Study Of The RAS Gene Function In The Apoptosis Of K562 Cell Line

One of the most frequently activated oncogenes in human cancer is the Ras gene family, which functions as a molecular switch in a large network of signaling pathways, mainly controlling the differentiation or proliferation of cells. Mutated RAS genes encode constitutively activated proteins, which have been implicated in tumorigenesis. Following activation, oncogenic RAS remains constitutively in the active form, which endows the auto proliferation of the tumor cells, which is the essential step in the tumor form.It has been known that point mutation is the most common way of RAS activation. RAS mutation can be found in numerous of human cancers, physical, chemical or biological factors all could contribute to such processes. In humans, about 30% of the tumors carry RAS mutations, of which K-RAS is the most frequently mutated. Be the important factor in tumor process, RAS gene has been widely studied both in clinic and basic researches. K-RAS mutations are often found in adenocarcinomas of the pancreas, colon and lung, H-RAS mutations are more familiar in urinary system tumors, and the N-RAS mutations are seen in the heamopoietic system.The research of the RAS pathway is helpful in the cancer genetic therapy. Problems arouses that the pathway is involved in a complicated network interactions consists of large amount of different signaling factors. Uncovering or elucidating suchcomplex network would have great implication in the mechanism study of carcinogenesis.RNAi has been developed recent years as the strategy to knock down the target gene expression, so as to investigate the structure, function or signal pathway. It is generally accepted that RNAi is an effective, feasible and stable approach in functional genomics studies. It has been demonstrated that specific and efficient RNA interference can be achieved in mammalian cells with duplexes of 21 nt RNAs that form a 19 bp region with a 2 nt overhang. Such a duplex is small enough to bypass the interferon responses of differentiated cultured cells, while achieving the expected gene silencing effect.It is now possible to generate siRNAs in cells from various expression vectors and, therefore, both synthetic and vector derived siRNAs can be used in the functional analysis of genes of interest in mammalian cells. Several studies exploiting RNAi have been performed. Although the dependence of the activities of siRNAs on their target sequences was reported recently, the selection of the best target site is often difficult. The published data suggest that the GC content of the target sequence, the position and accessibility of the target site, and the strength of terminal base-pairings of siRNAs are important.According to the reasons above, we decided to do study work in the N-RAS gene function in the apoptosis with RNAi. We chose human leukemia cell line K562 as the cellular model, which was originally derived from a patient with chronic myeloid leukemia (CML) in blast crisis. To complete the purpose, we designed experiments with three steps. Firstly, we investigated the N-RAS expression in K562 cells, including the mutation and the expression level compare with the white blood cells from the health individual. Secondly, the N-RAS gene expression is silenced by RNAi. Thirdly, the cellular phenotypes were studied prior and posterior of the RNAi silencing. Detection was performed both at the mRNA and the cellular level. We also worked on the RNAi experiment itself. We wanted to know if the secondary structure of the target gene mRNA influence the silence efficiency.The experimental study has demonstrated that:1. N-RAS expression in K562 cells was detected by RT-PCR and direct sequencing. Specific PCR primers were designed with the Primer Premier 5.0 to amplify the conserved regions of human N-RAS genome. Then the purified PCR products were cloned into the T vector and amplified by E.coli. Following that, the reconstructed plasmid was extracted to be sequenced in two directions. The sequencing result was compared with N-RAS genome by BLAST which is offered by NCBI online for point mutation detect. RT-PCR was performed using the GAPDH as standard and the peripheral mononuclear cells of healthy individual as the control. After the experiment, it was found that the there is no N-RAS gene mutation in K562 cell line, while the expression level of it is obviously higher than normal cells, which providing a satisfactory model for further siRNA studies.2. siRNA selecting and gene expression silencing of the N-RAS gene. We took the sequencing result as the template for siRNA design. Selected target sequence was analyzed with software RNA structure 3.5 for the secondary structure. The result was considered into the siRNA design. Finally 4 siRNAs were finalized aiming the secondary structure region and non- secondary structure region separately with the web tool Dharmacon siRNA design Center. 48 and 72 hours after transfection, the four siRNA experiment groups were detected separately by RT-PCR, cell viability assay and flow cytometric analysis of cell apoptosis to detect the varied effect. The comparison was performed by both groups and time to explore the effect of the different factors. It was found that the 72 hours treatment is better than that of 48 hours for the silencing effects, and the non-secondary structure region siRNAs were demonstrated to be better targeted in the RNAi experiment.It can be concluded from the first part result that the K562 cell line could be an excellent model for the RNAi experiment. The second part, both mRNA and cell level, demonstrated that the loss of the N-RAS gene expression result in apoptosis of tumor cells, which suggest the importance of N-RAS gene in tumor formation. Furthermore,the secondary structure of the target gene mRNA could be one of the influential factors that cause the diversified silence efficiency of the specific siRNAs, which should be taken into consideration into the design of the siRNA.In summary, we investigated the expression of the N-RAS gene in K562 cell line, and the results demonstrated RNAi could specifically knock down the expression of N-RAS expression, leading to cellular apoptosis of K562 cells. Subsequent study of the N-RAS function in apoptosis and signal pathway would contribute to gene therapy of leukemia both theoretically and practically.