JMed, 2005, 353(23):2477-2490.
8. Richard Pearson, Jacqueline Fleetwood, Sally Eaton, et al.Kruppel-like transcription factors: A functional family[J]. 2008, 40(10):1996-2001.
9. Kremer Ta S, Reeves HL, Naria G, et al. Frequent inactivation of the tumor suppressor Kruppel-like factor 6(KLF6) in hepatocellular carcinoma[J]. Hepatology, 2004, 40(5):1047-1052.
10. Reeves HL, Narla G, Ogunbiyi 0, et al. Kruppel-like factor 6(KLF6)is a tumor-suppressor gene frequently inactivated in colorectal cancer[J].Gastroenterology, 2004,126(4):1090-1103.
11. Jeng YM, Hsu HC. KLF6, a putative tumor suppressor gene, is mutated in astrocytic gliomas[J]. Int J Cancer, 2003, 105(5):625-629.
12. Kremer Ta S, Reeves HL, Naria G, et al. Frequent inactivation of the tumor suppressor Kruppel-like factor 6(KLF6) in hepatocellular carcinoma[J]. Hepatology, 2004, 40(5):1047-1052.
13. Van Vliet J, Turner J, Crossley, M, et al. Human Kruppel like factor 8: a CACCC-box binding protein that associates with CtBP and represses transcription[J]. Nucleic Acids Res, 2000, 28(9):1955-1962.
14. Zhao J, Bian ZC, Yee K, et al. Identification of transcription factor KLF8 as a downstreamtarget of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression[J].Mol Ce11,2003,11(6):1503-1515.
15.McLean GW,Carragher NO,Avizienyte E,et al.The role of focal-adhesion kinase in cancer-a new therapeutic opportunity[J].Nat Rev Cancer,2005,5(7):505-515.
16.Van Slambrouck S,Grijelmo C,De Wever O,et al.Activation of the FAK-src molecular scaffolds and p130Cas-JNK signaling cascades by alphal-integrins during colon cancer cell invasion[J].Int J Oncol,2007,31(6):1501-1508.
17.Golubovskaya VM,Kweh FA,Cance WG.Focal adhesion kinase and cancer[J].Histol Histopathol,2009,24(4):503-510.
1．付伟金，李甲初，丁强，等．KLF8在肾癌组织中的表达及临床意义[J]．中华泌尿外科杂志，2008，29(11)：16-18．
2.Katner AL,Gootam P,Hoang QB,et al.A recombinant adenovirus expressing p7(Kipl)induces cell cycle arrest and apoptosis in human 786-0 renal carcinoma cells[J].J Urol,2002,168(2):766-773.
3.Jemal A,Siegel R,Ward E,et al.Cancer Statistics 2007[J].CA Cancer J Clin,2007;57(1):43-66.
4.Motzer RJ,Bander NH,Nanus DM.Renal cell carcinoma[J].N Engl J Med 1996;335(12):865-875.
5. Guo S, Kemphues KJ. Par-1, a gene required for establishing polarity in C.elegans embryos, encodes, a putative Ser/Thr kinase that is asymmet rically distributed[J]. Cell, 1995, 81(4):611-620.
6. Fire A, Xu S, MontgomeryMK, et al. Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans [J]. Nature, 1998, 391 (6669) :806-811
7. Tuschl T. Functional genomics: RNA sets the standard [J]. Nature, 2003,421 (6920):220-221.
8. Wang X,Zhao J.KLF8 transcription factor participates in oncogenic transformation[J]. Oncogene, 2007, 26(6):456-461.
9. Wang X, Zheng M, Liu G, et al. Kruppel-Like Factor 8 Induces Epithelial to Mesenchymal Transition and Epithelial Cell Invasion[J]. Cancer Res, 2007, 67(15) :7184-7193.
10. Zhao J, Bian ZC, Yee K, et al. Identification of transcription factor KLF8 as a downstreamtarget of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression[J]. Mol Cell, 2003, 11 (6):1503-1515.
1．付伟金，李甲初，丁强，等．KLF8在肾癌组织中的表达及临床意义[J]．中华泌尿外科杂志，2008，29(11)：16-18．
2.Jemal A,Siegel R,Ward E,et al.Cancer Statistics 2007[J].CA Cancer J Clin,2007;57(1):43-66.
3.Motzer RJ,Bander NH,Nanus DM.Renal cell carcinoma[J].N Engl J Med 1996,335(12):865-875.
4.Wang X,Zhao J.KLF8 transcription factor participates in oncogenic transformation[J].Oncogene,2007,26(6):456-461.
5.Wang X,Zheng M,Liu G,et al.Kruppel-Like Factor 8 Induces Epithelial to Mesenchymal Transition and Epithelial Cell Invasion[J].Cancer Res,2007,67(15):7184-7193.
6.Zhao J,Bian ZC,Yee K,et al.Identification of transcription factor KLF8 as a downstreamtarget of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression[J].Mol Cell,2003,11(6):1503-1515.
7.Sandhu C,Slingerland J.Deregulation of cel cycle in cancer[J].Cancer detect Prev,2000,24(2):107-108.
8.Umekita Y,Ohi Y,Sagara Y,et al.Overexpression ofcyclinD1 predicts forpoor pmgnesis in estrogen receptor-negative breast cancer patients[J].Int J Cancer,2002,98(3):415-418
9.Umekita Y,Ohi Y,Sagara Y,et al.Overexpression of cyclinD1 predicts for poor prognosis in estrogen receptor-negative breast cancer patients[J].Int J Cancer,2002,98(3):415-418.
10.Bindra RS,Vasselli JR,Stearman R,et al.VHL-mediated hypoxia regulation of cyclin D1 in renal carcinoma cells[J].Cancer Res,2002,62(11):3014-3019.
11.Hedberg Y,Roos G,Ljungberg B,et al.Cyclin D3 protein content in human renal cell carcinoma in relation to cyclin D1 and clinico-pathological parameters[J].Acta Oncol,2002,41(2):175-181.
12. Naw shad A,L agamba D, Po lad A,et al. Transform inggrow th facto r2B signaling during epithelial mesenchymal transforation: Implication for embryogenesis and tumor metastasis [J]. Cells Tissues Organs, 2005, 179 (1) :11-23.
13. Thiery JP. Epithelial-mesenchymal transitions in tumor progression[J].Nat Rev Cancer, 2002, 2(6):442-454.
14. Masterson J, 0' Dea S. Posttranslational truncation of E-cadherin and significance for tumour progression[J]. Cells Tissues Organs, 2007, 185(1-3): 175-179.
15. Halbleib JM, Nelson WJ. Cadherins in development: cell adhesion, sorting,and tissue morphogenesis[J]. Genes Dev, 2006, 20(23):3199-3214.
16. Salon C, Lantuejoul S, Eymin B, et al. The E-cadherin-beta-catenin complex and its implication in lung cancer progression and prognosis[J]. Future Oncol, 2005, 1(5): 649-660.
1.O'Sullivan GC,Tangney M,Casey G,et al.Modulation of p21-activated kinase l alters the behavior of renal cell carcinoma[J].Int J Cancer,2007,121:1930-1940.
2.Zhao J,Wei XL,Jia YS,et al.Silencing of herg gene by shRNA inhibits SH-SY5Y cell growth in vitro and in vivo[J].Eur J Pharmacol.2008,28,579(1-3):50-57.
3.Hu X,Su F,Qin L,et al.Stable RNA interference of ErbB-2 gene synergistic with epirubicin suppresses breast cancer growth in vitro and in vivo[J].Biochem Biophys Res Commun.2006,4,346(3):778-785.
4.Wang S,Liu H,Ren L,et al.Inhibiting colorectal carcinoma growth and metastasis by blocking the expression of VEGF using RNA interference [J].Neoplasia, 2008,10:399-407.
