Hsa_circ_0003848 promotes gastric cancer via miR-144-3p/mesenchymal-epithelial transition factor and serves as a plasma biomarker in Helicobacter pylori infection

Figure 1 Identification and basic features of hsa_circ_0003848 in gastric cancer. A: Venn diagram showing overlapping differentially expressed circular RNAs across datasets; B: Schematic of hsa_circ_0003848 derived from exons 2-3 of PSEN1; C: Sanger sequencing confirming the back-splice junction; D: Real-time quantitative PCR using random and oligo (dT) primers showing the circular nature of hsa_circ_0003848; E: RNA stability assay indicating that hsa_circ_0003848 is more stable than linear PSEN1; F: Subcellular localization showing cytoplasmic enrichment of hsa_circ_0003848. n = 3.

Figure 2 Diagnostic potential of hsa_circ_0003848 in gastric cancer. A: Comparison of hsa_circ_0003848 expression between gastric cancer (GC) tissues and adjacent normal tissues, n = 72; B: Receiver operating characteristic curve analysis of hsa_circ_0003848 for distinguishing GC tissues from adjacent normal tissues (area under the receiver operating characteristic curve = 0.781); C: Expression levels of hsa_circ_0003848 in plasma samples collected before and 10 days after surgery, n = 72; D: Receiver operating characteristic curve analysis of hsa_circ_0003848 for detecting preoperative and postoperative GC (area under the receiver operating characteristic curve = 0.769); E: Expression of hsa_circ_0003848 in plasma samples from healthy controls and Helicobacter pylori (H. pylori)-infected individuals; F: Expression levels of hsa_circ_0003848 across different groups: Healthy, benign lesions + H. pylori (+), and early gastric cancer + H. pylori (+), showing progressive upregulation in H. pylori-related gastric conditions. Healthy, n = 30; precancerous lesions, n = 60; early GC, n = 60, ^cP < 0.001. AUC: Area under the receiver operating characteristic curve; BL: Benign lesions; EGC: Early gastric cancer; HP: Helicobacter pylori.

Figure 3 Effects of hsa_circ_0003848 on gastric cancer cell function. A: Real-time quantitative PCR shows higher expression of hsa_circ_0003848 in gastric cancer cells compared to normal GES-1 cells; B: Knockdown efficiency of hsa_circ_0003848 confirmed in GES-1, AGS, and HGC-27 cells using siRNAs; C: Cell counting kit-8 assay shows reduced proliferation in AGS and HGC-27 cells after knockdown; D: Colony formation assay indicates decreased clonogenic ability in AGS and HGC-27 after knockdown; E: Transwell assay shows reduced migration of gastric cancer cells after knockdown; F: Flow cytometry reveals increased apoptosis in AGS and HGC-27 cells after knockdown; G: Cell cycle analysis shows G1 phase arrest in AGS and HGC-27 cells after knockdown. n = 3, ^bP < 0.01, ^cP < 0.001. NC: Negative control.

Figure 4 Hsa_circ_0003848 regulates mesenchymal-epithelial transition factor expression by sponging hsa-miR-144-3p. A: Venn diagram showing hsa-miR-144-3p as a shared predicted target of hsa_circ_0003848 across three databases; B: Dual-luciferase reporter assay confirming the direct binding between hsa_circ_0003848 and hsa-miR-144-3p; C: Venn diagram identifying mesenchymal-epithelial transition factor (MET) among six candidate genes potentially regulated by hsa-miR-144-3p; D: Knockdown of hsa_circ_0003848 reduced MET mRNA expression; E: Hsa-miR-144-3p directly binds to MET 3′UTR as confirmed by luciferase assay; F: Positive correlation between hsa_circ_0003848 and MET expression in gastric cancer tissues; G: Western blot showing decreased MET protein levels after silencing hsa_circ_0003848. Western blot analysis of MET protein levels following siRNA-mediated knockdown of hsa_circ_0003848. The bar chart represents the quantitative densitometry analysis relative to GAPDH (loading control) from three independent measurements; H: RNA immunoprecipitation assay showing both hsa_circ_0003848 and hsa-miR-144-3p are enriched in argonaute2 complexes. n = 3, ^bP < 0.01, ^cP < 0.001. AGO2: Argonaute2; IgG: Immunoglobulin G; MET: Mesenchymal-epithelial transition factor; NC: Negative control.

Figure 5 Mesenchymal-epithelial transition factor overexpression reverses the anti-migratory effects of hsa_circ_0003848 knockdown in gastric cancer cells. A: Transwell migration assays showing that knockdown of hsa_circ_0003848 significantly reduces AGS cell migration; B: Overexpression of hsa-miR-144-3p inhibits AGS cell migration, while inhibition of hsa-miR-144-3p promotes cell motility. Mesenchymal-epithelial transition factor overexpression reverses the migratory inhibition caused by hsa_circ_0003848 knockdown. ns, no significance, n = 3, ^cP < 0.001; C: Schematic model illustrating the proposed regulatory axis, where hsa_circ_0003848 functions as a competing endogenous RNA, sponging hsa-miR-144-3p to derepress mesenchymal-epithelial transition factor expression and enhance gastric cancer cell migration. MET: Mesenchymal-epithelial transition factor; NC: Negative control.