Fig 1: TMEM161B-AS1 suppresses the proliferation, invasion and glycolysis of ESCC cells. A, qRT-PCR was performed to investigate the expression of TMEM161B-AS1 in a panel of ESCC cells (Eca109, KYSE30, KYSE70, KYSE150 and KYSE450) and normal oesophageal epithelial cell Het-1A. B, Three siRNAs against TMEM161B-AS1 significantly down-regulated the expression of TMEM161B-AS1 in Eca109 and KYSE30 cells at 48 h after transfection. C, pcDNA3.1-TMEM161B-AS1 promoted the expression of TMEM161B-AS1 in Eca109 and KYSE30 cells at 48 h after transfection. D, TMEM161B-AS1 overexpression significantly suppressed cell proliferation in Eca109 and KYSE30 cells at indicated time-points. E, TMEM161B-AS1 knockdown markedly promoted cell proliferation in Eca109 and KYSE30 cells at indicated time-points. F and G, TMEM161B-AS1 up-regulation inhibited cell invasion in Eca109 and KYSE30 cells at 48 h after transfection, bar = 50 µm. H and I, TMEM161B-AS1 depletion enhanced cell invasion in Eca109 and KYSE30 cells at 48 h after transfection, bar = 50 µm. J, TMEM161B-AS1 overexpression suppressed glucose consumption and lactate production in Eca109 and KYSE30 cells at 48 h after transfection. K, TMEM161B-AS1 knockdown promoted glucose consumption and lactate production in Eca109 and KYSE30 cells at 48 h after transfection. L and M, TMEM161B-AS1 overexpression elevated HIF1AN expression, but reduced the expressions of HIF-1a, HK2, PFKM and LDHA in Eca109 and KYSE30 cells at 48 h after transfection. N and O, TMEM161B-AS1 depletion suppressed HIF1AN expression, but increased the expressions of HIF-1a, HK2, PFKM and LDHA in Eca109 and KYSE30 cells at 48 h after transfection. *P < .05, **P < .01, ***P < .001 and ****P < .0001, indicating statistical significance
Fig 2: TMEM161B-AS1 suppresses cell proliferation, invasion and glycolysis by manipulating miR-23a-3p/HIF1AN signal axis. TMEM161B-AS1 exhibits the low level in ESCC tissues and cells, and its overexpression inhibits the expression of miR-23a-3p expression in ESCC cells and further results in the up-regulation of HIF1AN expression in ESCC cells, which further triggers the suppression of ESCC glycolysis
Fig 3: miR-23a-3p mimic and HIF1AN siRNA reversed the suppression function of TMEM161B-AS1 in ESCC cells. A, miR-23a-3p mimic and HIF1AN siRNA reversed the proliferation suppression of TMEM161B-AS1 in Eca109 and KYSE30 cells at 48 h after transfection. B and C, miR-23a-3p mimic and HIF1AN siRNA reversed the invasion suppression of TMEM161B-AS1 in Eca109 and KYSE30 cells at 48 h after transfection, bar = 50 µm. D, miR-23a-3p mimic and HIF1AN siRNA reversed the suppression of glucose consumption mediated by TMEM161B-AS1 in Eca109 and KYSE30 cells at 48 h after transfection. E, miR-23a-3p mimic and HIF1AN siRNA reversed the suppression of lactate production evoked by TMEM161B-AS1 in Eca109 and KYSE30 cells at 48 h after transfection. F and G, miR-23a-3p mimic and HIF1AN siRNA reversed the promotion of HIF1AN and suppression of glycolysis-related proteins HIF-1a, HK2, PFKM, LDHA regulated by TMEM161B-AS1 in Eca109 and KYSE30 cells at 48 h after transfection. H, miR-23a-3p inhibitor and pcDNA3.1-HIF1AN reversed the proliferation promotion of TMEM161B-AS1 siRNA#1 in Eca109 and KYSE30 cells at 48 h after transfection. I and J, miR-23a-3p inhibitor and pcDNA3.1-HIF1AN reversed the invasion promotion of TMEM161B-AS1 siRNA#1 in Eca109 and KYSE30 cells at 48 h after transfection, bar = 50 µm. K, miR-23a-3p inhibitor and pcDNA3.1-HIF1AN reversed