5. Jemal A, Siegel R, Ward E, et al. Cancer Statistics 2007[J]. CA Cancer J Clin, 2007, 57(1) :43-66.
6. Motzer RJ, Bander NH, Nanus DM. Renal-cell carcinoma[J]. N Engl J Med 1996,335(12):865-875.
7. Messing EM, Manola J, Wilding G, et al. Phase Ⅲ study of interferon alfa-NL as adjuvant treatment for resectable renal cell carcinoma: an Eastern Cooperative Oncology Group/Intergroup trial [J]. J Clin Oncol, 2003,21 (7):1214-1222.
8. Clark JI, Atkins MB, Urba WJ, et al. Adjuvant high-dose bolus interleukin-2 for patients with high-risk renal cell carcinoma: a cytokine working group randomized trial[J].J Clin Oncol, 2003, 21(16):3133-3140.
9. Wang X,Zhao J. KLF8 transcription factor participates in oncogenic transformation[J]. Oncogene, 2007, 26(6):456-461.
10. Wang X, Zheng M, Liu G, et al.Kruppel-Like Factor 8 Induces Epithelial to Mesenchymal Transition and Epithelial Cell Invasion[J]. Cancer Res, 2007, 67(15):7184-7193.
11. Zhao J, Bian ZC, Yee K, et al. Identification of transcription factor KLF8 as a downstreamtarget of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression[J]. Mol Cell,2003,11 (6):1503-1515.
1.Kaczynski J,Cook T,Urrutia R.Spl- and Kruppel-like transcription factors[J].Genome Biol,2003,4(2):206.
2.Shindo T,anabe I,Fukushima Y,et al.Kruppel-like zinc-finger transcription factor KLF5/BTEB2 is a target for angiotensin Ⅱ signaling and an essential regulator of cardiovascular remodeling[J].Nat Med,2002,8(8):856-863.
3.Funnell A P,Maloney C A,Thompson L,et al.Perkins,et al.Erythroid Kruppel-like factor directly activates the basic Kruppel-like factor gene in erythroid cells[J].Mol Cell Biol,2007,27(7):2777-2790.
4.Dang DT,Pevsner J,Yang V W.The biology of the mammalian Kruppel-like family of transcription factors[J]. Int J Biochem Cell Biol, 2000, 32(11-12):1103-1121.
5. Wani MA, Means RT Jr, Lingrel JB. Loss of LKLF function results in embryonic lethality in mice[J]. Transgenic Res, 1998, 7(4):229-238
6. Wani MA, Wert SE, Lingrel JB. Lung Kruppel-like factor, a zinc finger transcription factor, is essential for normal lung development [J]. J Biol Chem, 1999,274(30) : 21180-21185.
7. Kuo CT, Veselits ML, Barton KP,et al.The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilization during murine embryogenesis[J].Genes Dev, 1997, 11(22):2996-3006.
8. Carlson CM, Endrizzi BT, Wu J, et al. Kruppel-like factor 2 regulates thymocyte and T-cellmigration[J].Nature, 2006,442(7100):299-302.
9. Schober SL, Kuo CT, Schluns KS, et al. Expression of the transcription factor lung Kruppel-like factor is regulated by cytokines and correlates with survival of memory T cells in vitro and in vivo[J]. J Immunol, 1999, 163(7):3662-3667.
10. Grayson JM, Murali-Krishna K, Altman JD, et al.Gene expression in antigen-specific CD8~+ T cells during viral infection[J].J Immunol, 2001, 166(2) :795-799.
11. Wu J,Lingrel JB. Kruppel-like factor 2, a novel immediate-earlytranscriptional factor, regulates IL-2 expression in T lymphocyte activation[J]. J Immunol, 2005, 175(5):3060-3066.
12. Katz J P, Perreault N, Goldstein B G, et al. The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon[J]. Development, 2002, 129(11) :2619-2628.
13. Segre J A, Bauer C, Fuchs E. Klf4 is a transcription factor required for establishing the barrier function of the skin[J].Nat.Genet, 1999,22(4) : 356-360.
14. Wei D, Gong W, Kanai M, et al. Drastic down-regulation of Kruppel-like factor 4 expression is critical in human gastric cancer development and progression[J]. Cancer Res, 2005, 65(7): 2746-2754.
15. Wang N, Liu ZH, Ding F, et al. Down-regulation of gut-enriched Kruppel-like factor expression in esophageal cancer [J]. WorldJ Gastroenterol, 2002, 8(6):966-970.
16. Choi BJ, Cho YG, Song JW, et al. Altered expression of the KLF4 in colorectal cancers[J].Pathol Res Praet, 2006, 202(8):585-589.
17. Bianchi F, Hu J, Pelosi G, et al. Lung cancers detected by screening with spiral computed tomography have a malignant phenotype when analyzed by cDNA microarray[J].Clin Cancer Res, 2004,10(18):6023-6028.
18. Ohnishi S, Ohnami S, Laub F, et al. Downregulation and growth inhibitory effect of epithelial-type Kruppel-Like transcription factor KLF but not KL in bladder cancer [J]. Biochem Biophys Res Commun, 2003, 308(2): 251-256.
19. Shie JL, Chen ZY, Fu M, et al. Gut-enriched kruppel-like factor represses cyclin D1 promoter activity through Spl motif[J]. Nucleic Acidses, 2000, 28(15):2969-2975.
20. Chen ZY, Shie JL, Tseng CC. Gut-enriched Kruppel-like factor represses ornithine deearboxylase gene expression and functions as checkpoint regulator in colonic cancer cells[J]. JBiol Chem, 2002, 277(48):46831-46839.
21. Ghaleb AM, Nandan M0, Chanchevalap S, et al. Kruppel-like factors 4 and 5:the yin and yang regulators of cellular proliferation[J]. Cell Res, 2005,15 (2): 92-96.
22. Bateman NW, Tan D, Pestell RG, et al. Intestinal tumor progression is associated with altered function of KLF5[J].J Biol Chem, 2004, 279(13):12093-12101.
23. Nandan M0, Chanchevalap S, Dalton WB, et al. Kruppel like factor 5 promotesmitosis by activating the cyclin Bl/Cdc2 complex during oncogenic Ras-mediated transformation[J]. FEBS Lett, 2005, 579(21) :4757-4762.
24. Nandan M0, Yoon HS, Zhao W, et al. Kruppel-like factor 5 mediates the transforming activity of oncogenic H-Ras[J]. Oncogene 2004,23 (19):3404-3413.
25. Aizawa K, Suzuki T, Kada N, et al. Regulation of platelet-derived growth factor-A chain by Kruppel like factor 5: new pathway of cooperative activation with nuclear factor-kappaB[J]. J Biol Chem, 2004, 279(1) :70-76.
26. Shindo T, Manabe I, Fukushima Y, et al. Kruppel-like zinc-finger transcription factor KLF5/BTEB2 is a target for angiotensin II signaling and an essential regulator of cardiovascular remodeling[J]. Nat Med, 2002, 8 (8): 856-863.
27.OishiY,Manabe I,Tobe K,et al.Kruppel-like transcription factor KLF5 is a key regulator of adipocyte differentiation[J].Cell Metab,2005,1(1):27-39.
28.Matsumoto N,Kubo A,Liu H,et al.Developmental regulation of yolk sac hematopoiesis by Kruppel-like factor 6[J].Blood,2006,107(4):1357- 1365.
29.Narla G,Heath KE,Reeves HL,et al.A candidate tumor suppressor gene mutated in prostate cancer[J].Science,2001,294(5551):2563-2566.
30.Reeves HL,Narla G,Ogunbiyi O,et al.Kruppel-like factor 6(KLF6)is a tumor-suppressor gene frequently inactivated in colorectal cancer[J].Gastroenterology,2004,126(4):1090-1103.
