Effects Of Silencesing Phosphatase Of Regenerating Liver Cell-1by RNA Iinterferene On Proliferation And Invasion Of Human Tongue Cancer Cells

Objective:1.To construct a lentiviral vector for PRL-1gene RNA interference andscreening stable transfection cells of human tongue squamous cancer cellline TCA8113.2.To study the effects of phosphatase of regenerating liver cell-l（PRL-1）small interfering RNA（siRNA）on cell growth,invasion and MMP2andMMP9expression in TcaS l l3cells.Methods:1.According to the Gene bank of PRL-1mRNA sequence（NM₀03463）, According to the sequence designed fiver target sequenceof oligonucleotides.BLAST analysis the sequence specificity. The designedPRL-1gene-specific siRNA sequences were to construct lentiviralexpression vector GV248,While building PRL-1over-expression plasmidswere co-transfected293T cells, Western Blot screening of the most effectiveexogenous PRL-1RNAi target sequences of genes.The LV-PRL1siRNA, and the secondary packaging of the original plasmid pHelper1.0andpHelper2.0of these three plasmids using Lipofectamine2000according toInvitrogen’s instructions were co-transfected293T cells to produce lentiviralpackaging.The titer of virus was tested according to Puromycin drugscreening and the expression of green fluorescent protein. TCA8113cellswere infected with the recombinant lentivirus.The PRL-1expression levelwere assessed by Realtime-PCR experiment and Western blotassay.Screening stably transfected recombinant lentivirus and blank virusTca8113cell lines useing the Puromycin.2.Experimental design （1） control group （CON）: Tca8113cells withoutany treatment;（2）KD:stably transfected with LV-siRNA-PRL-1’s Tca8113cells （3） NC: blank virus vector transfected Tca8113cells.Total RNA and thetotal protein was extracted from cells in each group,Quantitative PCR andwestern blot assay Were used to detect the expression of PRL-1, MMP-9,MMP-2expression.Application Transwell chamber experiments to observethe PRL-1gene silencing on cell invasion of Tca8113impact.Colonyformation assay was observed PRL-1gene silencing on Tca8113cellproliferation activity.Established tumor model in nude mice, cells of eachexperimental group logarithmic growth phase were inoculatedsubcutaneously in nude mice, observed the tumor formation of each group.Results: We have Successfully constructed PRL-1gene interferencelentiviral vector and obtain the corresponding lentivirus, recombinant lentivirus can effectively silence the PRL-1gene expressing of Tca8113tongue squamous cell carcinoma cells.After transfection by PRL-1siRNA,the level of mRNA and protein of PRL-1which was Compared withthe control group has been down-regulated, and MMP2and MMP9expression level decreased obviously,the invasion ability of human tonguesquamous cancer cell was reduced（P<0.05）． Colony formation assay,compared with the number of clones formed of CON groups,KD groups（71.67±10.41） was significantly reduced, the difference was statisticallysignificance （P <0.01）.Conclusions:1.We have Successfully constructed PRL-1gene interference lentiviralvector and obtain the corresponding lentivirus,and Screening stablytransfected recombinant lentivirus and blank virus Tca8113cell lines.2.siRNA-PRL-1gene recombinant lentiviral vector can effectivelysilence the PRL-1gene expressing of Tca8113tongue squamous carcinomacells.3. RNA interference expression of PRL-1gene of Tca8113inhibit cellproliferation and invasion.4.While the level of mRNA and protein of PRL-1has beendown-regulated, the MMP2and MMP9expression level decreasedobviously.Down-regulation of PRL-1could inhibit invasion of humantongue squamous cancer cell. Which may be related to the lower expression of MMP2and MMP9.