Fig 2: TFPI-2 knockdown restores the alterations in Panc-1 cell apoptosis, proliferation, migration and invasion induced by miR-23a inhibition. (A) The transfection efficiency of si-TFPI-2 in Panc-1 cells was determined using reverse transcription-quantitative PCR. (B) MTT assay results indicated that TFPI-2 knockdown increased Panc-1 cell proliferation following transfection with miR-23a inhibitor. (C) Quantification of (D) flow cytometry results revealed that TFPI-2 knockdown reduced apoptosis in Panc-1 cells transfected with miR-23a inhibitor. (E) Migration assays and (F) quantification of the migrated cells suggested that TFPI-2 knockdown promoted the migration of Panc-1 cells transfected with miR-23a inhibitor. (G) Invasion assays and (H) quantification of the invaded cells indicated that TFPI-2 knockdown enhanced the invasiveness of Panc-1 cells transfected with miR-23a inhibitor. *P<0.05 vs. miR-23a inhibitor + TFPI-2 NC. TFPI-2, tissue factor pathway inhibitor 2; miR, microRNA; NC, negative control; si, small interfering RNA.

Fig 3: miR-23a directly targets and negatively modulates the expression of TFPI-2. (A) Prediction of the binding sequence between miR-23a and TFPI-2 and illustration of the mutated sequence of TFPI-2 that was used for the luciferase reporter assays. (B) Panc-1 cells co-transfected with miR-23a or NC mimics and reporter vectors containing TFPI-2-WT 3'-UTR or TFPI-2-MUT 3'-UTR were analyzed using luciferase reporter assays. (C) Representative western blots, (D) relative protein expression and (E) RT-qPCR analyses of TFPI-2 expression in Panc-1 cells following transfection with miR-23a inhibitor. (F) Representative western blots, (G) relative protein expression and (H) RT-qPCR analyses of TFPI-2 expression in MiaPaCa2 cells following transfection with miR-23a inhibitor. *P<0.05 vs. NC. TFPI-2, tissue factor pathway inhibitor 2; WT, wild-type; MUT, mutant; miR, microRNA; NC, negative control; UTR, untranslated region; RT-qPCR, reverse transcription-quantitative PCR.

Fig 4: LSD2 negatively regulates the expression of TFPI-2 through DNMT3B. (A) Expression of DNMT3B mRNA in 40 cases of clinical samples of SCLC. (B) Expression of DNMT3B mRNA in SCLC cell lines. BEAS-2B was used as the control group. (C) Expression of TFPI-2 mRNA in 40 cases of clinical samples of SCLC. (D) Expression of TFPI-2 mRNA in SCLC cell lines. BEAS-2B was used as the control group. (E) mRNA expression of DNMT3B and TFPI-2 in LSD2+ and/or si-DNMT3B H1417 cells. (F and G) Effect of LSD2+ and/or si-DNMT3B on the protein expression of TFPI-2. Control, H1417 cells; si-DNMT3B, H1417 cells transfected with si-DNMT3B; LSD2+, H1417 cells transfected with LSD2 overexpression plasmids; si-DNMT3B+LSD2+, H1417 cells transfected with si-DNMT3B and LSD2 overexpression plasmids. *P<0.05; **P<0.01 and ***P<0.001 vs. the BEAS-2B group or as indicated. DNMT3B, DNA methyltransferase 3B; TFPI-2, tissue factor pathway inhibitor-2; LSD2, lysine-specific demethylase 2; SCLC, small cell lung cancer; siRNA, small interfering RNA; si-DNMT3B, siRNA to target DNMT3B.

Fig 5: Histone modification H3K4me1 at the promoter region of TFPI-2 in SCLC cells. ChIP assays were performed with antibodies against H3K4me1 in SCLC cells. Region of the TFPI-2 promoter for quantitative ChIP-PCR assay were indicated via primers S1 and AS1. The binding of H3K4-me1 antibody to region of the TFPI-2 promoter was measured by the quantification of ChIP DNA against a standard curve generated from input DNA. (A) H3K4me1 enrichment level in the promoter region of TFPI-2 showed lower degree in H1417 cells than that in BEAS-2B cells, *P<0.05 vs. the BEAS-2B group. (B) TFPI-2 promoter region showed a significantly lower enrichment level of H3K4me1 in the cell line H1417 with LSD2 overexpression. (C) TFPI-2 promoter region showed a significantly higher enrichment level of H3K4me1 in the cell line H1417 with siRNA-LSD2. *P<0.05 and **P<0.01 as indicated. Control, H1417 cells; si-LSD2, H1417 cells transfected with si-LSD2; TFPI-2, tissue factor pathway inhibitor-2; siRNA-NC, H1417 cells transfected with siRNA-NC; si, small interfering; SCLC, small cell lung cancer; si-LSD2, siRNA to target LSD2; siRNA-NC, control siRNA of LSD2; H3K4me1, monomethylated histone H3 lysine 4; ChIP, chromatin immunoprecipitation.