31.Jeng YM,Hsu HC.KLF6,a putative tumor suppressor gene,is mutated in astrocytic gliomas[J].Int J Cancer,2003,105(5):625-629.
32.KremerTaS,Reeves HL,Naria G,et al.Frequent inactivation of the tumor suppressor Kruppel-like factor 6(KLF6)in hepatocellular carcinoma[J].Hepatology,2004,40(5):1047-1052.
33．潘修成，陈智，陈峰等．原发性肝癌组织中Kruppel样因子6(KLF6)的表达及其对肝癌细胞增殖的抑制作用[J]．实用肿瘤杂志，2007，22(2)：125-129．
34．孙明，杨宇如，黄健等．转染抑癌基因KLF6对前列腺癌PC-3细胞的作用研究[J]．中华男科学杂志，2006，12(6)：502-505．
35.Laub,F,Sumiyoshi H,Kajimura D,et al.Transcription factor KLF7 is important for neuronal morphogenesis in selected regions of the nervous system.Mol Cell Biol,2005,25(13):5699-5711.
36.KajimuraD,Dragomir C,Ramirez F,et al.Identification of genes regulated by transcription factor KLF7 in differentiating olfactory sensory neurons[J].Gene,2007,388(1-2):34-42.
37.Zhao J,Bian ZC,Yee K,et al.Identification of transcription factor KLF8 as a downstreamtarget of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression[J].Mol Cell,2003,11(16):1503-1515.
38.Wang X,Urvalek AM,Liu J,et al.Activation of KLF8 transcription by FAK in human ovarian epithelial and cancer cells[J].J biological chemistry,2008,283(20):13934-13942.
39.Wang X,Zhao J.KLF8 transcription factor participates in oncogenic transformation[J].Oncogene,2007,26(6):456-461.
40.Wang X,Zheng M,Liu G,et al.Kruppel-Like Factor 8 Induces Epithelial to Mesenchymal Transition and Epithelial Cell Invasion[J]. Cancer Res, 2007, 67(15): 7184-7193.
41. Morita M, Kobayashi A, Yamashita T, et al.Functional analysis of basic transcription element binding protein by gene targeting technology[J]. Mol Cell Biol, 2003, 3(7):2489-2500.
42. SimmenRC, Eason R R, McQuown J R, et al. Subfertility, uterine hypoplasia,and partial progesterone resistance in mice lacking the Kruppel-like factor 9/basic transcription element-binding protein-1(Bteb1)gene[J]. J Biol Chem, 2004, 279(28):29286-29294.
43. Subramaniam, M, Gorny G, Johnsen, S A, et al.TIEGl null mouse-derived osteoblasts are defective in mineralization and in support of osteoclast differentiation in vitro[J]. Mol Cell Biol, 2005, 25(3):1191-1199.
44. Subramaniam M, Hawse J R, Johnsen, SA,et al. Role of TIEG1 in biological processes and disease states[J]. J Cell Biochem, 2007, 102(3):539-548.
45. Johnsen SA, Subramaniam M, Janknecht R, et al. TGF beta inducible early gene enhances TGFbeta/Smad-dependent transcriptional responses[J]. Oncogene, 2002, 21(37):5783-5790.
46. Bieker JJ.Kruppel-like factors:three fingers in many pies[J].J Biol Chem, 2001, 276(37) :34355-34358.
47. Medina D, Ullrich R, Meyn R, et al. Environmental carcinogens and p53 tumor-suppressor gene interactions in a transgenic mouse model for mammary carcinogenesis[J]. Environ Mol Mutagen, 2002, 39(2-3):178-183.
48. Zhang JS, Moncrieffe MC, Kaczynski J, et al. A conserved alpha-helical motif mediates the interaction of Spl-like transcriptional repressors with the corepressor mSin3A[J]. Mol Cell Biol, 2001, 21(15):5041-5049.
49. Subramaniam M, Hefferan TE, Tau K, et al. Tissue, cell type, and breast cancer stage-specific expression of a TGF-beta inducible early transcription factor gene[J]. Cell Biochem, 1998, 68(2):226-236.
50. Cook T, Urrutia R. TIEG proteins join the Smads as TGF-beta-regulated transcription factors that control pancreatic cell growth, Am J Physiol Gastrointest Liver Physiol[J]. 2000, 278(4):513-521.
51. Asano H, Li XS, Stamatoyannopoulos G. FKLF-2: a novel Kruppel-like transcriptional factor that activates globin and other erythroid lineage genes[J]. Blood, 2000, 95(11):3578-3584.
52.Song A,Nikolcheva T,Krensky AM.Transcriptional regulation of RANTES expression in T lymphocytes[J].Immunity Rev,1999,10(10):93-103.
53.Fisch S,Gray S,Heymans S,et al.Kruppel-like factor 15 is a regulator of cardiomyocyte hypertrophy[J].Proc Natl Acad Sci,2007,104(17):7074 - 7079.
1.Jemal A,Siegel R,Ward E,et al.Cancer Statistics 2007[J].CA Cancer J Clin,2007,57(1):43-66.
2.Flanigan RC,Campbell SC,Clark JI,et al.Metastatic renal cell carcinoma[J].Curr Treat Options Oncol,2003,4:385-390.
3.Messing EM,Manola J,Wilding G,et al.Phase Ⅲ study of interferon alfa-NL as adjuvant treatment for resectable renal cell carcinoma:an Eastern Cooperative Oncology Group/Intergroup trial[J].J Clin Oncol,2003,21(7):1214-1222.
4.Clark JI,Atkins MB,Urba WJ,et al.Adjuvant high-dose bolus interleukin-2 for patients with high-risk renal cell carcinoma:a cytokine working group randomized trial[J].J Clin Oncol,2003,21(16):3133-3140.
5.Eisen T,Oudard S,Szczylik C,et al.Sorafenib for older patients with renal cell carcinoma: subset analysis from a randomized trial[J].J Natl Cancer Inst, 2008, 100(20): 1454-1463.
6. Escudier B, Eisen T, Stadler WM, et al. Sorafenib in advanced clear cell renal cell carcinoma[J]. N Engl J Med, 2007, 356(2):125-134.
7. Guo S, Kemphues KJ. Par-1, a gene required for establishing polarity in C.elegans embryos, encodes, a putative Ser/Thr kinase that is asymmet rically distributed[J]. Cell, 1995, 81(4):611-620.
8. Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans[J]. Nature, 1998(391):806-811.
9. Tuschl T. Functional genomics: RNA sets the standard [J]. Nature, 2003,421(6920):220-221.
10. Susan Parrish, Jamie Fleenor, Si Qun Xu, et al. Functional Anatomy of a dsRNA Trigger: Differential Requirement for the Two Trigger Strands in RNA Interference[J]. Molecular Cell, 2000, 6(5):1077-1087.
11. Hammond SM, Beach D, Beach D. An RNA-directed nuclease mediates post-transcriptonal gene silencing in drosophila cells [J]. Nature, 2000,404(6775): 293-296.
12. Gutwein P, Schramme A, Voss B, et al. Downregulation of junctional adhesion molecule-A is involved in the progression of clear cell renal cell carcinoma[J].Biochem Biophys Res Commun, 2009, 380(2):387-91.
13. Kanda S, Miyata Y, Kanetake H, et al. Downregulation of the c-Fes protein-tyrosine kinase inhibits the proliferation of human renal carcinoma cells[J].Int J Oncol, 2009, 34(1):89-96.
14. Zheng JN, Pei DS, Sun FH, et al. Inhibition of renal cancer cell growth by oncolytic adenovirus armed short hairpin RNA targeting hTERT gene[J]. Cancer Biol Ther,2009, 8(1). Epub ahead of print
15. Tang SW, Chang WH, Su YC, et al.MYC pathway is activated in clear cell renal cell carcinoma and essential for proliferation of clear cell renal cell carcinoma cells[J]. Cancer Lett, 2009, 273(1):35-43.
