Yin Yang 1 activates JAK-STAT3-mediated epithelial-mesenchymal transition in Helicobacter pylori-induced gastric cancer progression

Figure 1 Yin Yang 1 is upregulated in gastric cancer tissues and cells. ^bP < 0.01, ^cP < 0.001. A and B: Yin Yang 1 (YY1) expression in tissue samples of gastric cancer was predicted by the GEPIA and starBase websites; C: Kaplan-Meier survival curve of YY1 expression data retrieved from the Kaplan-Meier Plotter database; D and E: YY1 level in gastric cancer tissue samples (n = 6) detected by Western blotting and quantitative real-time polymerase chain reaction; F: Immunohistochemical staining of differential YY1 expression in gastric cancer and paired paracancerous tissues; G: Kaplan-Meier survival analysis comparing survival rates of gastric cancer patients with high and low YY1 expression from our study; H and I: Protein and mRNA expression of YY1 in normal gastric epithelial cell line and gastric cancer cell lines (SGC7901, MGC803, AGS, HGC27, MKN45) was examined by Western blotting and quantitative real-time polymerase chain reaction; J: Immunofluorescence staining was used to assess expression and location of YY1 expression in normal gastric epithelial cell line and gastric cancer cell lines (AGS and SGC7901). STAD: Stomach adenocarcinoma; YY1: Yin Yang 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 2 Yin Yang 1 is upregulated in Helicobacter pylori-induced gastritis. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. A: Protein expression of Yin Yang 1 (YY1) in GES-1 cell lines cocultured with Helicobacter pylori (H. pylori) strains ATCC26695, ATCC43504, and HPWT; B: Immunohistochemistry analysis of differential YY1 and N-cadherin expression in chronic nonatrophic gastritis, atrophic gastritis, and intestinal metaplasia tissues; C: Schematic diagram of a mouse model of H. pylori infection (gastritis); D: Representative images of gastric mucosa in mice with gastritis, assessed by hematoxylin and eosin staining, and H. pylori expression detected by Warthin-Starry staining; E and F: YY1 and N-cadherin expression in gastric tissues of mice 12 weeks and 36 weeks post-H. pylori infection was analyzed by Western blotting and immunohistochemistry. YY1: Yin Yang 1; AG: Atrophic gastritis; IM: Intestinal metaplasia; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 3 Helicobacter pylori enhances Yin Yang 1 expression in gastric cancer. ^aP < 0.05, ^cP < 0.001. A: Protein expression of Yin Yang 1 (YY1), N-cadherin, E-cadherin, Snail, and vimentin expression in AGS and SGC7901 cell lines cocultured with Helicobacter pylori (H. pylori) wild type (HPWT) and H. pylori strains ATCC26695 and ATCC43504 by Western blotting; B and C: Western blotting and quantitative real-time polymerase chain reaction were used to measure YY1 expression in H. pylori-negative and -positive tissues; D: Schematic diagram of the TFF1-KO mouse model (gastric cancer) infected with H. pylori; E: Pathological changes in the gastric mucosa of TFF1-KO mice were assessed by hematoxylin and eosin staining, and H. pylori expression was detected by Warthin-Starry staining; F: Immunohistochemical analysis of YY1 and N-cadherin expression in gastric precancerous lesions and gastric cancer tissues in mice. YY1: Yin Yang 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; N(HP-): Normal tissue without Helicobacter pylori infection; N(HP+): Normal tissue with Helicobacter pylori infection; T(HP-): Tumor tissue without Helicobacter pylori infection; T(HP+): Tumor tissue with Helicobacter pylori infection; HP: Helicobacter pylori.

Figure 4 Yin Yang 1 enhances malignant phenotypes in gastric cancer cells. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. A: Western blotting confirmed the transfection efficiency of Yin Yang 1 (YY1) lentivirus; B and C: Colony formation assays and 5-ethynyl-2’-deoxyuridine assessing the impact of YY1 knockdown and overexpression on proliferation of AGS cells; D and E: Wound healing and Transwell assays evaluating the effects of YY1 knockdown and overexpression on the invasion and migration of AGS cells. EdU: 5-Ethynyl-2’-deoxyuridine; YY1: Yin Yang 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; sh: Short hairpin.

Figure 5 Yin Yang 1 promotes epithelial-mesenchymal transition via activation of the Janus kinase 2/signal transducer and activator of transcription 3 pathway. ^aP < 0.001. A: Total and phosphorylated protein levels of Janus kinase (JAK) 2 and signal transducer and activator of transcription 3 (STAT3) in AGS and SGC7901 cells cocultured with Helicobacter pylori were analyzed by Western blotting; B: Expression of JAK2/STAT3 and epithelial-mesenchymal transition (EMT)-related proteins in AGS and SGC7901 cell lines with Yin Yang 1 (YY1) knockdown and overexpression; C: GEPIA database was used to analyze the relationship between YY1 and JAK2 expression in gastric cancer tissues; D: Joint Array for Public Research algorithm predicted the YY1 binding site in JAK2 promoter; E: Interaction between YY1 and JAK3 was detected using chromatin immunoprecipitation assay; F: Activation of the EMT pathway and phosphorylated Janus kinase 2/phosphorylated signal transducer and activator of transcription 3 expression following addition of coumermycin A1 in AGS cells with YY1 knockdown, and inhibition of the EMT pathway and phosphorylated Janus kinase 2/phosphorylated signal transducer and activator of transcription 3 expression with addition of AG490 in AGS cells with YY1 overexpression. YY1: Yin Yang 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; JAK: Janus kinase; STAT3: Signal transducer and activator of transcription 3; pJAK2: Phosphorylated Janus kinase 2; pSTAT3: Phosphorylated signal transducer and activator of transcription 3; JASPAR: Joint Array for Public Research; TPM: Transcript per million.

Figure 6 Subcutaneous tumor formation experiment in nude mice. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. A: Representative images of subcutaneous tumors; B and C: Tumor volume growth curve and tumor weight measurements; D: Hematoxylin and eosin staining of subcutaneous tumors from four groups; E: Immunohistochemistry of subcutaneous tumors from four groups (Ki-67); F: Protein expression of the epithelial-mesenchymal transition and Janus kinase 2/signal transducer and activator of transcription 3 pathway with addition of AG490 from subcutaneous tumors. YY1: Yin Yang 1; JAK2: Janus kinase 2; STAT3: Signal transducer and activator of transcription 3; pJAK2: Phosphorylated Janus kinase 2; pSTAT3: Phosphorylated signal transducer and activator of transcription 3; JASPAR: Joint Array for Public Research.