【作者】 解英俊；

【导师】 赵吉生；

【作者基本信息】 吉林大学 ， 外科学， 2010， 博士

【摘要】 目的：构建并转染针对肝癌细胞系沉默Cdc42的高效率RNA干扰载体。在体内外实验探讨Cdc42-shRNA重组质粒对肝癌细胞SMMC-7721增殖、迁移、侵袭等恶性生物学行为的影响。方法：根据Genebank报道的Cdc42的mRNA序列，利用Promega公司siRNA在线设计软件设计特征的靶序列，分别合成3对寡核苷酸链，克隆到含U6启动子的pBS/U6载体，进行酶切鉴定和测序。采用脂质体法将构建的载体转染至肝癌细胞SMMC-7721中，用Westernblot及免疫荧光染色检测对Cdc42蛋白表达的抑制情况。采用计数法绘制细胞生长曲线；MTT法检测细胞增殖；划痕愈合实验方法测定细胞迁移能力；穿孔迁移实验方法观察细胞的侵袭能力。Westernblot方法检测PCNA和AFP蛋白表达。建立裸鼠皮下人肝癌移植瘤模型，观察裸鼠成瘤情况，免疫组织化学方法检测裸鼠瘤块中Cdc42的表达情况。结果：酶切鉴定和测序结果证实Cdc42-shRNA重组质粒构建完全正确；转染肝癌细胞SMMC-7721后，Westernblot检测肝癌细胞内源性Cdc42表达明显抑制。免疫荧光方法结果显示肝癌细胞荧光强度显著下降。细胞计数法显示转染Cdc42-shRNA2细胞生长速度明显减慢。MTT实验显示转染重组质粒后吸光度显著降低，并且呈剂量依赖性。划痕实验显示转染Cdc42-shRNA2的SMMC-7721细胞划痕愈合能力显著减弱，相对迁移距离比较差异显著。穿孔迁移实验方法显示肝癌细胞侵袭能力明显减弱。显著地抑制肝癌细胞的肿瘤标志蛋白AFP的表达水平及细胞增殖抗原PCNA表达水平。裸鼠成瘤实验转染组肿瘤生长速度减慢，瘤块重量明显减小。瘤块免疫组化结果Cdc42表达明显减弱。结论：成功构建了Cdc42-shRNA表达载体；并成功转染肝癌SMMC-7721细胞，高效地抑制了肝癌细胞内源性Cdc42的表达。Cdc42干扰质粒成功地转染肝癌SMMC-7721细胞后，明显改变了人肝癌细胞的恶性生物学行为，细胞生长速度减慢，增殖能力降低，细胞的迁移和穿过Matrigel胶的侵袭能力均有不同程度的降低；发现RNAi沉默Cdc42表达导致了AFP蛋白和PCNA蛋白表达含量的明显改变，降低肝癌细胞的恶性程度；体内成瘤实验也证实RNA干扰技术可沉默肝细胞癌Cdc42蛋白的表达，并且明显抑制肿瘤生长。更多还原

【Abstract】 BACKGROUNDBACKGROUNDS:Primary hepatocellular carcinoma(HCC)is a commonmalignant tumor of digestive system with poor early diagnosis;the most patiens ofdiagnosis have reached the advanced or distant metastasis; In recent years, livercancer has been greatly improved with multidisciplinary treatment modalities,but theoverall curative effect is bad. Therefore, we must better understand the incidence anddevelopment mechanism of hepatocellular carcinoma. To find a new treatmentstrategies to improve the prognosis of liver cancer is the focus of the study.The occurrence and development of HCC is a multi-gene and multi-stepprocess,with the development of gene technology,the research of gene treatment toHCC become a hotspot worldwide.RNA interference(RNAi)means degradation ofhomologous mRNA induced by endogenous or exogenous short double-strandedRNA(dsRNA)molecule,its effector molecule is 21-23 nucleotide(nt)dsRNA calledsmall interfering RNA(siRNA).The gene knockout effect caused by RNAi technologycan inhibit the expression of oncogene,tumor suppression gene and gene mutatin etal.The short hairpin RNA(shRNA) expressed by vector transfected into target cellscan be recognized and splitted into siRNA.Use the plasmid vector system which cansteadily express shRNA can inhibit gene expression permanently.The current study found that, the most important function of Rho GTPases is toregulate the actin-related protein and cytoskeleton-mediated construction, and relatedclosely with the malignant transformation, growth, invasion and metastasis, cell cycleregulation and apoptosis. It is clear that Rho proteins was involved almost in everystage of tumor formation and played an important role of the pathophysiologicalprocess in the tumor,and Rho proteins is promising target for cancer treatment that hasbecome one of the research frontier.Cdc42(Cell division cycle42)，a member of RhoGTPases，Involved in actin reorganization, formation of cell polarity, pseudopodgeneration, membrane transport and transcriptional regulation of expression and otherphysiological processes,and acts as a GTP-binding Protein switch to regulate multiple signal transduction pathways that control key cellular proeesses such as cellproliferation，survival，cytoarehitecture，adhesion，migration，and transcriptionalregulation. Several studies found that Cdc42 is over-expression in breast cancer, lungcancer, esophagus cancer, colon cancer, bladder cancer,and other organizations andtumor cells. In hepatocellular carcinoma tissue Cdc42 molecules expression wassignificantly increased than non-tumor liver tissue,and in highly metastatichepatocellular carcinoma cell lines expressed more than in low metastatichepatocellular carcinoma cell lines. Therefore, speculated that the protein may be inhepatocellular carcinoma cell proliferation and transformation play a role.At present, the application RNA interference technology research Cdc42 affectthe biological behavior of hepatocellular