Fiyz̤ (Apr 2022)
In Silico design and in vitro validation for siRNA targeting PTX3 gene in glioma cell line
Abstract
Background: Identification of underlying mechanisms of gliomas pathogenesis is of particular importance. The overexpression of PTX3 gene is profoundly implicated in glioma development. The RNA interference (RNAi)-based knockdown of target gene by means of double-stranded RNA molecules including siRNA can be harvested as a therapeutic tool for oncogene silencing. The present study aimed to induce apoptosis in U-87 glioma cell line by knockdown of PTX3 gene. Materials and Methods: A variety of in silico methods were applied for siRNA design to silence PTX3 gene. Scoring of candidate siRNAs was performed according to design rules. The best siRNA against the PTX3 gene as well as scrambled siRNA were selected. The efficacy of PTX3 silencing in the U-87 cells was evaluated by Real‐time PCR assay. To assess the effect of PTX3 knock down on cellular apoptosis, the mortality rate of transfected cells was compared with control groups by flow cytometry. Results: Designed PTX3‐siRNAs (n=53) were assessed and scored from different aspects and the best one was suggested in PTX3 gene expression knockdown assay. The 72-hour treatment of U-87 cells with designed PTX3‐siRNA in 100 nM concentration affected the gene expression by decreasing to 69%. Flowcytometry results indicated the induction of apoptosis in 65% of the cellsConclusion: The efficiency of designed siRNA was approved in vitro, with significant effect on the downregulation of PTX3 gene and induction of apoptosis in U-87 glioma cells. Based on our finding, targeting PTX3 via siRNA can be considered as an anti-cancer strategy
