RNAi On SelW Gene In Mus Musculus Muscle Cell

Selenoprotein W was one of Selenoproteins which was discovered lately. There are indirect evidences to verify that its role in metabolism has relationship with the healthy of skeletal muscle tissue, but direct evidence still did not seen. The Selenoprotein Ws in the tissues of bovine, ovine, monkey, human and rodents are sensitive to the level of dietetic selenium and mainly accumulate in the skeletal muscle. Its structure and undetectable characteristic in selenium deficient muscle tissue all demonstrate that there is a mysterious relationship between selenoprotein W and selenium deficient muscle diseases. To investigate the role of selenoprotein W quantitatively and qualitatively is helpful to analysis its important meaning to skeletal muscle metabolism. Meanwhile, monitoring changes of cell caused by quantitative and qualitative down-regulating of Selenoprotein W to reflect the relationship between target protein and the healthy of muscle cell, and analogy the clinical pathological course of white muscle disease.The down-regulation of Selenoprotein W mRNA by RNAi in this research, which was performed by introduced synthesized 21bp double strands siRNA into mouse skeletal muscle cell, in which a complex contains lipofactamine. Plus reagents and siRNA will be transfected into cell by pinocytosis and van der waals force;after the complex get into cells successfully, a RNA-induced silencing complex will be generated by the specific mechanism of cell to degenerate Selenoprotein W mRNA. The dynamics of mRNAs and proteins of Selenoprotein W were monitored by real time PCR and western blot;while cell status was measured by Flow cytometry. Transfection effectiveness was reported by a florescent labeled siRNA without homology to the sequence of target mRNA. This research was performed on passage and primary cell parallel.The effectiveness of RNAi in C2C12 cell on selenoprotein W gene was assayed. The highest effectiveness of interfering can be seen 24 hours later after transfection. The transfection effectiveness in this research were from 50% to 70%.The 2 positive siRNAs were compared in passage cell under the frame of the effectiveness to cause cell changing. Cells were separated into 2 groups to transfect 2 different positive siRNAs separately as lug according to the instruction of lipofectamine reagent. The cells were assayed by flow cytometery at 24 and 48 hours after transfection.In dose reaction assay the No. 1 siRNA with superior effectiveness was selected for the experiments and cells were divided into 6 groups to correspondence with 6 dose gradients of the same siRNA so as to down-regulate the level of SelW mRNA step by step. Every groups were transfected with siRNA in the sequence of 2ug, 1.5ug, 1.2ug, lug, 0.5ug, 0.25ug, andwere marked as 1 ~6 . Assay was done 24 hours after transfection.On the basis of the comparing of 2 siRNAs and dose reaction assay, the assay of effectiveness of RNA interfering was performed, in which positive, negative and blank control groups were set to be transfected with the No. 1 positive siRNA, negative siRNA and nothing (just changing the medium).All the cells used in prior experiments were assayed by flow cytometery, real-time PCR and Western blot to report cell status, the changes of target genes and proteins.Results:1. Either of the 2 synthesized siRNAs can trigger RNA interfering;but there was difference of effectiveness between the 2 siRNAs according to the results of flow cytometery;the No. 1 siRNA (31.7%) is better than No.2 (14.5%), so the No. 1 siRNA was selected for later experiments. The most suitable time for assay is 24 hours later after transfection.2.In dose reaction assay in passage cell, except from 3 groups with excessive dose of siRNA transfection with a 50~70% cell layer detached and fail to be assayed, the results in other groups were: apoptosis rate 32.5%, 12.4%, 6.58%;the amounts of SelW mRNA 0.25, 0.39, and 0.524 by standard curve method of Real time PCR;In protein assay, set the value of group 6 as standard, the amounts of proteins were 17.26, 31.08 and 50. All evidences show that lug of siRNA is most suitable dose, which not only can trigger highest effectiveness of interfering, but also sufficient to sustain a balance between cell healthy and interfering.3. In RNAi effectiveness test experiment in passage cell, the result of Flow cytometry showed that positive group can cause a highest apoptosis rate of 37.5%. There were significant difference while comparing the positive group with negative (0.93%)and blank controls(0.7%): P=3.08xl0"9 < 0.05, P=2.95*10"9 < 0.05. Real time PCR result indicated that the mRNA of SelW in positive group(0.275) dropped approximately 72.4 % compare to blank group(0.996), and negative control group(0.9867) dropped 0.93%. In the assay for the changing of proteins, taking blank control as a standard (100), the amounts of proteins in positive, negative group were: 25.07, 98.75;to compare with blank control, the amounts of proteins in positive and negative group dropped 74.93% and 1.25%.One should be noted was that the reactions in primary cell in dose reaction assay, RNAi effectiveness assay were consistent with that in passage cell. But the changing caused by RNA interfering in primary cell was weakness than that in passage cell. In dose reaction assay, overdose siRNA caused cell death;When lug, 0.5ug, 0.25ug were transfected, apoptosis rate were 18.3%, 8.1%, 4.3%;the amount of mRNAs were 0.22, 0.25, 0.32;amounts of protein were 39.5,46.2,and 50. In RNA interfering effectiveness assay, results in positive, negative and blank control group: anontosis rate were 20.0%, 1.03%, 0.4%;the amounts of target gene were 0.534, 0.663, 0.67;the amounts of proteins were 41.2, 48.8, 50.Tests illustrated: 1 .In the course of skeletal muscle metabolism, the depletion of SelW induce to apoptosis, and one function of SelW protein is apoptic inhibitor, which illustrated from degeneration to necrosis were not the only pathological mechanism of selenium deficient muscle disease.2.There is a threshold in RNA interfering, i.e. Better result needs enough dose of siRNA. This is meaningful to anti-virus and gene therapy introduced by RNAi. 3. The effectiveness of RNAi is not influenced by the abundance of target gene. Though SelW is a low abundance gene in muscle cell, experiment demonstrated that a better result of RNAi could be triggered on it. This supported the theory that the resistances to RNAi of different genes are heredity. 4. RNAi in primary cell is more conservative. All results come from every experiments in primary cell showed the effectiveness in primary cell was lower than that in passage cell, and the reason needs further study.