carcinoma cells not been reported at homeand abroad. We studied hepatocellular carcinoma cell lines to explore the Cdc42protein in liver cancer cell proliferation, migration, invasion and so on role and theapplication of RNA interference inhibition of the Cdc42 protein and cell biologicalbehavior in order to study the role of Cdc42 in the occurrence and development ofhepatocellular carcinoma, and exploration of the feasibility of targeted Cdc42treatment of hepatocellular carcinoma.This study is to establish the Cdc42-shRNA vector and transfect the hepatocellularcarcinoma cells SMMC-7721,detect the inhibition effect of Cdc42 expression,andobserve the growth,proliferation,mobility of SMMC-7721 cells and the expression ofAFP ansd PCNA;establish the nude mice model transplanted with SMMC-7721 cellssubcutaneously to observe the growth of transplanted tumor.Thus to provide a neweffective target for the treatment of HCC. This study is divided into three parts:1 Construction and identification of Cdc42-shRNA and its inhibition effect onCdc42 expression of hepatoma cells.Objective: To construct high efficiency of RNA silencing Cdc42 interferingcarrier. for liver cancer cell linesMethods According to the Cdc42 mRNA sequence in Genebank,use the siRNAdesign software of Promega to find the right target sequence,BLAST to confirm nohomologous with known human gene.Three pair of oligonucleotide strand wassynthesized.The sequence is 19nt sense sequence,6nt loop and 19nt anti-sensesequence and terminal signal,cloned the sequence to pBS/U6 vector.Rename the recombinant plasmid Cdc42-shRNA1、Cdc42-shRNA2、Cdc42-shRNA3,andrestriction enzyme digestion and sequencing to confirm the correctness.Transfect theplasmid into hepatoma cells SMMC-7721 to detect the expression of Cdc42 byWesternblot and immunofluorescence.Results: The enzyme electrophoresis confirmed that Cdc42-shRNA plasmid wassuccessfully constructed, inserted fragment sequencing results with the expected resultsare consistent with the synthesis of siRNA confirmed the synthesis of recombinantplasmid completely correct; Westernblot detect Cdc42-shRNA inhibited SMMC-7721hepatocellular carcinoma cells expressed endogenous Cdc42. Cdc42-shRNAsrecombinant plasmid is completely correct comfirmed by restriction enzyme digestionand sequencing.All the plasmids have gene silencing effect and the Cdc42-shRNA2 ismost significant.It can inhibit the endogenous Cdc42 expression about 80%.Thefluorescence intensity of hepatoma cells transfected by Cdc42- shRNA2 is much lower.Conclusion: The successful construction of the high efficiency of RNAi silencingCdc42 expression vectors, the restriction enzyme digestion and sequencing confirmedthe plasmid completely correct; successfully transfected with recombinant plasmidliver cancer SMMC-7721 cells, liver cells efficiently inhibited endogenous Cdc42expression. Tips, RNAi technology can be used as Cdc42 silence the expression ofliver cancer in an effective way for further experiments laid the experimental basis.2.Influence of Cdc42-shRNA to proliferation and mobility and expression ofAFP and PCNA of hepatoma cells.Objective: To study the influence of Cdc42-shRNA plasmid to proliferation,migration, invasion and other malignant biological behavior in hepatoma SMMC-7721cells.Methods: PBS/U6 Cdc42-shRNA interfering vector transfected to SMMC-7721cells with liposome method,The growth curve of ransfected cells SMMC-7721、SMMC-7721/pBS/U6-control、SMMC-7721/Cdc42-shRNA2 was detected bycountnumber method;the cells proliferation was detected by MTT,and calculate theinhibition rate.The cells mobility was detected by wound healing experiment.Transwell chamber experiments to observe the cell migration and invasion.DetectAFP and PCNA expression level by Westernblot.Results:The growth of hepatoma cells transfected by Cdc42-shRNA2 isslower.The absorbance of SMMC-7721 cells transfected by10ng and 30ng Cdc42-shRNA2 is much lower in MTT experiment,and there is a dose-dependentrelationship,the maximum inhibition rate is 40%. The