16. Zheng JN, Pei DS, Mao LJ, et al. Inhibition of renal cancer cell growth in vitro and in vivo with oncolytic adenovirus armed short hairpin RNA targeting Ki-67 encoding mRNA[J]. Cancer Gene Ther, 2009, 16(1):20-32.
17. Mikami S, Oya M, Shimoda M, et al. Expression of heparanase in renal cell carcinomas: implications for tumor invasion and prognosis[J]. Clin Cancer Res,2008, 14(19):6055-6061.
18. Chung B I,Malkowicz SB. Expression of the p roto-oncogene Axl in renal cell carcinoma[J].DNA and Cell Biol, 2003, 22(8):533-540.
19. Himta E, Yah L, Tsunoda T, et al. Genome-wide gene expression profiles of renal: identification of molecular targets for treatment of renal cell carcinoma[J]. Int J Oncol, 2006,29(4):799-827.
20. Simon JA, Lange CA.Roles of the EZH2 histone methyltransferase in cancer epigenetics[J].Mutat Res. 2008,647(1-2):21-29.
21. Chen Y, Lin MC, Yao H, et al. Lentivirus-mediated RNA interference targeting enhancer of zeste homolog 2 inhibits hepatocellular carcinoma growth through down-regulation of stathmin[J]. Hepatology, 2007, 46(1):200-208.
22. Holland D, Hoppe-Seyler K, Schuller B, et al. Activation of the enhancer of zeste homologue 2 gene by the human papillomavirus E7 oncoprotein[J]. Cancer Res, 2008, 68 (23): 9964-9972.
23. Chen Y, Lin MC, Wang H, et al.Proteomic analysis of EZH2 downstream target proteins in hepatocellular carcinoma[J]. Proteomics, 2007, 7(17):3097-104
24. Varambally S, Dhanasekaran SM, Zhou M, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer[J]. Nature,2002 ,419(6907): 624-629.
25. Wagener N, Holland D, Bulkescher J, et al. The enhancer of zeste homolog 2 gene contributes to cell proliferation and apoptosis resistance in renal cell carcinoma cells[J].Int J Cancer, 2008, 123(7):1545-1550.
26. Sato H, Senba H, Virgona N, et al. Connexin 32 potentiates vinblastine-induced cytotoxicity in renal cell carcinoma cells[J].Mol Carcinog,2007, 46(3) :215-224.
27. Tamm I, Komblau SM, Segall H, et al.Expression and prognostic significance o XIAP family genes in human cancers and myeloid leukemias[J]. Clin Cancer Res, 2000, 6 (5): 1798-1803.
28. MansouriA ,Z hangO , R idgwaL D,et al.Cisplatin resistance in an ovarian carcinoma is associated with a defectin programmed cell death control through XIAP regulation[J]. Oncol Res, 2003, 13(6-10):399-404.
29. Kim EH, K im SU, S hin DY,et al. Roscovitine sensitizes glioma cells to TRAIL-mediated apoptosis by downregulation of survivin and XIAR[J]. Oncogens, 2004, 23(2):446-456.
30. Bilim V, Yuuki K, Itoi T, et al. Double inhibition of XIAP and Bcl-2 axis is beneficial for retrieving sensitivity of renal cell cancer to apoptosis[J].Br J Cancer, 2008, 98(5):941-949.
31. Wagener N, Crnkovi(?)-Mertens I, Vetter C, et al. Expression of inhibitor of apoptosis protein Livin in renal cell carcinoma and non-tumorous adult kidney[J]. Br J Cancer, 2007, 97(9):1271-1276.
32. Wang L, Zhang Q, Liu B, et al. Challenge and promise: roles for Livin in progression and therapy of cancer. Mol Cancer Ther, 2008, 7(12):3661-3669.
33. Wang R, Lin F, Wang X, et al.Silencing Livin gene expression to inhibit proliferation and enhance chemosensitivity in tumor cells[J]. Cancer Gene Ther, 2008, 15(6): 402-412.
34. Crnkovic-Mertens I, Wagener N, Semzow J, et al. Targeted inhibition of Livin resensitizes renal cancer cells towards apoptosis[J]. Cell Mol Life Sci.2007 ,64(9):1137-1144.
35. Hirofumi Kishi, Mikio Igawa, Nobuyuki Kikuno, et al. Expression of the survivin gene in porstate cancer: Corelation with chmcopathological character istics,proilferative activity and apoptosis[J]. The journal of Urology,2004,171(5):1855-1860.
36. NamN H, Parang K. Current targets for anitcancerd rug discovery[J]. Cur Drug Targets, 2003, 4(2) : 159-179.
37. Altieri DC. Survivin, versatile modulation of cell division and apoptosis in cancer[J]. Oncogene, 2003, 22(53):8581-8589.
38. Sato A, Oya M, Ito K, et al. Survivin associates with cell proliferation in renal cancer cells: regulation of survivin expression by insulin like growth factor 1, Interferon-gamma and a novel NF-kappaB inhibiton[J].Int J Oncol, 2006, 28(4):841-846.
2.Motzer RJ,Bander NH,Nanus DM.Renal-cell carcinoma[J].N Engl J Med 1996;335(12):865-875.
3.Flanigan RC,Campbell SC,Clark JI,et al.Metastatic renal cell carcinoma[J].Current treatment options Oncology,2003,4(5):385-390.
4.Martel CL,Lara PN.Renal cell carcinoma:current status and future directions[J].Crit Rev Oncol Hematol,2003,45(2):177-190.
5.Bukowski RM.Natural history and therapy of metastatic renal cell carcinoma:the role of interleukin[J].Cancer,1997,80(7):1198-1120.
6.Messing EM,Manola J,Wilding G,et al.Phase Ⅲ study of interferon alfa-NL as adjuvant treatment for resectable renal cell carcinoma:an Eastern Cooperative Oncology Group/Intergroup trial[J].J Clin Oncol,2003,21(7):1214-1222.
7.Clark JI,Atkins MB,Urba WJ,et al.Adjuvant high-dose bolus interleukin-2 for patients with high-risk renal cell carcinoma:a cytokine working group randomized trial[J].J Clin Oncol,2003,21(16):3133-3140.
8.Eisen T,Oudard S,Szczylik C,et al.Sorafenib for older patients with renal cell carcinoma:subset analysis from a randomized trial[J].J Natl Cancer Inst,2008,100(20):1454-1463.
9.Escudier B,Eisen T,Stadler WM,et al.Sorafenib in advanced clear-cell renal-cell carcinoma.N Engl J Med,2007,356(2):125-134.
10. Tschoep K, Kohlmann A, Schlemmer M, et al. Gene expression profiling in sarcomas[J]. Crit Rev Oncol Hematol, 2007, 63(2):111-124.
11. Masiero M, Nardo G, Indraccolo S, Favaro E. RNA interference: implications for cancer treatment[J]. Mol Aspects Med,2007,28(1):143-166.
12. Imyanitov EN, Moiseyenko VM. Molecular-based choice of cancer therapy:realities and expectations[J]. Clin Chim Acta, 2007, 379(1-2):1-13.
13. McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs[J]. Nat Rev Genet,2002,3(10):737-747.
14. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J]. Nature 2001,411 (6836):494-498.
15. Wang X,Zhao J. KLF8 transcription factor participates in oncogenic transformation[J]. Oncogene, 2007,26(6):456-461.
16. Wang X, Zheng M, Liu G, et al. Kruppel-Like Factor 8 Induces Epithelial to Mesenchymal Transition and Epithelial Cell Invasion[J]. Cancer Res, 2007, 67(15) :7184-7193.
