Research Paper Volume 13, Issue 13 pp 17155—17176

Hypoxia induces chemoresistance of esophageal cancer cells to cisplatin through regulating the lncRNA-EMS/miR-758-3p/WTAP axis

WTAP is a downstream target of miR-758-3p in EC cells, and is required for hypoxia-mediated resistance to DDP. (A) Prediction with multi-database identified 14 possible target mRNAs of miR-758-3p. (B) Higher expression of WTAP in esophageal cancer cells was validated using the starBase. n=162 for the cancer group; n=11 for the control group. (C) The WTAP mRNA expression levels in ECA-109 cells under the hypoxic and normoxic conditions were determined by RT-qPCR. (D) Expression of WTAP and Glut1 in EC tissue determined using immunofluorescence. (E) Sequence comparison of miR-785-3p and wild type or mutated 3’UTR of WTAP. (F) Dual luciferase assay validated the interaction between miR-758-3p and WTAP in ECA-109 cells. (G, H) qPCR assays and western blot assays were used to determine the mRNA levels (G) and protein levels (H) of WTAP in ECA-109 cells after transfection of miR-758-3p mimic/inhibitor or the corresponding controls. (I) Western blot results show the protein levels of WTAP in hypoxic ECA-109 cells with the indicated treatments. (J) Ectopic WTAP expression reversed the growth inhibitory effect of DDP in combination with miR-758-3p mimic under hypoxic condition. The proliferation of hypoxic ECA-109 cells after the indicated treatment was evaluated by CCK-8 assays. (K, L) Ectopic WTAP expression reversed the apoptosis-promoting effect of DDP in combination with miR-758-3p mimic under hypoxic condition. The apoptosis rates (K) and western blot results on WTAP/BCL2/cleaved-caspase-3 levels (L) are shown. Representative band images from 5 independent experiments with similar results are shown in J. n=5 for each group.

Figure 3. WTAP is a downstream target of miR-758-3p in EC cells, and is required for hypoxia-mediated resistance to DDP. (A) Prediction with multi-database identified 14 possible target mRNAs of miR-758-3p. (B) Higher expression of WTAP in esophageal cancer cells was validated using the starBase. n=162 for the cancer group; n=11 for the control group. (C) The WTAP mRNA expression levels in ECA-109 cells under the hypoxic and normoxic conditions were determined by RT-qPCR. (D) Expression of WTAP and Glut1 in EC tissue determined using immunofluorescence. (E) Sequence comparison of miR-785-3p and wild type or mutated 3’UTR of WTAP. (F) Dual luciferase assay validated the interaction between miR-758-3p and WTAP in ECA-109 cells. (G, H) qPCR assays and western blot assays were used to determine the mRNA levels (G) and protein levels (H) of WTAP in ECA-109 cells after transfection of miR-758-3p mimic/inhibitor or the corresponding controls. (I) Western blot results show the protein levels of WTAP in hypoxic ECA-109 cells with the indicated treatments. (J) Ectopic WTAP expression reversed the growth inhibitory effect of DDP in combination with miR-758-3p mimic under hypoxic condition. The proliferation of hypoxic ECA-109 cells after the indicated treatment was evaluated by CCK-8 assays. (K, L) Ectopic WTAP expression reversed the apoptosis-promoting effect of DDP in combination with miR-758-3p mimic under hypoxic condition. The apoptosis rates (K) and western blot results on WTAP/BCL2/cleaved-caspase-3 levels (L) are shown. Representative band images from 5 independent experiments with similar results are shown in J. n=5 for each group.