the increase of glucose consumption mediated by TMEM161B-AS1 siRNA#1 in Eca109 and KYSE30 cells at 48 h after transfection. L, miR-23a-3p inhibitor and pcDNA3.1-HIF1AN reversed the increase of lactate production evoked by TMEM161B-AS1 siRNA#1 in Eca109 and KYSE30 cells at 48 h after transfection. M and N, miR-23a-3p inhibitor and pcDNA3.1-HIF1AN reversed the down-regulation of HIF1AN and up-regulation of glycolysis-related proteins HIF-1a, HK2, PFKM and LDHA regulated by TMEM161B-AS1 in Eca109 and KYSE30 cells at 48 h after transfection. *P < .05, **P < .01, ***P < .001 and ****P < .0001, indicating statistical significance
Fig 4: miR-23a-6p promotes glycolysis by suppressing HIF1AN expression in ESCC cells. A, TargetScan online tool was performed to predict the possible binding sites of miR-23a-3p in HIF1AN 3’-UTR transcript. B, The dual-luciferase reporter assay system was conducted to determine the interaction of miR-23a-3p with HIF1AN 3’-UTR in Eca109 and KYSE30 cells. C, Pearson correlation assay was performed to detect the correlation of miR-23a-3p expression with HIF1AN expression in 63 cases of ESCC tissues. D, miR-23a-3p inhibitor promoted HIF1AN expression in Eca109 and KYSE30 cells. E, miR-23a-3p mimic suppressed HIF1AN expression in Eca109 and KYSE30 cells. F, miR-23a-3p inhibitor suppressed glucose consumption in Eca109 and KYSE30 cells. G, miR-23a-3p inhibitor suppressed lactate production in Eca109 and KYSE30 cells. H, miR-23a-3p mimic promoted glucose consumption in Eca109 and KYSE30 cells. G, miR-23a-3p mimic improved lactate production in Eca109 and KYSE30 cells. J, The effects of miR-23a-3p down-regulation or up-regulation on glycolysis-related proteins HIF1AN, HIF-1a, HK2, PFKM and LDHA in Eca109 and KYSE30, and ß-actin was used as loading control. K, The relative level of HIF1AN, HIF-1a, HK2, PFKM and LDHA in Eca109 and KYSE30 after treatment with miR-23a-3p inhibitor or mimic. **P < .01, ***P < .001 and ****P < .0001, indicating statistical significance
Fig 5: Reduced expressions of TMEM161B-AS1 and HIF1AN in ESCC tissues and their associations with prognosis of ESCC patients. A, StarBase v3.0 online tool was performed to investigate the TMEM161B-AS1 expression in 162 cases of ESCA samples and 11 normal samples. B, qRT-PCR assay for the TMEM161B-AS1 expression in 63 cases of ESCC samples and paired normal samples. C, The expression of TMEM161B-AS1 in ESCC samples with I + II stage and III + IV stage. D, The expression of TMEM161B-AS1 in ESCC samples with lymph node metastasis and without lymph node metastasis. E, Log-rank test was used to determine the association of TMEM161B-AS1 expression with the prognosis of ESCC patients. F, qRT-PCR assay for the HIF1AN expression in 63 cases of ESCC samples and paired normal samples. G, The expression of HIF1AN in ESCC samples with I + II stage and III + IV stage. H, The expression of HIF1AN in ESCC samples with lymph node metastasis and without lymph node metastasis. I, Log-rank test was used to determine the association of HIF1AN expression with the prognosis of ESCC patients. J, StarBase v3.0 online tool was carried out to examine the correlation of TMEM161B-AS1 expression with HIF1AN expression in 162 cases of ESCA samples. K, Pearson correlation assay was performed to detect the correlation of TMEM161B-AS1 expression with HIF1AN expression in 63 cases of ESCC tissues. **P < .01, ***P < .001 and ****P < .0001, indicating statistical significance
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