Effect Of FASN Gene Silencing By MiRNA Interference On The Biological Behaviors Of Osteosarcoma Cells In Vitro

Objective:Construction and screening of the best recombinant plasmid recombinantplasmid of FASN gene. To investigate whether down-regulation of FASN affects theproliferation, invasion and migration in human osteosarcoma cell line U2-OS in vitro.Methods:①Four shRNA sequences were designed and synthesized based on the FASNcDNA sequence obtained from NCBI. The annealed four double-strand oligos werecloned into the pcDNA6.2-GW/EmGFP-miR vector.The four recombinant plasmids(named X197-1，2，3，4) were confirmed by DNA sequencing and the U2-OS cell wascotransfected with recombinant plasmids and Lipofectamine2000. The efficiency oftransfection was detected by an inverted fluorescence microscope. RT-PCR andWestern blotting were performed to detect FASN gene expression.②Optimized plasmid transfection conditions.Transfect U2-OS cells in theoptimized transfection conditions (plasmid: Lipofectamine2000=1:3) with theselected recombinant recombinant plasmid plasmid targeting FASN. MTT methodwas used to measure cell proliferation, Detect the cell invasion、migration capabilitiesby wound healing and Transwell invasion assay.Results:①FASN-recombinant plasmid was successfully constructed. The fluorescencerate of recombinant recombinant plasmid (X197-1、X197-2) is greater than50%48hours after transfection. RT-PCR and Western blot showed that the recombinantplasmid vectors (X197-1、X197-4) have significant inhibitory effects on FASNmRNA (53.2±4%、47.5±3%、41.9±5%、52.8±2%), and protein expression（inhibitionrate，9.4%、47.3%、64.5%、64.2%）(P<0.05). Compared with the other recombinantplasmids, X197-1is identified as the best plasmid.②After transfection by X197-1for24,48,72,96hours, the OD values are（0.1183±0.0408、0.1567±0.0476、0.3033±0.0121、0.4783±0.0107）, significantlylower than that in both negative control group and blank control group. Wound healing assays showed that the migration rate of U2-OS cells transfected by X197-1recombinant plasmid was (13.7±8.7)%, significantly lower than that in both negativecontrol group and blank control group which was (74.8±4.5)%and (77.6±6.4)%respectively (p＜0.05).Transwell invasion assay showed that the number oftransmembrane cells transfected with recombinant plasmid X197-1, targeting FASNgene, was (55±4.8), significantly less than that in both negative control group andblank control group which was (135±7.4) and (146±6.7)(p＜0.05). The resultsindicated that inhibition of FASN gene could inhibit U2-OS cell invasion andmigration in vitro.Conclusion:FASN recombinant plasmids were constructed successfully. Silencing FASNgene expression can effectively inhibit the proliferation, migration and invasion ofhuman osteosarcoma cell line U2-OS cells which laid a solid foundation for thefurther research.