The Study About The Function Of Cyclin E In K562 Cells With Methods Of RNAi And DNA Microarray

The cell cycle is controlled by numerous mechanisms ensuring correct cell division. In cancer, there are fundamental alterations in the genetic control of cell division, resulting in an unrestrained cell proliferation. Mutations mainly occur in two classes of genes: proto-oncogenes and tumour suppressor genes. In normal cells, the products of proto-oncogenes act at different levels along the pathways that stimulate cell proliferation. Mutated versions of proto-oncogenes or oncogenes can promote tumour growth. Different Cyclins are required at different phases of the cell cycle . Cell cycle deregulation associated with cancer occurs through mutation of proteins important at different levels of the cell cycle. In cancer, mutations have been observed in genes encoding CDK, Cyclins, CDK-activating enzymes, CKI, CDK substrates, and checkpoint proteins.Cyclin E, located on chromosome 19q12-13, produces a 395 amino acid protein that contributes to normal cell proliferation and development. As a cell cycle-regulating factor, Cyclin E is essential for progression through the G| phase of the cell cycle and initiation of DNA replication by interacting with, and activating its catalytic partner, the Cyclin-dependent kinase 2 (CDK2). Over expression of Cyclin E accelerates the G₁ phase of the cell cycle. This is accomplished by phosphorylating the product of the retinoblastoma gene (PRB). The ability of Cyclin E to phosphorylate PRB is dependent on a group of molecules termed Cyclin-dependent kinase inhibitors(CKIs). Cyclin E which associates with CDK2 regulate progression from G₁ into S phase.Cyclin E was identified originally by complementation of a yeast G1 Cyclin mutant and it was later shown to be deregulated and overexpressed in several tumors including breast, colon, and prostate carcinomas. Overexpression of Cyclin E correlates with advanced grades and stages of tumour and has prognostic significance in many tumour types. Expression of Cyclin E is elevated in premalignant lesions ofthe breast and skin, indicating an early event in the development of these tumours. The Cyclin E mRNA levels increase sharply in late G\ followed by accumulation of Cyclin E protein levels. The levels of Cyclin E and its associated CDK2 kinase activity reach a maximum at the Gi/S boundary. Cyclin E protein is regulated at the transcriptional level and subject to ubiquitin-dependent degradation. Cells with defect pRb and overexpression of Cyclin E show noticeable similarities in cell cycle kinetic parameters suggesting that the growth promoting properties conferred by pRb inactivation could be mediated by induction of Cyclin E. In a recent study, however, it was demonstrated that despite loss of periodic transcriptional repression of Cyclin E, pRb defective cells still had distinct cell cycle specific variations in Cyclin E mRNA level, protein expression and associated CDK2 activity. Cyclin E participates in abnormal cell cycle regulation of leukemic cells. Given the critical role of Cyclin E gene in the occurrence and development of leukemia, drug therapy targeting at this gene has attracted increasing attention in current research of leukemia.Studies have suggested that overexpression of Cyclin E gene promotes DNA synthesis in the leukemic cells, resulting in excessive proliferation of these cells. Further study indicates that Cyclin E, due to its close association with the progression and prognosis of leukemia, may potentially provide a valuable marker for diagnoses of some types of leukemia.The siRNAs can be effectively transcribed by Pol III promoters in human cells and elicit target-specific mRNA degradation. These siRNA-encoded genes have been transiently transfected into human cells using plasmid or episomal viral backbones for delivery. Transient siRNA expression can be useful for rapid phenotypic determinations preliminary to making constructs designed to obtain long-term siRNA expression. Long before RNA interference had been established as operating in mammalian cells, researchers working on worms had recognised the great power that it promised as a research tool. The sequencing of the genomes of humans and most commonly studied model organisms has led to a situation in which the identities of very large numbers of genes are known but little is understood about their function. A cheap and easy way of ablating gene function holds out massive hope for improving our ability to untangle the complex regulatory pathways that control cellular behaviour in health and disease.The ability of RNA interference to provide relatively easy ablation of gene expression has opened up the possibility of using collections of siRNAs to analyse the significance of hundreds or thousands of different genes whose expression is known to be up-regulated in a disease, given an appropriate tissue culture model of that disease. The libraries of RNA interference reagents can be used in one of two ways. One is in a high throughput manner, in which each gene in the genome is knocked down one at a time and the cells or organism scored for a desired outcome—for example, death of a cultured cancer cell but not a normal cell. The other approach isto use large pools of RNA interference viral vectors and apply a selective pressure that only cells with the desired change in behaviour can