Targeting Polypeptide Lhrh - Mpg < Sup > Delta Nls < / Sup > As A Carrier Rna Interference Therapy Of Liver Cancer Research

Cancer has become a threat to human health, but so far there is no very effective treatment. Traditional chemotherapy drugs have very serious side effects, resulting in deterioration of patients’life quality. RNA interfering provides new thoughts and methods for cancer treatment. The major limitations for the use of siRNA are the instability of naked siRNA in physiological conditions and the bloodstream, and the inability to cross the cellular membrane to gain access to the intracellular environment. Cell-penetrating peptides (CPPs) are short sequences of amino acids, usually30or less amino acids, covalently linked through an amide or peptide bond. Considering the safety concerns and efficacy issues, peptide-based drug/gene vectors are emerging as alternatives for safe and efficient delivery means.The main strategy to improve tumor targeting therapy is modifying tumor-associated antigen on the carrier material. The present work extends showed that the luteinizing hormone-releasing hormone (LHRH) receptor is over expressed in liver cancer cells. In contrast, LHRH receptors are not expressed, or expressed at a low level in normal liver cells.In our research, we use LHRH modified MPG△NLS, which is one kind of CPPs, formatting a new fusion peptide:LHRH-MPG△NLS. To investigate the preparation method of LHRH-MPG△NLS/siRNA complexes, their stability and transfected efficiency and targeting human hepatoma cell line. The research work includes the following five parts.1. The biding capacity of LHRH-MPG△NLS with siRNA was detected by agarose gel electrophoresis. Results:the results of agarose gel electrophoresis confirmed that LHRH-MPG△NLS and siRNA could be totally integrated when N/P ratio was more than10:1.2. To investigate the preparation method of LHRH-MPG△NLS/siRNA nanoparticles and their stability. Result:When N/P is10:1～20:1and the pH is7.0～8.5, the size of the complexes formed150nm-250nm. The formation of complexes under this condition the complexes could keep stable for20days.3. Fluorescently labeled siRNA was complexed with LHRH-MPG△NLS at different N/P ratios, and then the LHRH-MPG△NLS/siRNA complexes were used to transfect human hepatoma cells (HepG2) and human normal liver cells (L02) with concentration of50nmol/L,100nmol/L,200nmol/L, respectively. In Cell Analyzer2000was employed to explore the optimal transfection conditions and investigated whether the complexes can target hepatoma cells. Results showed the best transfection conditions:N/P ratios was20:1, the concentration was200nmol/L and the time for transfection is24hours, the transfenction effiency in HepG2cells is greater than L02cells.4. The siRNA designed to silence GAPDH was complexed with LHRH-MPG△NLS, and then LHRH-MPG ANLS/siRNA complexes were used to transfect HepG2cells and L02cells respectively. The effects of gene silence were detected by reverse transcription-polymerase chain reaction (RT-PCR). Results:LHRH-MPG△NLS/siRNA complexes showed a greater levels of silencing in HepG2cells than L02cells.5. Toxicity of LHRH-MPG△NLS/siRNA complexes was detected by WST-1test, results showed that living cells ratio is more than90%when transfection concentration is between30nmol/L to200nmol/L.