The Study Of CRISPR/Cas9 System-mediated NF-κB Knockout Effect On The Immune Function Of Human Nasopharyngeal Carcinoma Cell 2(CNE-2)

Objectives:To confirm vIL-10 can inhibit the expression of "MHC-I antigen processing the operon" via NF-B pathwayMethods:1.Use the NF-κB p65 CRISPR/Cas9 KO Plasmid (buy from santa cruz company) to transfect the CNE-2,CNE2 cells without transfection of fluorescent plasmid as control, over-flow analyzers, sorting out the transfected p65 CRISPR/Cas9 KO Plasmid NF-κB cells (With green fluorescence). Select the single cell clones with green fluorescence, and collect the cell after these cells were cultured. The expression of NF-κB p65 in monoclonal knockout was detected by western blot, and establish the NF-KB p65 knockout stable cell lines. The protein and mRNA levels ofTAP1, TAP2, LMP2, LMP7, HLA-I in the cells were detected by western blotting and RT-PCR.2.Design and construction the over-expression vector of BCRF-1.To transfect the PCMV-N-Flag plasmid and PCMV-N-Flag-BCRF plasmid into the CNE-2 cells and CNE-2 cells which wasknockouted the NF-κB p65.The protein and mRNA levels of TAP1, TAP2, LMP2, LMP7, HLA-1 in the cells were detected by western blotting and RT-PCR.3.①Set CNE2 group (the untreated group) to A group;②Set CNE2 PCMV-N-flag group (the empty vector group) to B group;③Set CNE2 PCMV-N-flag-BCRF group (the BCRF-1 overexpression group) to C group;④Set CNE2-NF-κB-KO group (the NF-κB knockout group) to D group;⑤Set CNE2-NFκB-KO-PCMV-N-flag group(the NF-κB knockout and the empty vector group) to E group;⑥Set CNE2-NFκB-KO-PCMV-N-flag-BCRF (the NF-κB knockout and BCRF-1 overexpression group) to F group.Results:1. mRNA expression:①TAP-1 and TAP2:Compared with the’CNE2 PCMV-N-flag’group (the control group), the expression of TAP-1 and TAP-2 mRNA in CNE-2 cell of the’CNE2 PCMV-N-flag-BCRF’group (the BCRF-1 overexpression group) did not change significantly(P>0.05). Compared with the ’CNE2 PCMV-N-flag-BCRF’group (the BCRF-1 overexpression group), the expression of TAP-1 and TAP-2 mRNA in CNE-2 cell of the ’CNE2-NFicB-KO-PCMV-N-flag-BCRF’(the NF-κB knockout and BCRF-1 overexpression group) group was significantly down-regulated (P<0.05)②LMP-2 and LMP-7:Compared with the ’CNE2 PCMV-N-flag’ group (the control group), the mRNA expression of LMP-2 and LMP-7 in CNE-2 cell of the’CNE2 PCMV-N-flag-BCRF’ group (the BCRF-1 overexpression group)did not change significantly(P>0.05). Compared with the’CNE2 PCMV-N-flag-BCRF’group (the BCRF-1 overexpression group), the expression of LMP-2 and LMP-7 mRNA in CNE-2 cell of the’CNE2-NFκB-KO-PCMV-N-flag-BCRF’group (the NF-κB knockout and BCRF-1 overexpression group) was significantly down-regulated (P< 0.05)③ HLA-I:Compared with the ’CNE2 PCMV-N-flag’ group(the control group), the mRNA expression of HLA-I in CNE-2 cell of the ’CNE2 PCMV-N-flag-BCRF’ group (the BCRF-1 overexpression group) did not change significantly (F>0.05) Compared with the’CNE2 PCMV-N-flag-BCRF’group (the BCRF-1 overexpression group), the mRNA expression of HLA-I in CNE-2 cell of the ’CNE2-NFκB-KO-PCMV-N-flag-BCRF’group (the NF-κB knockout and BCRF-1 overexpression group) did not change significantly (P>0.05)2. protein expression:①TAP-1 and TAP2:Compared with the’CNE2 PCMV-N-flag’group (the control group), the expression of TAP-1 and TAP-2 protein in CNE-2 cell of the’CNE2 PCMV-N-flag-BCRF’group (the BCRF-1 overexpression group) was significantly down-regulated (P<0.05). Compared with the’CNE2 PCMV-N-flag-BCRF’group (the BCRF-1 overexpression group), the expression of TAP-1 and TAP-2 protein in CNE-2 cell of the ’CNE2-NFκB-KO-PCMV-N-flag-BCRF’group (the NF-κB knockout and BCRF-1 overexpression group) was significantly up-regulated (P<0.05)(2)LMP-2 and LMP-7:Compared with the’CNE2 PCMV-N-flag’group (the control group), the expression of TAP-1 and TAP-2 protein in CNE-2 cell of the ’CNE2 PCMV-N-flag-BCRF’group (the BCRF-1 overexpression group) was up-regulated (P<0.05). Compared with the’CNE2 PCMV-N-flag-BCRF’group (the BCRF-1 overexpression group), the expression of TAP-1 and TAP-2 protein in CNE-2 cell of the’CNE2-NFKB-KO-PCMV-N-flag-BCRF’group (the NF-κB knockout and BCRF-1 overexpression group) was significantly down-regulated (P <0.05)③ HLA-I:Compared with the’CNE2 PCMV-N-flag’group (the control group), the expression of HLA-I protein in CNE-2 cell of the’CNE2 PCMV-N-flag-BCRF’ group (the BCRF-1 overexpression group) was significantly down-regulated (P< 0.05). Compared with the’CNE2 PCMV-N-flag-BCRF’ group (the BCRF-1 overexpression group), the expression of HLA-I protein in CNE-2 cell of the ’CNE2-NFκB-KO-PCMV-N-flag-BCRF’group (the NF-κB knockout and BCRF-1 overexpression group) was significantly down-regulated (P<0.05)Conclusions:vIL-10 can inhibit the expression of "MHC-I antigen processing the operon" in CNE-2 cell, and this inhibition may occur at the transcriptional and post transcriptional level. Knockdown of NF-kappa B p65 can enhance the CNE-2 cell antigen presenting ability, and can provide a new method for the treatment of nasopharyngeal carcinoma.