A study on mechanism of splicing factor DDX3Y promoting proliferation of gastric cancer cells and growth of organoids from males through down-regulating IL-6/JAK/STAT3 signaling pathway

ZHOU Yajing; LI Dongbao; WANG Pengbo; DUAN Kaipeng; DONG Chao; LI Weikang; SUN Xiaotong; XU Chengxiang; ZHOU Jin

doi:10.3969/j.issn.1674-3806.2025.06.11

Abstract：Objective To analyze the mechanism of splicing factor DEAD-box helicase 3 Y-linked(DDX3Y)promoting the proliferation of gastric cancer cells and growth of organoids from males through down-regulating IL-6/JAK/STAT3 signaling pathway.Methods The expression levels of DDX3Y in gastric cancer tissues and their adjacent tissues,as well as the relationship between the expressions of DDX3Y and the patients' prognosis were analyzed by using bioinformatics methods.The gastric cancer tissues and their adjacent tissues(＞5 cm away from the gastric cancer tissues)of the male gastric cancer patients who were admitted to the First Affiliated Hospital of Soochow University were collected for immunohistochemical staining.Human immortalized gastric mucosal epithelial cells(GES-1)and male-derived gastric cancer cells(SNU-1,NUGC-3,KATOⅢ,MKN74,HGC-27 and NCI-N87)were cultured.The expression levels of DDX3Y in different cell lines were detected by using Western blot method.SNU-1 and HGC-27 cells were transfected with lentivirus to construct DDX3Y-high-expression/DDX3Y-low-expression cells(OE-DDX3Y/shDDX3Y),and EdU cell proliferation assays were performed.The organoids were established by using tumor tissues of the male gastric cancer patients,and lentiviral transfection was used to further generate organoids with DDX3Y-high-expression/DDX3Y-low-expression.The effects of inhibiting IL-6/JAK/STAT3 signaling pathway on organoid diameter and viability was analyzed.The possibility of DDX3Y binding to IL-6 precursor mRNA was predicted by using the RBPmap online website.Results DDX3Y mRNA expression level in the adjacent cancer tissues was significantly higher than that in the gastric cancer tissues(P＜0.05).The survival prognosis of the patients with high expression of DDX3Y mRNA was significantly better than that of the patients with low expression of DDX3Y mRNA(log-rank test:x2=7.856,P=0.005).Compared with that in GES-1 cells,the expression of DDX3Y protein in HGC-27 cells was significantly higher(P＜0.05).Compared with that in HGC-27 cells,expression of DDX3Y protein in SNU-1 cells was significantly lower(P＜0.05).In the SNU-1 cells,the expression level of DDX3Y protein in the OE-DDX3Y group was significantly higher than that in the high expression control group(P＜0.05),and the proportion of EdU staining positive cells in the OE-DDX3Y group was significantly lower than that in the high expression control group(P＜0.05).In the HGC-27 cells,the expression level of DDX3Y protein in the shDDX3Y-1 group was significantly lower than that in the low expression control group(P＜0.05),and the proportion of EdU staining positive cells in the shDDX3Y group was significantly higher than that in the low expression control group(P＜0.05).Compared with that in the high expression control group,the organoid diameter in the OE-DDX3Y group was significantly decreased(P＜0.05),and the vitality in the OE-DDX3Y group was significantly reduced(P＜0.05).After inhibiting the IL-6/JAK/STAT3 signaling pathway,the organoid diameter in the OE-DDX3Y group was further decreased(P＜0.05),and the vitality in the OE-DDX3Y group was further reduced(P＜0.05).Compared with that in the low expression control group,the organoid diameter in the shDDX3Y group was significantly increased(P＜0.05),and the vitality in the shDDX3 Y group was significantly increased(P＜0.05).After inhibiting the IL-6/JAK/STAT3 signaling pathway,the organoid diameter in the shDDX3Y group was significantly decreased(P＜0.05),and the viability in the shDDX3Y group was significantly reduced(P＜0.05).The prediction results of the RBPmap online website suggested that DDX3Y was very likely to bind to the IL-6 precursor mRNA(Z-score＞3,with a significant binding signal).Conclusion Downregulation of splicing factor DDX3Y promotes male gastric cancer proliferation and organoid growth.The alternative splicing of IL-6 regulated by the splicing factor DDX3Y can further promote tumor progression by influencing the IL-6/JAK/STAT3 signaling pathway.