MiR-27b-5p promotes gastric cancer progression by targeting CREBZF to regulate the STAT3 pathway

Figure 1 Comparative expression of miR-27b-5p in gastric cancer tissue and its effect for gastric cancer cell lines. A: Expression of miR-27b-5p in 15 gastric cancer and adjacent non-cancer tissue; B and C: Expression of miR-27b-5p and overall survival in gastric cancer based on The Cancer Genome Atlas-STAD dataset; D: Expression of miR-27b-5p and proliferation of AGS cell line following the miR-27b-5p interference; E: Expression of miR-27b-5p and proliferation of NCI-N87 cell line following the miR-27b-5p interference. ^bP < 0.01. NC: Negative control; OD: Optical density.

Figure 2 Flow cytometric analyses of the cell cycle after miR-27b-5p interference. A: Flow cytometry detection of cell cycle changes in NCI-N87 cells after miR-27b-5p interference; B: Bar graph showing cell cycle changes in NCI-N87 cells; C: Flow cytometry detection of cell cycle changes in AGS cells after miR-27b-5p interference; D: Bar graph showing cell cycle changes in AGS cells. ^bP < 0.01. NC: Negative control.

Figure 3 MiR-27b-5p promotes migration of gastric cancer cells based on wound healing and Transwell assays. A: Scratch experiments to explore migration of AGS cell line after miR-27b-5p interference; B: Scratch experiments to explore migration of NCI-N87 cell line after miR-27b-5p interference; C: Transwell assays to detect migration of AGS cell line after miR-27b-5p interference; D: Transwell assay to detect migration of NCI-N87 cell line after miR-27b-5p interference. ^aP < 0.05, and ^bP < 0.01. NC: Negative control.

Figure 4 Influence of miR-27b-5p on the mRNA and protein expression of cAMP response element-binding protein zinc finger. A: Dual-luciferase reporter experiment demonstrated that miR-27b-5p targeted cAMP response element-binding protein zinc finger (CREBZF); B and C: Relative mRNA and protein expression of CREBZF in 15 gastric cancer and adjacent noncancerous tissue; D: Overexpression or inhibition of miR-27b-5p affected mRNA and protein expression of CREBZF in AGS cell line; E: Overexpression or inhibition of miR-27b-5p affected mRNA and protein expression of CREBZF in NCI-N87 cell line. ^aP < 0.05, and ^bP < 0.01. MUT: Mutant-type; WT: Wild-type; NC: Negative control; CREBZF: cAMP response element-binding protein zinc finger.

Figure 5 Effects of miR-27b-5p and cAMP response element-binding protein zinc finger overexpression and inhibition on gastric cancer proliferation. A: MRNA and protein expression of cAMP response element-binding protein zinc finger (CREBZF) after CREBZF interference in AGS cell line; B: MRNA and protein expression of CREBZF after CREBZF interference in NCI-N87 cell line; C: Impact of overexpression or inhibition of miR-27b-5p and CREBZF on proliferation of AGS cell line; D: Impact of overexpression or inhibition of miR-27b-5p and CREBZF on proliferation of NCI-N87 cell line. ^aP < 0.05, and ^bP < 0.01. NC: Negative control; CREBZF: cAMP response element-binding protein zinc finger; OD: Optical density.

Figure 6 Flow cytometry analysis of gastric cancer cell cycle after cAMP response element-binding protein zinc finger and miR-27b-5p interference. A: Cell cycle of AGS cells transfected by miR-27b-5p mimic or miR-27b-5p inhibitor or negative control (NC), in combination with OE-cAMP response element-binding protein zinc finger (CREBZF) or sh-CREBZF or NC; B: Cell cycle of NCI-N87 cells following transfection with miR-27b-5p mimic, miR-27b-5p inhibitor, or NC, in combination with OE-CREBZF, sh-CREBZF, or NC. ^bP < 0.01. NC: Negative control; CREBZF: cAMP response element-binding protein zinc finger.

Figure 7 Wound healing of gastric cancer cells after cAMP response element-binding protein zinc finger and miR-27b-5p interference. A: Scratch assays to examine migration of AGS cells after cAMP response element-binding protein zinc finger and miR-27b-5p interference; B: Scratch experiment to detect migration of NCI-N87 cells after cAMP response element-binding protein zinc finger and miR-27b-5p interference; C: Bar graph showing migration of AGS cells; D: Bar graph showing migration of NCI-N87 cells. ^aP < 0.05, and ^bP < 0.01. NC: Negative control; CREBZF: cAMP response element-binding protein zinc finger.

Figure 8 Transwell assay to evaluate the impact of overexpression and inhibition of miR-27b-5p and cAMP response element-binding protein zinc finger on transmembrane migration of gastric cancer cells. A: Transwell migration assay following overexpression and inhibition of miR-27b-5p and cAMP response element-binding protein zinc finger in AGS cells; B: Transwell migration assay following overexpression and inhibition of miR-27b-5p and cAMP response element-binding protein zinc finger in NCI-N87 cells. ^bP < 0.01. NC: Negative control; CREBZF: cAMP response element-binding protein zinc finger.

Figure 9 Western blotting of gastric cancer cells for miR-27b-5p and cAMP response element-binding protein zinc finger regulation of signal transducer and activator of transcription 3 pathway-related protein expression. A: Overexpression or inhibition of miR-27b-5p and cAMP response element-binding protein zinc finger (CREBZF) on protein status of phosphorylated signal transducer and activator of transcription 3 (STAT3), c-myc, B-cell lymphoma 2, survivin, STAT3, CREBZF, cyclin D1, and Bcl-2-associated X protein in AGS cells; B: Overexpression or inhibition of miR-27b-5p and CREBZF on protein status of phosphorylated STAT3, c-myc, B-cell lymphoma 2, survivin, STAT3, CREBZF, cyclin D1, and Bcl-2-associated X protein in NCI-N87 cells. Bcl-2: B-cell lymphoma 2; Bax: Bcl-2-associated X protein; p-STAT3: Phosphorylated signal transducer and activator of transcription 3; CREBZF: cAMP response element-binding protein zinc finger.

Figure 10  Changes in xenograft tumors in nude mice after overexpression or inhibition of miR-27b-5p. A: Tumor images of subcutaneous tumors in nude mice constructed by lentivirus transfection overexpression of miR-27b-5p in AGS cells (LV-miR-27b-5p and negative control group LV-negative control), and knockdown of miR-27b-5p (anti-LV-miR-27b-5p and negative control group anti-LV-negative control); B and C: Changes in subcutaneous tumor weight and volume of nude mice injected with AGS cells overexpression or inhibition of miR-27b-5p; D: Growth curve of subcutaneous tumors in nude mice that have been injected with AGS cells exhibiting either overexpression or inhibition of miR-27b-5p; E: Expression status of cAMP response element-binding protein zinc finger/signal transducer and activator of transcription 3 associated proteins in xenograft tumors of nude mice following overexpression or inhibition of miR-27b-5p. ^bP < 0.01. NC: Negative control; STAT3: Signal transducer and activator of transcription 3; CREBZF: cAMP response element-binding protein zinc finger; Bcl-2: B-cell lymphoma 2; Bax: Bcl-2-associated X protein; p-STAT3: Phosphorylated signal transducer and activator of transcription 3.