Studies On Effect And Mechanism Of KLE Cell Apoptosis Induced By Silencing F10 Gene

Gestational trophoblastic diseases include complete,partial and invasive moles, placental site trophoblastic tumours and choriocarcinomas,occurrence and development of GTD involves multifactorial pathological processes with multiple genetic alterations including activation of oncogenes and inactivation of tumour suppressor genes.Because gestational trophoblasts are special cells which are different from other somatic cells,they have specific biological and differentiational characteristics.Therefore it becomes very important to study the mechanism of GTD evolution and to search for novel effective methods of diagnosis and therapy.RNA interference was initially discovered in C.elegans(roundworm).RNAi is a sequence-specific posttranscriptional gene silencing mechanism,triggered by double-stranded RNA(dsRNA) and causes degradation of mRNAs homologous in sequence to the dsRNA.In 2001,Tuschl and his colleagues initially reported that chemically synthesized 21 nt siRNA(small interfering RNA)duplexes specifically suppress the expression of endogenous and heterologeous genes in different mammalian cell lines,and no non-specific gene silencing effects were observed.It is now well known that RNAi has become a powerful tool for studying gene function. Antisense oligonucleotide and ribozyme technologies,have been useful in reverse genetics,but only to a limited degree.At present,reverse genetics is the most effective way to assess the function of a gene,but so far there has been no general method for reverse genetics other than gene targeting by homologous recombination, which is costly and slow.Techniques such as antisense oligonucleotide and ribozyme technologies,have been useful in reverse genetics,but only to a limited degree.RNAi has been widely used to study gene function owing the following advantages: (ⅰ) DsRNA is much more stable than single-stranded RNA and more resistant to be degraded;(ⅱ)RNAi is a potent method,requiring only a few molecules of dsRNA per cell to silence gene expression;(ⅲ)High sequence specificity—Only one nucleotide mutation in the target mRNA will make the dsRNA ineffective;(ⅳ) Most of the genes in the genome of various organisms can be knocked down by RNAi. Therefore,RNA interference has attracted the attention of many researchers.The overexpression of oncogenes are genomic features of human cancers.Inhibition of abnormal expression of these oncogenes has been a common idea for studying the function of F10 gene.In 2005,Suppressive subtractive hybridization method was applied.Subtractive cDNA library was created between hydatidiform mole and normal early-pregnancy villous tissues.New genes were screened and full-length sequence were cloned.Totally 28 clones were selected from the cDNA library,10 gene sequences were identified,and 3 genes were found to be new genes.The full length sequence of one gene—F10 was cloned.The aim was to show that F10 gene might be closely associated with the pathogenesis of hydatidiform mole.Hybridization was used to study the expression of F10 in 12 cases of hydatidiform mole,6 cases of invasive mole,and 8 cases of choriocarcinoma.F10 mRNA was positive in all cases of hydatidiform mole,invasive mole,and choriocarcinoma,and the expression intensity significantly increased in ascending order with hydatidiform mole,invasive mole and choriocarcinoma.Analysis of results suggested that the expression of F10 gene may relate to the occurrence and invasiveness of trophoblastic tumor,with possible involvement in the invasion or malignant changes of trophoblastic cells.In 2006,In situ hybridization was used to study the expression of F10mRNA in different tumor tissues and normal tissues.F10 mRNA was shown to be positive in adenocarcinoma of ovarian carcinoma, endometrium carcinoma,mammary carcinoma,hepatocarcinoma and gastric carcinoma.There was no significant difference at the expression intensity of different adenocarcinomas.F10 mRNA was negative in normal tissues and hepatocarcinoma para-tumor tissues.Higher expression of F10 gene may be closely associated with trophoblastic tumors,suggesting an important role in the development of the adenocarcinoma.A novel related gene F10 expressed highly in HM was detected and subtractive cDNA library between normal villus,early pregnancy,choriocarcinoma and HM tissues was achieved in our study using suppression subtractive hybridization technique.An investigation of the features of F10 expression and essential functions were carried out with analysis of the gene silencing technique based on RNA interference in this study.Chapter 1 Knock-down of F10 expression by RNAi induces apoptosis of KLE cellContents and MethodsTo explore the small member a chain RNA to the KLE cell the silent effect of the F10 genes,and confirm the influence of F10 gene silencing on KLE cell apoptosis;The dsRNA aim to F10 genes was produced by in vitro transcription;plasmid was transfected into KLE cells.The expression level of F10 gene was detected by fluorescence quantitative RT-PCR in different time after KLE cell was transfected. The difference of apoptosis of KLE cell was assayed by FCM(flow cytometry) after F10 gene silencing.Results1,Sufficient dsRNA can be prepared by in vitro transcription.Molecular weight of dsRNA is about 21 bp by crylicacyl-amine gel electrophoresis,and the concentration of dsRNA is 740 ng/μl(52.85μmol/L).2 The results by fluorescence quantitative RT-PCR of the Taqman probe method indicate that compared with the control group,the copy count of F10 gene decreases after the KLE cells were transfected by dsRNA with different concentration,including 10nM,20nM and 50nM,and the decrease is remarkable when using dsRNA with 20nM and 50nM.The phenomena had been maintained till 72 hours after transfection.Among these nanomolars,the most significant