| Prion protein（PrP）is a glycoprotein composed of 253 amino acids encoded by the PRNP gene,which is widely expressed in various tissues and organs in vivo,and which is consistent with prion(PrPSC)in primary structure.Although PrPSC had been studied in terms of transmission pathways and pathogenic mechanisms,the physiological functions and specific mechanisms of PrPC are still unclear.In this study,we developed cell lines（HT29 and U937）with low expression of PrP using technologies of CRISPR-Cas9 and lentiviral vector RNAi.Then the effects of PrP on cell growth and inflammatory response were studied in these knock-out/down cell lines.Firstly,western-blotting results suggested the expression of PrP in the cell line constructed by CRISPR-Cas9 and lentiviral vector RNAi was significantly lower than the control group.Therefore,the PrP knocking out/down cell lines were successfully constructed,including HT29-PrP-/-,HT29-siPrP and U937-siPrP.Then the effects of PrP on cell growth and inflammatory response were studied using these cell lines.For cell growth study,MTT results showed that compared to WT,PrP kockong-out/down cell lines had slower rates of cell growth and proliferation.Considering U937 could be used as immunocells,we conducted co-culture systems with tumor cells（HT29,MDA-MB-435 and B16）and U937.Interestingly,U937-siPrP cell line could inhibit the growth of tumor cells more siginificantly,as compared to U937-siLuc;whereas the proliferation rate of U937-siPrP itself was higher than U937-siLuc.For inflammatory response study,three cell lines were treated with LPS and the expression of cytokines were measured.RT-qPCR results suggested that the PrP knocking out/down cells had less expression of cytokines at 8 h,while more expression at 24 h.Furthrmore,NF-κB activation,demonstrated by p65 translocation to nuclei,was inhibited within 1h stimulation of LPS in HT29-PrP-/-.Therefore,PrP may affect inflammatory response by affecting the activation of NF-κB signaling pathway. |
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