Research Paper

Figure 3. LncRNA CASC15 up-regulates MMP7 expression in ESCC by binding to miR-140-5p. (A) Subcellular localization of lncRNA CASC15 in EC109 cells (× 400). (B) A heat map of down-regulated miRNAs in GSE97049 dataset (the X label indicates the sample number, while the Y label indicates the miRNA name, and each small square indicates the expression of one miRNA in one sample). (C) The intersection of predicted miRNAs regulated by lncRNA CASC15 from RNA 22 database, RAID database and GSE97049 dataset (three circles represent the miRNAs in the RNA 22 database, RAID database and GSE97049 dataset respectively, and the middle part indicates the intersection). (D) RT-qPCR to detect the expression of miR-140-5p in ESCC tissues and cells (*p < 0.05 vs. the adjacent normal tissues or HEEC cells). (E) The correlation analysis of lncRNA CASC15 and miR-140-5p expression in ESCC tissues. (F) The correlation between lncRNA CASC15 and miR-140-5p expression in EC109 cells (*p < 0.05 vs. EC109 cells transfected with oe-NC, #p < 0.05 vs. EC109 cells transfected with sh-NC). (G) A heat map of differentially expressed genes in GSE45168 dataset (the X axis indicates the sample number, while the Y axis indicates the miRNA name, and each small square indicates the expression of one miRNA in one sample). (H) The intersection of predicted downstream target genes of miR-140-5p from RNA22 database and differentially expressed genes obtained from GSE45168 dataset (two circles indicate up-regulated genes in the predicted results from RNA22 database and GSE45168 dataset, and the middle part indicates their interaction). (I) The correlation analysis of the predicted downstream target genes of miR-140-5p (each circle refers to one gene, and the ligature between two circles represents interaction between two genes). (J) RT-qPCR and Western blot analyses to detect the expression of MMP7 and other candidate target genes (KCNA2 and S100A14) at mRNA and protein levels in ESCC tissues and cells (*p < 0.05 vs. the adjacent normal tissues or HEEC cells). (K) RT-qPCR to detect the expression of lncRNA CASC15 and MMP7 measured after upregulating or downregulating miR-140-5p (*p < 0.05 vs. EC109 cells transfected with NC mimic, #p < 0.05 vs. EC109 cells treated with NC inhibitor). (L) The correlation analysis between lncRNA CASC15 and MMP7 expression in ESCC. (M) A bioinformatic prediction website to predict the putative binding sites between miR-140-5p and lncRNA CASC15 or MMP7. (N) Dual-luciferase reporter assay to examine the luciferase activity after the co-transfection of miR-140-5p mimic with wt-CASC15 and mut-CASC15. (O) Dual-luciferase reporter assay to test the luciferase activity after the co-transfection of miR-140-5p mimic with wt-MMP7 and mut-MMP7. (*p < 0.05 vs. EC109 cells transfected with NC mimic). (P) RIP to assess the enrichment of lncRNA CASC15, miR-140-5p, and MMP7 (*p < 0.05 vs. normal mouse IgG). (Q) RIP to study the effect of lncRNA CASC15 on the binding of miR-140-5p and MMP7 (*p < 0.05 vs. the treatment of oe-NC anti-Ago2; #p < 0.05 vs. the treatment of sh-NC anti-Ago2). (R) RT-qPCR to detect the mRNA expression of MMP7 in ESCC cells (*p < 0.05 vs. the treatment of oe-NC and NC mimic; #p < 0.05 vs. the treatment of oe-CASC15 and NC mimic). (S) Dual-luciferase reporter assay to test the luciferase activity after the co-transfection of oe-CASC15 + miR-140-5p mimic with wt-MMP7 and mut-MMP7 (*p < 0.05 vs. EC109 cells transfected with oe-NC + NC mimic; #p < 0.05 vs. EC109 cells transfected with oe-CASC15 + NC mimic). The measurement data were presented as the mean ± standard deviation. Data in panel (D) (left) and (J) (left) were analyzed by paired t test and those in panel (D) (right) and (F, J) (right) and (K, N, O, P, Q, R, S) by unpaired t test; the experiment was repeated 3 times. NC mimic, EC109 cells transfected with negative control plasmids of mimic; miR-140-5p mimic, EC109 cells transfected with miR-140-5p mimic; oe-NC, EC109 cells transfected with negative control plasmids of overexpression vector; sh-NC, EC109 cells transfected with negative control plasmids of short hairpin RNA; wt-CASC15, EC109 cells transfected with luciferase reporter plasmids containing wild-type lncRNA CASC15; mut-CASC15, EC109 cells transfected with luciferase reporter plasmids containing mutant lncRNA CASC15; wt-MMP7, EC109 cells transfected with luciferase reporter plasmids containing wild-type MMP7; mut-MMP7, EC109 cells transfected with luciferase reporter plasmids containing mutant MMP7.