Supplementary Material for: c-Rel Promotes Invasion of Choriocarcinoma Cells via PI3K/AKT Signaling

<p><b><i>Objective:</i></b> Choriocarcinoma is the most common epithelial cancer among gestational trophoblastic diseases (GTDs); the mechanism of trophoblastic carcinogenesis is unknown. This study aimed to examine the expression of NF-κB family proteins in GTDs and placental tissues as well as the roles of c-Rel in choriocarcinoma. <b><i>Methods:</i></b> We examined the expression of NF-κB family proteins in normal placenta and hydatidiform mole tissues as well as extravillous trophoblast (EVT) and choriocarcinoma cell lines by Western blot and immunohistochemistry. Immunoprecipitation was performed to determine which proteins can bind with c-Rel in choriocarcinoma cells. To investigate the roles of c-Rel in choriocarcinoma, we examined the effects of c-Rel knockdown and overexpression on cell proliferation, migration, and invasion using small interfering RNAs and gene activation plasmid. <b><i>Results:</i></b> The expression of c-Rel was strong in choriocarcinoma and EVTs, but very weak in villi of normal placenta and hydatidiform mole. Immunoprecipitation suggested that c-Rel heterodimerizes with p65 in choriocarcinoma. c-Rel knockdown reduced invasion, migration, and AKT phosphorylation in choriocarcinoma cells. c-Rel overexpression in choriocarcinoma increased migratory and invasive abilities, and the effect on invasion was inhibited by a PI3K inhibitor. <b><i>Conclusion:</i></b> These findings suggest that c-Rel might play a role in promoting the invasion of choriocarcinoma cells through PI3K/AKT signaling.</p

Supplementary Material for: Reduced DEFA5 expression and STAT3 activation underlie the submucosal invasion of early gastric cancers

Introduction Submucosal invasion is a core hallmark of early gastric cancer (EGC) with poor prognosis. However, the molecular mechanism of the progression from intramucosal gastric cancer (IMGC) to early submucosal-invasive gastric cancer (SMGC) is not fully understood. The objective of this study was to identify genes and pathways involved in the submucosal invasion in EGC using comprehensive gene expression analysis. Methods Gene expression profiling was performed for eight cases of IMGC and eight cases of early SMGC with submucosal invasion ≥ 500 μm. To validate the findings of gene expression analysis and to examine the gene expression pattern in tissues, immunohistochemical (IHC) staining was performed for 50 cases of IMGC and SMGC each. Results Gene expression analysis demonstrated that the expression levels of small intestine-specific genes were significantly decreased in SMGC. Among them, defensin alpha 5 (DEFA5) was the most downregulated gene in SMGC, which was further validated in SMGC tissues by IHC staining. Gene set enrichment analysis showed a strong association between SMGC, the JAK-STAT signaling pathway, and the upregulation of STAT3-activating cytokines. The expression of phosphorylated STAT3 was significant in the nucleus of tumor cells in SMGC tissues but not in areas expressing DEFA5. Conclusion The results of this study strongly suggest that the downregulation of DEFA5 and the activation of STAT3 play a significant role in the submucosal invasion of EGC

Supplementary Material for: Gastroesophageal Reflux Disease-Related Disorders of Systemic Sclerosis Based on the Analysis of 66 Patients

<b><i>Background/Aims:</i></b> Gastroesophageal reflux disease (GERD)-related disorders of systemic sclerosis (SSc) patients have not been adequately investigated. <b><i>Methods:</i></b> Sixty-six SSc patients (5 males and 61 females; 56.6 ± 14.6 years old) who underwent esophagogastroduodenoscopy were analyzed on the basis of 16 background factors. They were additionally compared with 116 matched non-SSc subjects controlling age, sex, and use of proton pump inhibitors (PPIs). <b><i>Results:</i></b> The mean disease duration of 66 patients was 5.1 ± 8.1 years, and their breakdown was as follows: 53 (80.3%) with GERD, 38 (57.6%) with GERD-related symptoms, and 20 (30.3%) with reflux esophagitis (RE; LA-A: 10, LA-B: 5, LA-C: 4, LA-D: 1). Use of PPI (<i>p</i> = 0.0455), complication of interstitial lung disease (<i>p</i> = 0.0242), and history of cyclophosphamide therapy (<i>p</i> = 0.0184) denoted significant association with GERD-related symptoms. Older age (<i>p</i> = 0.0211) was significantly associated with RE. None of GERD-related disorders showed any difference between 37 diffuse cutaneous SSc and 29 limited cutaneous SSc patients. The matched analysis indicated that SSc patients had higher prevalence of GERD (<i>p </i>< 0.0001), GERD-related symptoms (<i>p</i> = 0.0034), and RE (<i>p </i>= 0.0002). <b><i>Conclusion:</i></b> SSc patients tend to have worse GERD symptoms and severer RE. However, most SSc-associated factors did not show significant association with GERD-related disorders, indicating the difficulty in predicting GERD-related disorders among SSc patients