wound healing capatility ofSMMC-7721 cells transfected by Cdc42-shRNA2 is weaker than pBS/U6-controlgroup with comparison of relative mobility distance significantly different.Transwellchamber experimental methods showed Cdc42 protein expression after the silence,SMMC-7721 decreased significantly invasion ability.The AFP and PCNA expressionof hepatoma cells is siginificantly inhibited after the Cdc42-shRNA2 was transfectedcompared with pBS/U6-control group,the inhibition rate is about 75%-80%.Conclusion: Cdc42 interfere with plasmid successfully transfected hepatomaSMMC-7721 cells, significant changes in human malignant biological behavior ofhepatocellular carcinoma cells, cell growth slowed down to reduce proliferation, cellcycle arrest, cell migration and the invasive ability of plastic have different degrees ofreduced through the Matrigel.RNAi silencing Cdc42 led expression level of AFPprotein expression to significant changes in content, and reduce liver cancer cellinvasion and metastasis of capacity;3.Effect of Cdc42-shRNA vector on nude mice model transplanted with hepatomacells subcutaneously.Objective: To evaluate the in vivo gene silencing technology, the impact ofinterference with Cdc42 on the hepatocellular carcinoma.Methods:15 BALB/c-nu/nu mice was randomly divided into 3 groups,5 eachgroup;group A was inoculated with SMMC-7721,and B group with SMMC- 7721/pBS/U6-control and C group SMMC-7721/Cdc42-shRNA2.The nude mice modelwasestablished with 6×106/0.2ml cells inoculated subcutaneously in the armpit.Afterthe tumor grown,measure the length and width and calculate the volume of tumorevery 7 days.Execute the nude mice on the 28th day and cut the tumor and weigh,calculate the inhibition rate.Detect the expression of Cdc42 of the tumor by immunohistochemistry.Results Nude mice suffered from tumor blocks after inoculation of tumor cells in.In which A group and B group can be the 6th day after inoculation into the tumor,while the C composition of tumors was delayed until 8-11 days after inoculation. Withthe extension of feeding time, the tumor block increased gradually, without thephenomenon of spontaneous remission. Tumor volume growth curves show that thetumor volume of group A and group B is about same otherwise group C is siginificantly smaller.All the nude mice were executed on 28th day and the tumor wereweight,the tumor weight of group C is much lighter than group A and B,the inhibitionrate is 63.7%.The Cdc42 expression is strong positive in group A and B,but weakpositive in group C.Conclusion:In vivo tumor experiments also confirmed that RNA interferencetechnology can be silent Cdc42 protein expression in hepatocellular carcinoma andsilencing Cdc42 expression could significantly inhibit tumor growth.In conclusion, a high efficiency of RNAi silencing Cdc42 expression vectors wassuccessfully constructed, transfected hepatoma SMMC-7721 cells and inhibitedefficiently the expression of endogenous Cdc42 protein. RNAi technology can be usedas silence the Cdc42 expression of liver cancer in an effective way for furtherexperiments to lay an experimental basis; Cdc42 interfere with plasmid successfullytransfected hepatoma SMMC-7721 cells, significant changes in human malignantbiological behavior of hepatocellular carcinoma cells, cell growth slowed down toreduce proliferation, cell cycle arrest, cell migration and the invasive ability of plastichave different degrees of reduced through the Matrigel.RNAi silencing Cdc42 ledexpression level of AFP protein expression to significant changes in content, and reduceliver cancer cell invasion and metastasis of capacity; In vivo tumor experiments alsoconfirmed that RNA interference technology can be silent Cdc42 protein expression inhepatocellular carcinoma and silencing Cdc42 expression could significantly inhibittumor growth.This test verified Cdc42 is a important elements of development and progressionof liver cancer. Cdc42 liver cancer is most likely a potential therapeutic target, with abroad clinical application.更多还原