Figure 1
Design of sequence-specific shRNAs targeting LMP1. TW03-LMP1 and HNE1-LMP1 cells were treated with sequence-specific shRNAs targeting LMP1. A, Diagrammatic representation of the mRNA encoding full-length LMP1 sequence (CDS). The shRNA targeting the 117-135 base pair (bp) of LMP1 CDS is named LMP1-shRNA1, while the shRNA targeting 1135-1153 bp of LMP1 CDS is named LMP1-shRNA2. B, mRNA expression of LMP1 in TW03-LMP1 and HNE1-LMP1 cells under the administration of lentivirus containing shcontrol, LMP1-shRNA1, and LMP1-shRNA2 sequences. C, LMP1 protein expression in TW03-LMP1 and HNE1-LMP1 cells after the administration of lentivirus containing shcontrol, LMP1-shRNA1, and LMP1-shRNA2 sequences. GAPDH was also assayed as the loading control.
Figure 2
Sequence-specific shRNA targeting LMP1 retains the nasopharyngeal carcinoma (NPC)-LMP1 immunogenicity and provokes T cell immunity. A, Schematic illustration of the generation of NPC-LMP1-antigen-specific cytotoxic T-lymphocyte (CTLs) from peripheral blood mononuclear cells (PBMCs) of healthy donors by co-culturing with the irradiated TW03-LMP1 cells under the treatment of shcontrol, LMP1-shRNA1, and LMP1-shRNA2 in interleukin (IL)-2 medium for 14 days and re-stimulated with the irradiated TW03-LMP1 cells at day 7. B, In vitro cytotoxicity measurement of NPC-LMP1-antigen-specific CTLs induced by the above indicated condition against TW03-LMP1 cells at different ratios for 6 h. The killing capacity of CTLs was determined via LDH Cytotoxicity Assay Kit. C and D, NPC-LMP1-antigen-specific CTLs induced by the above condition co-cultured with TW03-LMP1 cells for 4 h and harvested for FACS staining. The FACS plot (C) and the statistical graph (D) show the releasing level of interferon (IFN)-γ and granzyme (GrB) from CD8+ T cells. Representative FACS plots are shown from 1 of 3 independent experiments. E, Cytotoxicity measurement of NPC-LMP1-antigen-specific CTLs induced by the above condition against TW03-LMP1 cells at 50:1 and 100:1 ratios in the presence of MHC-class I blocking antibody (W6/32). F and G, The NPC-LMP1-antigen-specific CTLs induced by the above condition were co-cultured with TW03-LMP1 cells in the presence of MHC-class I blocking antibody (W6/32) for 4 h and harvested for FACS staining. The FACS plot (F) and statistical graph (G) of 1 of 3 independent experiments show the level of IFN-γ and granzyme (GrB) in CD8+ T cells. H and I, CD33+cells were isolated from healthy PBMCs using human CD33 microbeads and co-cultured with TW03-LMP1 cells under the treatment of shcontrol, LMP1-shRNA1, and LMP1-shRNA2 in a Transwell system for 48 h. The percentage of myeloid-derived suppressor cells (MDSCs) was measured by FACS staining. Representative density plots are shown as the CD33+CD11b+cells in the HLA-DR cell population (H) and statistical graph of the percentage of MDSCs induced by NPC-LMP1 cells under the indicated treatment (I) are shown from at least three independent experiments. Data are reported as means±SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test).
Figure 3
LMP1 targeting by sequence-specific shRNA inhibited nasopharyngeal carcinoma (NPC)-LMP1 cell growth and migration. A, Cell growth curve showing the proliferation of TW03-LMP1 cells after the treatment of shcontrol, LMP1-shRNA1, and LMP1-shRNA2, according to MTT cell proliferation assay. Statistical analysis was done with data from at least three independent experiments. B, Wound healing assays of TW03-LMP1-shcontrol, TW03-LMP1-shRNA1, and TW03-LMP1-shRNA2 cells. Wound closures were photographed at 0, 12, and 24 h after removing the scratched cells. C, Migration rates were calculated with data from at least three independent experiments. Data are reported as means±SD. **P<0.01, ***P<0.001 (Student's t-test).
Figure 4
Sequence-specific shRNAs targeting LMP1 inhibited tumor growth in vivo. A, Protocol of subcutaneous injection of xenograft tumor and plasmid treatment (by intravenous injection) procedure in nude mice. B, Growth curves of xenograft tumor volumes of the nude mice in three groups: G1: PBS 100 µL, G5: LMP1-shRNA1 plasmid (100 µL, 1.5 mg/kg), and G6: LMP1-shRNA2 plasmid (100 µL, 1.5 mg/kg). All groups were treated by caudal vein injection. C, Plasmids and nanomaterials were mixed in vitro in accordance with the requirements of the use of nanomaterials. PCR results show the nanomaterial and plasmid binding. D and E, Growth curves of tumor volumes of the nude mice in several groups: G2: Nano-LDH + shcontrol (100 µL, 1.5 mg/kg), G3: Nano-LDH + LMP1-shRNA1 (100 µL, 1.5 mg/kg), G4: Nano-LDH + LMP1-shRNA2 (100 µL, 1.5 mg/kg), G5: LMP1-shRNA1 plasmid (100 µL, 1.5 mg/kg), and G6: LMP1-shRNA2 plasmid (100 µL, 1.5 mg/kg). All groups were treated by caudal vein injection. F, Protocol of subcutaneous injection of xenograft tumor following the NPC-LMP1 antigen-specific T cells treatment (by intravenous injection) procedure in nude mice. G, Growth curves of xenograft tumor volumes of the nude mice in each group, including G7: CTL1 (NPC-LMP1-sh-control cells induced T cells) (200 µL, 2.5*106 T cells), G8: CTL2 (NPC-LMP1-shRNA1 cells induced T cells) (200 µL, 2.5*106 T cells), and G9: CTL3 (NPC-LMP1-shRNA2 cells induced T cells) (200 µL, 2.5*106 T cells). Data are reported as means±SD (n=3). *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). H, Schematic diagram of the mechanisms of specific-sequence shRNAs targeting LMP1 and inhibiting NPC growth.