Fig 1: PRDX1 mediated the function of LINC00460 to promote the proliferation and metastasis of HNSCC cells. a, b The growth curves were detected by CCK-8 assay when PRDX1 was knocked down in CAL-27 cells stably transduced with LINC00460 (a) and when LINC00460 was knocked down in CAL-27 cells stably transduced with PRDX1 vector (b). c, d The colonizing abilities were detected by colony formation assays when PRDX1 was knocked down in CAL-27 cells stably transduced with LINC00460 (c) and when LINC00460 was knocked down in CAL-27 cells stably transduced with PRDX1 vector (d). e, f The migration abilities were assessed by transwell assays when PRDX1 was knocked down in CAL-27 cells stably transduced with LINC00460 (e) and when LINC00460 was knocked down in CAL-27 cells stably transduced with PRDX1 vector (f). *p < 0.05, ***p < 0.001, ****p < 0.0001, ns: no significance
Fig 2: Upregulation of LINC00460 and PRDX1 was correlated with poor clinicopathologic features in HNSCC patients. a The relative expression of LINC00460 in HNSCC tissues (Tumor) and their adjacent normal tissues (Adjacent) was detected by qRT-PCR. ****p < 0.0001, n = 123. b LINC00460 expression was classified into two groups according to the expression ratio (T/N) of tumor tissue (T) to adjacent normal tissue (N) in HNSCC tissue. c The relative expression of PRDX1 in HNSCC tissues (Tumor) and their adjacent normal tissues (Adjacent) was detected by qRT-PCR. *p < 0.05, n = 123. d PRDX1 expression was classified into two groups according to the expression ratio (T/N) of HNSCC tumor tissue (T) to adjacent normal tissue (N). e The associations between the relative expression of LINC00460 and lymph metastasis/pathological differentiation were investigated in patients with HNSCC. p < 0.05. f The association between the relative expression of PRDX1 and tumor size was investigated in patients with HNSCC. p < 0.05. g Images of IHC staining of PRDX1 in normal oral mucosa and oral squamous cell carcinoma are shown. h Pearson correlation analysis of the expression of LINC00460 and PRDX1 in HNSCC tissue. p < 0.0001, r = 0.322, n = 143. i A proposed model illustrating the modulatory role of LINC00460 and PRDX1 in regulating HNSCC cell proliferation and metastasis
Fig 3: IDH1 and ME1 play a key role in maintaining redox balance and resisting cell death in 4T1 cells under glucose-deprivation with or without lactic acidosis(A) Cellular GSH/GSSG ratios of 4T1 control, IDH1 knockout, ME1 knockout and IDH1&ME1 knockout cells under glucose deprivation with or without lactic acidosis. (B) Cellular ROS levels of 4T1 control, IDH1 knockout, ME1 knockout and IDH1&ME1 knockout cells under glucose deprivation with or without lactic acidosis. ROS was probed by DCFH-DA. Bar = 25 µm. (C) The redox state of thioredoxin 1 (Trx1) in 4T1 control, IDH1 knockout, ME1 knockout and IDH1&ME1 knockout cells under glucose deprivation with or without lactic acidosis. The reduced form of Trx1 was derivatized with IAA, hence it had more negative charge than oxidized form and moved faster in the native PAGE. The reduced form/oxidized form ratios were calculated based on the gray values of the bands. (D) The redox state of peroxiredoxin 1 (Prx1) in 4T1 control, IDH1 knockout, ME1 knockout and IDH1&ME1 knockout cells under glucose deprivation with or without lactic acidosis. The reduced form of Prx1 is monomer and the oxidized form is dimer, hence they can be separated by non-reducing SDS-PAGE. The reduced form/oxidized form ratios were calculated based on the gray values of the bands.(E) Survival curves of 4T1 control, IDH1 knockout, ME1 knockout and IDH1&ME1 knockout cells under glucose deprivation with or without lactic acidosis. (F) The necrosis and apoptosis rates of 4T1 control, IDH1 knockout, ME1 knockout and IDH1&ME1 knockout cells under glucose deprivation with or without lactic acidosis. PI (+), Annexin V-FITC(+): necrosis or late apoptosis; PI (-), Annexin V-FITC(+): early apoptosis. Glc (-): Glucose-free RPMI-1640 medium; Glc (+) + LA: Glucose-free RPMI-1640 medium supplemented with 20 mM lactic acid, pH 6.7. Data are shown as means ± SD, with n = 3 biological replicates in (A) (C) (D) (F), n = 5 biological replicates in (E), and the results were all confirmed by three independent experiments. n. s. not significant, **p < 0.01, ***p < 0.001 (Student's test). See also Supplementary Figs. 4 and 5.