17. Zhao J, Bian ZC, Yee K, et al. Identification of transcription factor KLF8 as a downstreamtarget of focal adhesion kinase in its regulation of cyclin Dl and cell cycle progression[J].Mol Cell, 2003, 11 (6):1503-1515.
18. Naw shad A, L agamba D, Po lad A , et al. Transforming growth factor-β signaling during epithelial-mesenchymal transforation: Implication for embryogenesis and tumor metastasis[J].Cells Tissues Organs,2005,179(1) : 11-23.
19. Thiery JP. Epithelial mesenchymal transitions intumor progression [J]. Nat Rev Cancer, 2002, 2(6):442-454.
20. McLean GW, Carragher NO, Avizienyte E, et al. The role of focal-adhesion kinase in cancer-a new therapeutic opportunity[J].Nat Rev Cancer,2005, 5 (7): 505-515.
21. Van Slambrouck S, Grijelmo C, De Wever 0, et al. Activation of the FAK-src molecular scaffolds and pl30Cas-JNK signaling cascades by alphal-integrins during colon cancer cell invasion[J].Int J Oncol,2007, 31(6):1501-1508.
22. Golubovskaya VM, Kweh FA, Cance WG. Focal adhesion kinase and cancer[J].Histol Histopathol, 2009, 24(4):503-510.
1.Wang X,Zhao J.KLF8 transcription factor participates in oncogenic transformation[J].Oncogene,2007,26(6):456-461.
2.Wang X,Zheng M,Liu G,et al.Kruppel-Like Factor 8 Induces Epithelial to Mesenchymal Transition and Epithelial Cell Invasion[J]. Cancer Res, 2007, 67(15): 7184-7193.
3. Storkel S, Eble JN, Adlakha K, et al. Classification of renal cell carcinoma[J]. Cancer, 1997, 80(5):987-989.
4. Tuna B, Yorukoglu K, Gurel D, et al. Significance of COX-2 expression in human renal cell carcinoma[J]. Urology, 2004, 64(6):1116-1120.
5. Pootrakul L, Datar RH, Shi SR, et al. Expression of stress response protein Grp78 is associated with the development of castration-resistant prostate cancer[J]. Clin Cancer Res. 2006,12(20Pt1):5987-5993.
6. Linehan WM, Vasselli J, Srinivasan R, et al. Genetic basis of cancer of thekidney: disease-specific approaches to therapy[J]. Clin Cancer Res,2004,10(18 Pt 2): 6282-6289.
7. CohenHT, McGovern FJ. Renal cell carcinoma [J]. New Eng JMed, 2005, 353(23):2477-2490.
8. Richard Pearson, Jacqueline Fleetwood, Sally Eaton, et al.Kruppel-like transcription factors: A functional family[J]. 2008, 40(10):1996-2001.
9. Kremer Ta S, Reeves HL, Naria G, et al. Frequent inactivation of the tumor suppressor Kruppel-like factor 6(KLF6) in hepatocellular carcinoma[J]. Hepatology, 2004, 40(5):1047-1052.
10. Reeves HL, Narla G, Ogunbiyi 0, et al. Kruppel-like factor 6(KLF6)is a tumor-suppressor gene frequently inactivated in colorectal cancer[J].Gastroenterology, 2004,126(4):1090-1103.
11. Jeng YM, Hsu HC. KLF6, a putative tumor suppressor gene, is mutated in astrocytic gliomas[J]. Int J Cancer, 2003, 105(5):625-629.
12. Kremer Ta S, Reeves HL, Naria G, et al. Frequent inactivation of the tumor suppressor Kruppel-like factor 6(KLF6) in hepatocellular carcinoma[J]. Hepatology, 2004, 40(5):1047-1052.
13. Van Vliet J, Turner J, Crossley, M, et al. Human Kruppel like factor 8: a CACCC-box binding protein that associates with CtBP and represses transcription[J]. Nucleic Acids Res, 2000, 28(9):1955-1962.
14. Zhao J, Bian ZC, Yee K, et al. Identification of transcription factor KLF8 as a downstreamtarget of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression[J].Mol Ce11,2003,11(6):1503-1515.
15.McLean GW,Carragher NO,Avizienyte E,et al.The role of focal-adhesion kinase in cancer-a new therapeutic opportunity[J].Nat Rev Cancer,2005,5(7):505-515.
16.Van Slambrouck S,Grijelmo C,De Wever O,et al.Activation of the FAK-src molecular scaffolds and p130Cas-JNK signaling cascades by alphal-integrins during colon cancer cell invasion[J].Int J Oncol,2007,31(6):1501-1508.
17.Golubovskaya VM,Kweh FA,Cance WG.Focal adhesion kinase and cancer[J].Histol Histopathol,2009,24(4):503-510.
1．付伟金，李甲初，丁强，等．KLF8在肾癌组织中的表达及临床意义[J]．中华泌尿外科杂志，2008，29(11)：16-18．
2.Katner AL,Gootam P,Hoang QB,et al.A recombinant adenovirus expressing p7(Kipl)induces cell cycle arrest and apoptosis in human 786-0 renal carcinoma cells[J].J Urol,2002,168(2):766-773.
3.Jemal A,Siegel R,Ward E,et al.Cancer Statistics 2007[J].CA Cancer J Clin,2007;57(1):43-66.
4.Motzer RJ,Bander NH,Nanus DM.Renal cell carcinoma[J].N Engl J Med 1996;335(12):865-875.
5. Guo S, Kemphues KJ. Par-1, a gene required for establishing polarity in C.elegans embryos, encodes, a putative Ser/Thr kinase that is asymmet rically distributed[J]. Cell, 1995, 81(4):611-620.
6. Fire A, Xu S, MontgomeryMK, et al. Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans [J]. Nature, 1998, 391 (6669) :806-811
7. Tuschl T. Functional genomics: RNA sets the standard [J]. Nature, 2003,421 (6920):220-221.
8. Wang X,Zhao J.KLF8 transcription factor participates in oncogenic transformation[J]. Oncogene, 2007, 26(6):456-461.
9. Wang X, Zheng M, Liu G, et al. Kruppel-Like Factor 8 Induces Epithelial to Mesenchymal Transition and Epithelial Cell Invasion[J]. Cancer Res, 2007, 67(15) :7184-7193.
10. Zhao J, Bian ZC, Yee K, et al. Identification of transcription factor KLF8 as a downstreamtarget of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression[J]. Mol Cell, 2003, 11 (6):1503-1515.
1．付伟金，李甲初，丁强，等．KLF8在肾癌组织中的表达及临床意义[J]．中华泌尿外科杂志，2008，29(11)：16-18．
2.Jemal A,Siegel R,Ward E,et al.Cancer Statistics 2007[J].CA Cancer J Clin,2007;57(1):43-66.
3.Motzer RJ,Bander NH,Nanus DM.Renal cell carcinoma[J].N Engl J Med 1996,335(12):865-875.
4.Wang X,Zhao J.KLF8 transcription factor participates in oncogenic transformation[J].Oncogene,2007,26(6):456-461.
5.Wang X,Zheng M,Liu G,et al.Kruppel-Like Factor 8 Induces Epithelial to Mesenchymal Transition and Epithelial Cell Invasion[J].Cancer Res,2007,67(15):7184-7193.
6.Zhao J,Bian ZC,Yee K,et al.Identification of transcription factor KLF8 as a downstreamtarget of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression[J].Mol Cell,2003,11(6):1503-1515.
7.Sandhu C,Slingerland J.Deregulation of cel cycle in cancer[J].Cancer detect Prev,2000,24(2):107-108.