effect of F10 gene silencing happened in KLE cell transfected with 20 nM dsRNA for 48h,the expression of F10 mRNA down regulated to 83%.3 The DNA content in KLE is detected by FCM(flow cytometry) and the results show that when compared with control group,the apoptosis index of KLE cells transfected with dsRNA increased from 0.36%to 8.91%,which is 24.75 times the control.Summary1,The dsRNA aim to F10 genes was produced by in vitro transcription and plasmid transfected into KLE cells.Expression level of F10 gene was detected by fluorescence quantitative RT-PCR in different time after KLE cell was transfected.The result to show that F10 gene in KLE cells can be specifically knocked-down with dsRNA produced by in vitro transcription.2,The DNA content in KLE is detected by FCM(flow cytometry) and the results show that the down regulation of F10 gene induces apoptosis of KLE cells. Chapter 2 The establishment of silent vector and setting up the stable cloneContents and MethodsDesign siRNA oligonucleotide snippet with hair card kind aim at the F10 gene. Set up pRetroQ-i vector aim at the F10 and establish the down -regulation of KLE cell line under the F10 gene expression stability.Depending on the F10 gene sequence in GenBank(Genbank NO.gi:5641046),design the candidate sequence by the soft and confirm the siRNA sequence aim to F10 gene.Synthesize siRNA oligonucleotide with hair card kind.The recombinant plasmid pRetroQ-i F10 producing short hairpin RNA of F10 gene was constructed by legating the double DNA annealed with two synthesizing oligonucleotides to pRetroQ,pRetroQ-i F10 plasmid was transfected into KLE cells,then the cells were screened by puromycine, and a single clone was established.Results1 The sequencing is correct and the established F10 plasmid showed that shRNA expression template successfully cloned to the pRetroQ/i F10 vector.The plasmid with the right insert sequences was chosen to make the single clone.2 The detection of F10 and GAPDH gene in single clone by fluorescence quantitative RT-PCR displayed that the expression level of F10 was not significantly differente between two RetrQ/negative cell clones,while it had down-regulated with different degrees among the five pRetrQ/RNAi.The expression levels of F10 descended by over 70%in two clones,and in the No.5 clone it descended to adjust to 74%.It is doubtlessly the effective F10 knocking-down positive clone.Summary1,Design siRNA oligonucleotide snippet with hair card kind aim at the F10 gene.Set up pRetroQ-i vector aim at the F10.The results sequencing is correct and the established F10 plasmid showed that shRNA expression template successfully cloned to the pRetroQ/i F10 vector.2,Depending on the F10 gene sequence in GenBank(Genbank NO.gi:5641046), design the candidate sequence by the soft and confirm the siRNA sequence aim to F10 gene.Synthesize siRNA oligonucleotide with hair card kind.The recombinant plasmid pRetroQ-i F10 producing short hairpin RNA of F10 gene was constructed by legating the double DNA annealed with two synthesizing oligonucleotides to pRetroQ.pRetroQ-i F10 plasmid was transfected into KLE cells,then the cells were screened by puromycine,and single clone was established.3 The detection of F10 gene in single clone by fluorescence quantitative RT-PCR displayed that the No.5 clone it down regulated to adjust to 74%.It is doubtlessly the effective F10 knocking-down positive clone.Chapter 3 Effect and mechanism of KLE cell aapoptosis induced by silencing F10 geneContents and MethodsTo explore the effect and mechanism of F10 silencing on apoptosis of KLE by setting up a cell line in which F10 gene is knocked-down..The apoptosis of KLE cell was detected by FCM(flow cytometry).and Fluorescence quantitative RT-PCR together with western blot were used to examine the expression of cytochrome and Bcl-2 mRNAResults1 The apoptosis of KLE cell by FCM displays that the rate of cell apoptosis from negatively matched control is 1.8%,while in cell clone of F10 knocked-down gene,it increases to 16.8%,and is distinctly higher than the matched control.2 The results form fluorescence quantitative RT-PCR show that the expression of cytochrome in F10 knocked-down gene cell clone increases to 85%,while that of KLE cell has no significant difference compared to the matched control.3 The results from Weston blot detection show that the relative expression of cytochrome C is 0.46,0.38 and 0.13,corresponding to in KLE cell,control group cell and in mitochondrion of experimental group.The relative expression of cytochrome C in cytoplasm is 0.15,0.15 and 0.44 respectively,which is significantly higher in the group of disturbed F10 gene.The expression of bcl-2 m-RNA was significantly down-regulated to 64%,and the relative expression of protein descended to 0.11 from 0.44,which is the control group.SummaryExplore the effect and mechanism of F10 silencing on apoptosis of KLE by setting up a cell line in which F10 gene is knocked-down.1,The cell clone of knocked-down F10 gene induced decreased expression of BCL-2 illuminating mitochondria participated in the mechanism of F10 gene silencing effect on cell apoptosis.2,The release of cytochrome C may be one of the pathways by which F10 gene knocking-down induced KLE cell apoptosis.Conclusion1 F10 gene in KLE cells can be specifically knocked-down with dsRNA produced by in vitro transcription2 The silencing of F10 gene induces the apoptosis of KLE cells.3 The expression of F10 is stably down-regulated By building a cell clone with F10 gene silencing and this is the bases of the further experiment.4 The cell clone of F10 gene knocking-down induced decreased in expression of BCL-2 and illuminated mitochondria have participated in the mechanism of F10 gene silencing effect on cell apoptosis.5 the release of cytochrome C may be one of the pathways by which F10 gene knocking-down induced KLE cell apoptosis.