survive. RNA interference allows analysis in a matter of days of the effect of loss of gene function at the cellular level that would have taken several months or even years by previous methods such as homologous recombination. RNA interference is already proving to be an invaluable research tool, allowing much more rapid characterisation of the function of known genes. More importantly, the technology considerably bolsters functional genomics to aid in the identification of novel genes involved in disease processes.Someone have previously taken advantage of an oligo-nucleotide /RNAse H procedure in cell extracts on native mRNA transcripts originally designed for identifying ribozyme accessible sites, and have found that this approach can be applied to siRNA site accessibility as well. Unfortunately, this process can be time consuming, and, in the end, it is still necessary to synthesize the siRNA genes for final testing. Cloning the siRNAs in the expression vector of choice can also be a time-consuming process, although, if the unusually compact HI promoter is used, it can be facilitated by the annealing and extension of two oligo nucleotides containing the hairpin and promoter sequences. In this report, we describe a PCR-based approach for rapid synthesis of Pol III promoter-siRNA gene constructs and their subsequent transfection into cells. This procedure can be utilized for the facile screening of siRNA encoding genes to identify those with the best functional activity for a given target. The PCR products are used directly, without subsequent cloning, by transfecting them into cells followed by functional assays. The method described here can also be used for screening siRNA gene libraries. The method is fast and inexpensive, allowing several different siRNA gene candidates to be rapidly screened for efficacy. Our approach can be utilized with siRNAs expressed independently from Pol III promoters or siRNAs that are transcribed as hairpin precursors.DNA chip technology was originated from the idea of making integrated computer chip for parallel detection of biological agents. With the advent of DNA microarray technology, groups of genes or even the whole genome could be studied simultaneously in parallel, ensuring more and more studies conducted systematically. Microarray technology continues to improve in performance aspects regarding sensitivity and selectivity and in becoming a more economical research tool. The use of DNA microarrays will continue to revolutionize genetic analysis and many important diagnostic areas. Additionally, microarray technology that has been developed for DNA analysis is now also being applied to new areas of proteomic and cellular analysis. The following is a compilation of microarray publications on some of the broad areas of application: general applications of microarrays for gene expression analysis; gene expression analysis on high-density microarrays; gene expression analysis for cancer; gene expression analysis for drug discovery, metabolism, and toxicity; gene expression analysis for neuroscience applications:gene expression analysis for microbiological and infectious disease; use of microarrays for genotyping; general articles on use of microarrays for cancer; infectious disease diagnostics; use of microarrays for disease diagnostics; and applications of microarrays for plant biology. In addition to microarrays for DNA hybridization analysis, considerable efforts are now being directed at the development and use of microarrays for proteomic applications and microarrays for the immobilization of cell and tissue samples.In our study, three Cyclin ^-specific siRNAs designed were linked at the downstream of U6 promoter and amplified by PCR, and transfected into K562 cells via liposome for transcription to produce the inhibitory effect. The effect of RNA interference is directly correlated to the density of transfected cells, and as K562 cells proliferate at a fast rate, the high cell density at transfection is not advisable. According to our repeated experiment, the cell density at (45-55) proved optimal. Inhibitory effect was observed 16 h after the transfection, and at 48 h, cell growth arrest occurred with 61.9% of the cells arrested at Gl stage, and Cyclin E mRNA expression was significantly weakened as shown by RT-PCR, suggesting that Cyclin ^-specific siRNA can significantly inhibit Cyclin E mRNA and the proliferation of K562 cells.The second part of the study, we used DNA microarray technique to study the tumor genes present in the genome of K562 cell of RNA interference. We applied the Restriction Display (RD) technique combined with labeling by extension in corporate Cy-dNTP. Cy3/Cy5 (two different fluorescence dyes) separately labeled the control and the treated samples. The gene expression microarray was scanned by using a dual-laser scanner (GenePix 4100A) and the data were statistically analyzed with the Quantarray software package. Among the 578 target genes, 68 down-regulated genes (Cy3/Cy5>2.0) and 12 up-regulated genes (Cy3/Cy5<2.0) were identified after RNA interference 1.In summary, we currently blended the DNA miroarray and RNAi methods together to study to the differentially expressed genes after interference in K562 cells. This study can provide reference to the therapeutic approaches in genetic level as well as to find new trains of thoughts against erythroleukemia.