8.Umekita Y,Ohi Y,Sagara Y,et al.Overexpression ofcyclinD1 predicts forpoor pmgnesis in estrogen receptor-negative breast cancer patients[J].Int J Cancer,2002,98(3):415-418
9.Umekita Y,Ohi Y,Sagara Y,et al.Overexpression of cyclinD1 predicts for poor prognosis in estrogen receptor-negative breast cancer patients[J].Int J Cancer,2002,98(3):415-418.
10.Bindra RS,Vasselli JR,Stearman R,et al.VHL-mediated hypoxia regulation of cyclin D1 in renal carcinoma cells[J].Cancer Res,2002,62(11):3014-3019.
11.Hedberg Y,Roos G,Ljungberg B,et al.Cyclin D3 protein content in human renal cell carcinoma in relation to cyclin D1 and clinico-pathological parameters[J].Acta Oncol,2002,41(2):175-181.
12. Naw shad A,L agamba D, Po lad A,et al. Transform inggrow th facto r2B signaling during epithelial mesenchymal transforation: Implication for embryogenesis and tumor metastasis [J]. Cells Tissues Organs, 2005, 179 (1) :11-23.
13. Thiery JP. Epithelial-mesenchymal transitions in tumor progression[J].Nat Rev Cancer, 2002, 2(6):442-454.
14. Masterson J, 0' Dea S. Posttranslational truncation of E-cadherin and significance for tumour progression[J]. Cells Tissues Organs, 2007, 185(1-3): 175-179.
15. Halbleib JM, Nelson WJ. Cadherins in development: cell adhesion, sorting,and tissue morphogenesis[J]. Genes Dev, 2006, 20(23):3199-3214.
16. Salon C, Lantuejoul S, Eymin B, et al. The E-cadherin-beta-catenin complex and its implication in lung cancer progression and prognosis[J]. Future Oncol, 2005, 1(5): 649-660.
1.O'Sullivan GC,Tangney M,Casey G,et al.Modulation of p21-activated kinase l alters the behavior of renal cell carcinoma[J].Int J Cancer,2007,121:1930-1940.
2.Zhao J,Wei XL,Jia YS,et al.Silencing of herg gene by shRNA inhibits SH-SY5Y cell growth in vitro and in vivo[J].Eur J Pharmacol.2008,28,579(1-3):50-57.
3.Hu X,Su F,Qin L,et al.Stable RNA interference of ErbB-2 gene synergistic with epirubicin suppresses breast cancer growth in vitro and in vivo[J].Biochem Biophys Res Commun.2006,4,346(3):778-785.
4.Wang S,Liu H,Ren L,et al.Inhibiting colorectal carcinoma growth and metastasis by blocking the expression of VEGF using RNA interference [J].Neoplasia, 2008,10:399-407.
5. Jemal A, Siegel R, Ward E, et al. Cancer Statistics 2007[J]. CA Cancer J Clin, 2007, 57(1) :43-66.
6. Motzer RJ, Bander NH, Nanus DM. Renal-cell carcinoma[J]. N Engl J Med 1996,335(12):865-875.
7. Messing EM, Manola J, Wilding G, et al. Phase Ⅲ study of interferon alfa-NL as adjuvant treatment for resectable renal cell carcinoma: an Eastern Cooperative Oncology Group/Intergroup trial [J]. J Clin Oncol, 2003,21 (7):1214-1222.
8. Clark JI, Atkins MB, Urba WJ, et al. Adjuvant high-dose bolus interleukin-2 for patients with high-risk renal cell carcinoma: a cytokine working group randomized trial[J].J Clin Oncol, 2003, 21(16):3133-3140.
9. Wang X,Zhao J. KLF8 transcription factor participates in oncogenic transformation[J]. Oncogene, 2007, 26(6):456-461.
10. Wang X, Zheng M, Liu G, et al.Kruppel-Like Factor 8 Induces Epithelial to Mesenchymal Transition and Epithelial Cell Invasion[J]. Cancer Res, 2007, 67(15):7184-7193.
11. Zhao J, Bian ZC, Yee K, et al. Identification of transcription factor KLF8 as a downstreamtarget of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression[J]. Mol Cell,2003,11 (6):1503-1515.
1.Kaczynski J,Cook T,Urrutia R.Spl- and Kruppel-like transcription factors[J].Genome Biol,2003,4(2):206.
2.Shindo T,anabe I,Fukushima Y,et al.Kruppel-like zinc-finger transcription factor KLF5/BTEB2 is a target for angiotensin Ⅱ signaling and an essential regulator of cardiovascular remodeling[J].Nat Med,2002,8(8):856-863.
3.Funnell A P,Maloney C A,Thompson L,et al.Perkins,et al.Erythroid Kruppel-like factor directly activates the basic Kruppel-like factor gene in erythroid cells[J].Mol Cell Biol,2007,27(7):2777-2790.
4.Dang DT,Pevsner J,Yang V W.The biology of the mammalian Kruppel-like family of transcription factors[J]. Int J Biochem Cell Biol, 2000, 32(11-12):1103-1121.
5. Wani MA, Means RT Jr, Lingrel JB. Loss of LKLF function results in embryonic lethality in mice[J]. Transgenic Res, 1998, 7(4):229-238
6. Wani MA, Wert SE, Lingrel JB. Lung Kruppel-like factor, a zinc finger transcription factor, is essential for normal lung development [J]. J Biol Chem, 1999,274(30) : 21180-21185.
7. Kuo CT, Veselits ML, Barton KP,et al.The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilization during murine embryogenesis[J].Genes Dev, 1997, 11(22):2996-3006.
8. Carlson CM, Endrizzi BT, Wu J, et al. Kruppel-like factor 2 regulates thymocyte and T-cellmigration[J].Nature, 2006,442(7100):299-302.
9. Schober SL, Kuo CT, Schluns KS, et al. Expression of the transcription factor lung Kruppel-like factor is regulated by cytokines and correlates with survival of memory T cells in vitro and in vivo[J]. J Immunol, 1999, 163(7):3662-3667.
10. Grayson JM, Murali-Krishna K, Altman JD, et al.Gene expression in antigen-specific CD8~+ T cells during viral infection[J].J Immunol, 2001, 166(2) :795-799.
11. Wu J,Lingrel JB. Kruppel-like factor 2, a novel immediate-earlytranscriptional factor, regulates IL-2 expression in T lymphocyte activation[J]. J Immunol, 2005, 175(5):3060-3066.
12. Katz J P, Perreault N, Goldstein B G, et al. The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon[J]. Development, 2002, 129(11) :2619-2628.
13. Segre J A, Bauer C, Fuchs E. Klf4 is a transcription factor required for establishing the barrier function of the skin[J].Nat.Genet, 1999,22(4) : 356-360.
14. Wei D, Gong W, Kanai M, et al. Drastic down-regulation of Kruppel-like factor 4 expression is critical in human gastric cancer development and progression[J]. Cancer Res, 2005, 65(7): 2746-2754.
15. Wang N, Liu ZH, Ding F, et al. Down-regulation of gut-enriched Kruppel-like factor expression in esophageal cancer [J]. WorldJ Gastroenterol, 2002, 8(6):966-970.
16. Choi BJ, Cho YG, Song JW, et al. Altered expression of the KLF4 in colorectal cancers[J].Pathol Res Praet, 2006, 202(8):585-589.
17. Bianchi F, Hu J, Pelosi G, et al. Lung cancers detected by screening with spiral computed tomography have a malignant phenotype when analyzed by cDNA microarray[J].Clin Cancer Res, 2004,10(18):6023-6028.
18. Ohnishi S, Ohnami S, Laub F, et al. Downregulation and growth inhibitory effect of epithelial-type Kruppel-Like transcription factor KLF but not KL in bladder cancer [J]. Biochem Biophys Res Commun, 2003, 308(2): 251-256.
