Vector Construction And Silencing Effect Of MTA1 Gene Targeted Small Interfering RNA

Background and aims : Malignant tumor is a difficulty in clinical therapy due to its invasion and metastasis, which can lead to death. The metastasis of tumor is a complicated procedure which involves interaction of many genes and its products: tumor cells detaching from primary focus; invading blood vessel and lympgatic vessel; attaching to the endothelial cell for infiltrating far organ; inducing neovascularization and avoiding the host anti-tumor reaction, et al. Therefore understanding the gene and its products which involves in the tumor invasion and metastasis is an important study on domestic and abroad at present. Many studies about the relationship of expression of tumor metastasis associated gene and tumor metastasis have proceeded in order to look for the safe and effective proposal in treating malignant tumor and extending patients life cycle.Metastasis associated gene 1(MTA1) is a new tumor metastasis related gene, which locates 14q32.3 in chromosome. MTA1 is the protein coded by human metastasis associated gene 1 which includes 715 amino acid residue and molecular weight is 82kD. It is not cell surface protein and seceret protein due to its visible hydrophilicity. The C-terminal of protein is rich of pyrrolidinecarboxylic acid which can matched to SH3 binding domain completely. While SH3 binding domain participates in the interaction of protein and protein in the signal transduction pathway, relating to the tumor invasion and metastasis genes, belonging to the composition of cystoskeleton. MTA1 has nine protien kinase C sites(PKCs), two tyrosine kinase sites(TKs), seven casein kinase II(CK II) phosphorylation sites and four N2 glycosylation sites, which means MTA1 perhaps participating in the interaction of other proteins that sustain cells' normal function in the signal transduction pathway. MTA1 was thought as a components of nucleosome remodeling and histone deacetylase(NuRD), which have nucleosome reconstitution and histone deacetylation enzyme activity. These characteristics can regulate gene transcription and replication procedure by ATP-dependent NuRD acetylation and deacetylation influencing chromatinic state. So it indicates that MTA1 maybe has major contribution in regulating proteins which relate to tumor metastasis and invasion in signal transduction and gene expression regulation. Many studies show that high expression of MTA1 has close relation to some epithelium tumor invasion and metastasis. The MTA1 gene will be a effective target and control method in regulating malignant tumor invasion and metastasis.RNAi which develops in recent years open up a new door for molecule biological therapy of tumor. Compared with traditional gene silencing, it has effect and lasting a long period of time. At present, the technique has taken on an attracting perspective in gene therapy of tumor. So this experiment design and construct expression vector of siRNA to aim directly at MTA1, transfect it into human osteosarcoma cell line MG-63, observe its silencing effect.Methods: According to the MTA1 sequence GenBank has been adopted, we used the siRNA design and analysis software which were provided by following Web address (http://www.ambion.com/techlib/misc/siRNA_design.html;http://www2.takara-bio.co.jp/sirna-d/top.php) to design the target oligonucleotides. Then the oligonucleotides of target siRNAs were indexed through National Center for Biotechnology Information (http://www.ncbi.nlm.nih. gov/Blast) to compare their homologization, in order to identify the best siRNA oligonucleotides of MTA1. Afterwards, the siRNA oligonucleotides of MTA1 which has palindrome and loop structure was synthesized. The DNA was cloned into T vector. Then,α-complementation test and T7/SP6 PCR were used to screen the positive clones(pGEM-T-MTAl); After this, the target DNA was cut down from pGEM-T-MTA1 by Bgl II and HindIII restriction enzyme, and linked with siRNA express vector(pSuperneo); The positive ones were selected by Bgl II and HindIII restriction enzyme cutting test; Then the target DNA was sequenced. Eventually, the siRNA express vector pSuperneo-MTA1 was transfected into human osteosarcoma cell line (MG-63). RT-PCR was used to detected the express level of MTA1 mRNA in MG-63 after cultured and screened by G418. Result:1. The 116 target siRNA oligonucleotides were gotten. After BLAST in NCBI, an oligonucleotide was ensured, which had 19 bases GACCCTGCTGGCAGATAAA(即481-499nt).2. After the hairpin DNA of siRNA annealing, the results of agar-gel electrophoresis analysis showed that there was an obvious stripe whose molecular weight was similar to the expected.3. The annealing products was recombined with pGEM-T Easy, which was transformed into JM109. After screened and identified, the positive clone (pGEM-T-MTA1) was gotten.4. After sub-clone, the pSuperneo-MTA1 was gotten and identified through Bgl II and HindIII restriction enzyme cutting test.5. After sequenced, the DNA sequences inserted the recombination pSuperneo-MTA1 was conformed completely with the design.6. The MTA1 mRNA level in MG-63 was significant different among transfected, pSuperneo-C,pSuperneo and untransfected. The expression in experimental group which transfected pSuperneo-MTA1 was nearly inhibited. Express level of others was still high.Conclusion1. The siRNA oligonucleotide liked hairpin target MTA1 gene was design.2. The express vector pSuperneo-MTA1 was constructed successfully3. The express of MTA1 mRNA in osteosarcoma cell line (MG-63) was obviously silenced after pSuperneo-MTA1 transfected.