Fig 4: PRDX1 physically interacted with LINC00460 and affected HNSCC cell proliferation and migration. a Western blot analysis of PRDX1 following RNA pull-down assays with LINC00460 chromatin isolation by LINC00460 probes in CAL-27 cells stably transduced with LINC00460. b, c Relative expression of PRDX1 in CAL-27 cells infected with the PRDX1 lentiviral expression vector with HA-Tag (PRDX1-HA) as detected by Western blot analysis (b) and qRT-PCR (c). d qRT-PCR analysis of LINC00460 enriched by anti-HA in CAL-27 cells stably transduced with PRDX1-HA in RIP assays. Ten specific primers for LINC00460 were used to detect the results of RNA enrichment. e Construction of the PRDX1-Mut vector, which changed AAG to AGG from nucleotides 358 to 360, and the relative expression of PRDX1-WT and PRDX1-Mut vectors as detected by Western blot analysis in CAL-27 cells. f qRT-PCR analysis of LINC00460 enriched by anti-HA in CAL-27 cells transfected with PRDX1-HA-WT (WT) and PRDX1-HA-Mut (Mut) vectors in RIP assays. g Construction of the LINC00460-Mut vector, which changed TTGTGGC into GGAGAAT from nucleotides 320 to 326. h Western blot of PRDX1 following RNA pull-down assays retrieved by LINC00460 probes in CAL-27 cells transfected with LINC00460-WT (WT) and LINC00460-Mut (Mut) vectors. i, j The relative expression of PRDX1 in 7 HNSCC cell lines and normal oral epithelial cells (Normal cell) as determined by qRT-PCR (i) and Western blot analysis (j). k Silencing efficiency of si-PRDX1 in CAL-27 cells as detected by qRT-PCR. l The effect of PRDX1 expression on cell proliferation was evaluated with CAL-27 cells transfected with si-PRDX1 by CCK-8 assays. m The colonizing ability of CAL-27 cells transfected with si-PRDX1 was determined by colony formation assays. n The cell migration abilities of CAL-27 cells transfected with si-PRDX1 were determined by transwell assays, Scale bar: 1000 μm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: no significance
Fig 5: LINC00460 facilitated PRDX1 entry into the nucleus, and PRDX1 promoted the transcription of LINC00460 and EMT-related genes. a, b The expression of EMT markers (E-cadherin, N-cadherin, Vimentin, ZEB1 and ZEB2) was detected by Western blot analysis in CAL-27 cells when PRDX1 was knocked down or overexpressed. c, d The expression of EMT-associated genes (E-cadherin, N-cadherin, Vimentin, ZEB1 and ZEB2) was detected by qRT-PCR analysis in CAL-27 cells when PRDX1 was knocked down (c) or overexpressed (d). e qRT-PCR analysis of LINC00460 expression in CAL-27 cells when PRDX1 was knocked down or overexpressed. f The protein levels of PRDX1 in nuclear and cytoplasmic fractions were analyzed by Western blotting in CAL-27 cells transfected with SS-LINC00460 or LINC00460 vector. g ChIP-PCR analysis of anti-HA- or IgG- immunoprecipitated LINC00460 promoter fragments from CAL-27 cells stably transduced with PRDX1-HA. h ChIP-PCR analysis of anti-HA- or IgG- immunoprecipitated ZEB1, ZEB2 and VIM promoter fragments from CAL-27 cells stably transduced with PRDX1-HA when LINC00460 was overexpressed (LINC00460) or not overexpressed (NC). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: no significance
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