19. Shie JL, Chen ZY, Fu M, et al. Gut-enriched kruppel-like factor represses cyclin D1 promoter activity through Spl motif[J]. Nucleic Acidses, 2000, 28(15):2969-2975.
20. Chen ZY, Shie JL, Tseng CC. Gut-enriched Kruppel-like factor represses ornithine deearboxylase gene expression and functions as checkpoint regulator in colonic cancer cells[J]. JBiol Chem, 2002, 277(48):46831-46839.
21. Ghaleb AM, Nandan M0, Chanchevalap S, et al. Kruppel-like factors 4 and 5:the yin and yang regulators of cellular proliferation[J]. Cell Res, 2005,15 (2): 92-96.
22. Bateman NW, Tan D, Pestell RG, et al. Intestinal tumor progression is associated with altered function of KLF5[J].J Biol Chem, 2004, 279(13):12093-12101.
23. Nandan M0, Chanchevalap S, Dalton WB, et al. Kruppel like factor 5 promotesmitosis by activating the cyclin Bl/Cdc2 complex during oncogenic Ras-mediated transformation[J]. FEBS Lett, 2005, 579(21) :4757-4762.
24. Nandan M0, Yoon HS, Zhao W, et al. Kruppel-like factor 5 mediates the transforming activity of oncogenic H-Ras[J]. Oncogene 2004,23 (19):3404-3413.
25. Aizawa K, Suzuki T, Kada N, et al. Regulation of platelet-derived growth factor-A chain by Kruppel like factor 5: new pathway of cooperative activation with nuclear factor-kappaB[J]. J Biol Chem, 2004, 279(1) :70-76.
26. Shindo T, Manabe I, Fukushima Y, et al. Kruppel-like zinc-finger transcription factor KLF5/BTEB2 is a target for angiotensin II signaling and an essential regulator of cardiovascular remodeling[J]. Nat Med, 2002, 8 (8): 856-863.
27.OishiY,Manabe I,Tobe K,et al.Kruppel-like transcription factor KLF5 is a key regulator of adipocyte differentiation[J].Cell Metab,2005,1(1):27-39.
28.Matsumoto N,Kubo A,Liu H,et al.Developmental regulation of yolk sac hematopoiesis by Kruppel-like factor 6[J].Blood,2006,107(4):1357- 1365.
29.Narla G,Heath KE,Reeves HL,et al.A candidate tumor suppressor gene mutated in prostate cancer[J].Science,2001,294(5551):2563-2566.
30.Reeves HL,Narla G,Ogunbiyi O,et al.Kruppel-like factor 6(KLF6)is a tumor-suppressor gene frequently inactivated in colorectal cancer[J].Gastroenterology,2004,126(4):1090-1103.
31.Jeng YM,Hsu HC.KLF6,a putative tumor suppressor gene,is mutated in astrocytic gliomas[J].Int J Cancer,2003,105(5):625-629.
32.KremerTaS,Reeves HL,Naria G,et al.Frequent inactivation of the tumor suppressor Kruppel-like factor 6(KLF6)in hepatocellular carcinoma[J].Hepatology,2004,40(5):1047-1052.
33．潘修成，陈智，陈峰等．原发性肝癌组织中Kruppel样因子6(KLF6)的表达及其对肝癌细胞增殖的抑制作用[J]．实用肿瘤杂志，2007，22(2)：125-129．
34．孙明，杨宇如，黄健等．转染抑癌基因KLF6对前列腺癌PC-3细胞的作用研究[J]．中华男科学杂志，2006，12(6)：502-505．
35.Laub,F,Sumiyoshi H,Kajimura D,et al.Transcription factor KLF7 is important for neuronal morphogenesis in selected regions of the nervous system.Mol Cell Biol,2005,25(13):5699-5711.
36.KajimuraD,Dragomir C,Ramirez F,et al.Identification of genes regulated by transcription factor KLF7 in differentiating olfactory sensory neurons[J].Gene,2007,388(1-2):34-42.
37.Zhao J,Bian ZC,Yee K,et al.Identification of transcription factor KLF8 as a downstreamtarget of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression[J].Mol Cell,2003,11(16):1503-1515.
38.Wang X,Urvalek AM,Liu J,et al.Activation of KLF8 transcription by FAK in human ovarian epithelial and cancer cells[J].J biological chemistry,2008,283(20):13934-13942.
39.Wang X,Zhao J.KLF8 transcription factor participates in oncogenic transformation[J].Oncogene,2007,26(6):456-461.
40.Wang X,Zheng M,Liu G,et al.Kruppel-Like Factor 8 Induces Epithelial to Mesenchymal Transition and Epithelial Cell Invasion[J]. Cancer Res, 2007, 67(15): 7184-7193.
41. Morita M, Kobayashi A, Yamashita T, et al.Functional analysis of basic transcription element binding protein by gene targeting technology[J]. Mol Cell Biol, 2003, 3(7):2489-2500.
42. SimmenRC, Eason R R, McQuown J R, et al. Subfertility, uterine hypoplasia,and partial progesterone resistance in mice lacking the Kruppel-like factor 9/basic transcription element-binding protein-1(Bteb1)gene[J]. J Biol Chem, 2004, 279(28):29286-29294.
43. Subramaniam, M, Gorny G, Johnsen, S A, et al.TIEGl null mouse-derived osteoblasts are defective in mineralization and in support of osteoclast differentiation in vitro[J]. Mol Cell Biol, 2005, 25(3):1191-1199.
44. Subramaniam M, Hawse J R, Johnsen, SA,et al. Role of TIEG1 in biological processes and disease states[J]. J Cell Biochem, 2007, 102(3):539-548.
45. Johnsen SA, Subramaniam M, Janknecht R, et al. TGF beta inducible early gene enhances TGFbeta/Smad-dependent transcriptional responses[J]. Oncogene, 2002, 21(37):5783-5790.
46. Bieker JJ.Kruppel-like factors:three fingers in many pies[J].J Biol Chem, 2001, 276(37) :34355-34358.
47. Medina D, Ullrich R, Meyn R, et al. Environmental carcinogens and p53 tumor-suppressor gene interactions in a transgenic mouse model for mammary carcinogenesis[J]. Environ Mol Mutagen, 2002, 39(2-3):178-183.
48. Zhang JS, Moncrieffe MC, Kaczynski J, et al. A conserved alpha-helical motif mediates the interaction of Spl-like transcriptional repressors with the corepressor mSin3A[J]. Mol Cell Biol, 2001, 21(15):5041-5049.
49. Subramaniam M, Hefferan TE, Tau K, et al. Tissue, cell type, and breast cancer stage-specific expression of a TGF-beta inducible early transcription factor gene[J]. Cell Biochem, 1998, 68(2):226-236.
50. Cook T, Urrutia R. TIEG proteins join the Smads as TGF-beta-regulated transcription factors that control pancreatic cell growth, Am J Physiol Gastrointest Liver Physiol[J]. 2000, 278(4):513-521.
51. Asano H, Li XS, Stamatoyannopoulos G. FKLF-2: a novel Kruppel-like transcriptional factor that activates globin and other erythroid lineage genes[J]. Blood, 2000, 95(11):3578-3584.
52.Song A,Nikolcheva T,Krensky AM.Transcriptional regulation of RANTES expression in T lymphocytes[J].Immunity Rev,1999,10(10):93-103.
53.Fisch S,Gray S,Heymans S,et al.Kruppel-like factor 15 is a regulator of cardiomyocyte hypertrophy[J].Proc Natl Acad Sci,2007,104(17):7074 - 7079.
1.Jemal A,Siegel R,Ward E,et al.Cancer Statistics 2007[J].CA Cancer J Clin,2007,57(1):43-66.
2.Flanigan RC,Campbell SC,Clark JI,et al.Metastatic renal cell carcinoma[J].Curr Treat Options Oncol,2003,4:385-390.
3.Messing EM,Manola J,Wilding G,et al.Phase Ⅲ study of interferon alfa-NL as adjuvant treatment for resectable renal cell carcinoma:an Eastern Cooperative Oncology Group/Intergroup trial[J].J Clin Oncol,2003,21(7):1214-1222.
4.Clark JI,Atkins MB,Urba WJ,et al.Adjuvant high-dose bolus interleukin-2 for patients with high-risk renal cell carcinoma:a cytokine working group randomized trial[J].J Clin Oncol,2003,21(16):3133-3140.
5.Eisen T,Oudard S,Szczylik C,et al.Sorafenib for older patients with renal cell carcinoma: subset analysis from a randomized trial[J].J Natl Cancer Inst, 2008, 100(20): 1454-1463.
6. Escudier B, Eisen T, Stadler WM, et al. Sorafenib in advanced clear cell renal cell carcinoma[J]. N Engl J Med, 2007, 356(2):125-134.
7. Guo S, Kemphues KJ. Par-1, a gene required for establishing polarity in C.elegans embryos, encodes, a putative Ser/Thr kinase that is asymmet rically distributed[J]. Cell, 1995, 81(4):611-620.
8. Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans[J]. Nature, 1998(391):806-811.
9. Tuschl T. Functional genomics: RNA sets the standard [J]. Nature, 2003,421(6920):220-221.
10. Susan Parrish, Jamie Fleenor, Si Qun Xu, et al. Functional Anatomy of a dsRNA Trigger: Differential Requirement for the Two Trigger Strands in RNA Interference[J]. Molecular Cell, 2000, 6(5):1077-1087.
11. Hammond SM, Beach D, Beach D. An RNA-directed nuclease mediates post-transcriptonal gene silencing in drosophila cells [J]. Nature, 2000,404(6775): 293-296.
12. Gutwein P, Schramme A, Voss B, et al. Downregulation of junctional adhesion molecule-A is involved in the progression of clear cell renal cell carcinoma[J].Biochem Biophys Res Commun, 2009, 380(2):387-91.
13. Kanda S, Miyata Y, Kanetake H, et al. Downregulation of the c-Fes protein-tyrosine kinase inhibits the proliferation of human renal carcinoma cells[J].Int J Oncol, 2009, 34(1):89-96.
14. Zheng JN, Pei DS, Sun FH, et al. Inhibition of renal cancer cell growth by oncolytic adenovirus armed short hairpin RNA targeting hTERT gene[J]. Cancer Biol Ther,2009, 8(1). Epub ahead of print
15. Tang SW, Chang WH, Su YC, et al.MYC pathway is activated in clear cell renal cell carcinoma and essential for proliferation of clear cell renal cell carcinoma cells[J]. Cancer Lett, 2009, 273(1):35-43.
16. Zheng JN, Pei DS, Mao LJ, et al. Inhibition of renal cancer cell growth in vitro and in vivo with oncolytic adenovirus armed short hairpin RNA targeting Ki-67 encoding mRNA[J]. Cancer Gene Ther, 2009, 16(1):20-32.
17. Mikami S, Oya M, Shimoda M, et al. Expression of heparanase in renal cell carcinomas: implications for tumor invasion and prognosis[J]. Clin Cancer Res,2008, 14(19):6055-6061.
18. Chung B I,Malkowicz SB. Expression of the p roto-oncogene Axl in renal cell carcinoma[J].DNA and Cell Biol, 2003, 22(8):533-540.
19. Himta E, Yah L, Tsunoda T, et al. Genome-wide gene expression profiles of renal: identification of molecular targets for treatment of renal cell carcinoma[J]. Int J Oncol, 2006,29(4):799-827.
20. Simon JA, Lange CA.Roles of the EZH2 histone methyltransferase in cancer epigenetics[J].Mutat Res. 2008,647(1-2):21-29.
21. Chen Y, Lin MC, Yao H, et al. Lentivirus-mediated RNA interference targeting enhancer of zeste homolog 2 inhibits hepatocellular carcinoma growth through down-regulation of stathmin[J]. Hepatology, 2007, 46(1):200-208.
22. Holland D, Hoppe-Seyler K, Schuller B, et al. Activation of the enhancer of zeste homologue 2 gene by the human papillomavirus E7 oncoprotein[J]. Cancer Res, 2008, 68 (23): 9964-9972.
23. Chen Y, Lin MC, Wang H, et al.Proteomic analysis of EZH2 downstream target proteins in hepatocellular carcinoma[J]. Proteomics, 2007, 7(17):3097-104
24. Varambally S, Dhanasekaran SM, Zhou M, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer[J]. Nature,2002 ,419(6907): 624-629.
25. Wagener N, Holland D, Bulkescher J, et al. The enhancer of zeste homolog 2 gene contributes to cell proliferation and apoptosis resistance in renal cell carcinoma cells[J].Int J Cancer, 2008, 123(7):1545-1550.
26. Sato H, Senba H, Virgona N, et al. Connexin 32 potentiates vinblastine-induced cytotoxicity in renal cell carcinoma cells[J].Mol Carcinog,2007, 46(3) :215-224.
27. Tamm I, Komblau SM, Segall H, et al.Expression and prognostic significance o XIAP family genes in human cancers and myeloid leukemias[J]. Clin Cancer Res, 2000, 6 (5): 1798-1803.
28. MansouriA ,Z hangO , R idgwaL D,et al.Cisplatin resistance in an ovarian carcinoma is associated with a defectin programmed cell death control through XIAP regulation[J]. Oncol Res, 2003, 13(6-10):399-404.
29. Kim EH, K im SU, S hin DY,et al. Roscovitine sensitizes glioma cells to TRAIL-mediated apoptosis by downregulation of survivin and XIAR[J]. Oncogens, 2004, 23(2):446-456.
30. Bilim V, Yuuki K, Itoi T, et al. Double inhibition of XIAP and Bcl-2 axis is beneficial for retrieving sensitivity of renal cell cancer to apoptosis[J].Br J Cancer, 2008, 98(5):941-949.
31. Wagener N, Crnkovi(?)-Mertens I, Vetter C, et al. Expression of inhibitor of apoptosis protein Livin in renal cell carcinoma and non-tumorous adult kidney[J]. Br J Cancer, 2007, 97(9):1271-1276.
32. Wang L, Zhang Q, Liu B, et al. Challenge and promise: roles for Livin in progression and therapy of cancer. Mol Cancer Ther, 2008, 7(12):3661-3669.
33. Wang R, Lin F, Wang X, et al.Silencing Livin gene expression to inhibit proliferation and enhance chemosensitivity in tumor cells[J]. Cancer Gene Ther, 2008, 15(6): 402-412.
34. Crnkovic-Mertens I, Wagener N, Semzow J, et al. Targeted inhibition of Livin resensitizes renal cancer cells towards apoptosis[J]. Cell Mol Life Sci.2007 ,64(9):1137-1144.
35. Hirofumi Kishi, Mikio Igawa, Nobuyuki Kikuno, et al. Expression of the survivin gene in porstate cancer: Corelation with chmcopathological character istics,proilferative activity and apoptosis[J]. The journal of Urology,2004,171(5):1855-1860.
36. NamN H, Parang K. Current targets for anitcancerd rug discovery[J]. Cur Drug Targets, 2003, 4(2) : 159-179.
37. Altieri DC. Survivin, versatile modulation of cell division and apoptosis in cancer[J]. Oncogene, 2003, 22(53):8581-8589.
38. Sato A, Oya M, Ito K, et al. Survivin associates with cell proliferation in renal cancer cells: regulation of survivin expression by insulin like growth factor 1, Interferon-gamma and a novel NF-kappaB inhibiton[J].Int J Oncol, 2006, 28(4):841-846.