Circular RNAs (circRNAs) are a group of non-coding, covalently uninterrupted loop transcripts, most of which remain to be functionally characterized. Here, we identified circPDIA4 as an oncogenic circRNA in gastric cancer. Clinically, circPDIA4 was significantly upregulated in malignant tissues and associated with poor survival of gastric cancer patients. The biogenesis of circPDIA4 was mediated by the RNA binding protein Quaking, which bound intron 2 and 4 of PDIA4 pre-mRNA to promote backsplicing of exons 3 and 4. Elevated expression of circPDIA4 promoted distant metastasis in various mouse xenograft models in vivo and accelerated cancer cell invasion in vitro. CircPDIA4 functioned through distinct oncogenic mechanisms in the cytoplasm and the nucleus. Cytoplasmic circPDIA4 bound to ERK1/2 and sustained hyper-activation of the MAPK pathway by preventing DUSP6-mediated ERK1/2 dephosphorylation. Notably, circPDIA4 depletion enhanced the sensitivity of gastric cancer cells to ERK inhibitors. In the nucleus, circPDIA4 interacted with DHX9 as a decoy and repressed its inhibitory functions on circRNA biogenesis to boost expression of multiple oncogenic circRNAs, which promoted gastric cancer progression. These findings reveal a dual tumor-promoting mechanism for circPDIA4 by regulating oncogenic circRNA biogenesis and increasing MAPK activity. CircPDIA4 should be investigated further as a potential prognostic biomarker and therapeutic target in gastric cancer.
<p>Table S1 shows primers for RT-qPCR. Table S2 shows sequences of shRNAs and siRNAs. Table S3 shows antibodies used in the study. Table S4 shows the sequence of the biotin-labeled probes for circPDIA4 and controls. Table S5 shows the overlap circRNAs of the three gastric cancer cell lines. Table S6 shows the relevant clinic-pathological characteristics of gastric cancer cases stratified by circPDIA4 levels in Combined cohort. Table S7 shows the relevant clinic-pathological characteristics of gastric cancer cases stratified by QKI levels in Combined cohort. Table S8 shows Mass Spectrometry of proteins pulled-down by circPDIA4 in HGC-27 cells.</p>
<div>Abstract<p>Circular RNAs (circRNA) are a group of noncoding, covalently uninterrupted loop transcripts, most of which remain to be functionally characterized. Here, we identified circPDIA4 as an oncogenic circRNA in gastric cancer. Clinically, circPDIA4 was significantly upregulated in malignant tissues and was associated with poor survival of patients with gastric cancer. The biogenesis of circPDIA4 was mediated by the RNA-binding protein Quaking, which bound introns 2 and 4 of <i>PDIA4</i> pre-mRNA to promote backsplicing of exons 3 and 4. Elevated expression of circPDIA4 promoted distant metastasis in various mouse xenograft models <i>in vivo</i> and accelerated cancer cell invasion <i>in vitro</i>. CircPDIA4 functioned through distinct oncogenic mechanisms in the cytoplasm and the nucleus. Cytoplasmic circPDIA4 bound to ERK1/2 and sustained hyperactivation of the MAPK pathway by preventing DUSP6-mediated ERK1/2 dephosphorylation. Notably, circPDIA4 depletion enhanced the sensitivity of gastric cancer cells to ERK inhibitors. In the nucleus, circPDIA4 interacted with DHX9 as a decoy and repressed its inhibitory functions on circRNA biogenesis to boost expression of multiple oncogenic circRNAs, which promoted gastric cancer progression. These findings reveal a dual tumor-promoting mechanism for circPDIA4 by regulating oncogenic circRNA biogenesis and increasing MAPK activity. CircPDIA4 should be investigated further as a potential prognostic biomarker and therapeutic target in gastric cancer.</p>Significance:<p>Quaking-regulated circPDIA4 mediates different mechanisms in the nucleus and cytoplasm that coordinate to promote progression and drug resistance in gastric cancer.</p></div>
<p>Figure S1 shows elevated expression of circPDIA4 in gastric cancer tissues and impacts on viability of gastric cancer cells. Figure S2 shows that circPDIA4 enhanced invasive activities of gastric cancer cells. Figure S3 shows that circPDIA4 enhanced invasive activities of gastric cancer cells. Figure S4 shows that transcriptional factor SP1 plays an important role in up-regulating QKI expression in gastric cancer. Figure S5 shows that ERK1/2 interacts with circPDIA4 and phosphorylated ERK1/2 protein co-localizes with DUSP6 in gastric cancer. Figure S6 shows impacts of circPDIA4 on effects of different ERK inhibitors in gastric cancer. Figure S7 shows that silenced circPDIA4 promoted the suppression of cell viability induced by silencing of MAPK1 expression. Figure S8 shows that circPDIA4 facilitated oncogenic circRNAs formation depending on the RNA helicase DHX9 in gastric cancer.</p>
<p>Table S1 shows primers for RT-qPCR. Table S2 shows sequences of shRNAs and siRNAs. Table S3 shows antibodies used in the study. Table S4 shows the sequence of the biotin-labeled probes for circPDIA4 and controls. Table S5 shows the overlap circRNAs of the three gastric cancer cell lines. Table S6 shows the relevant clinic-pathological characteristics of gastric cancer cases stratified by circPDIA4 levels in Combined cohort. Table S7 shows the relevant clinic-pathological characteristics of gastric cancer cases stratified by QKI levels in Combined cohort. Table S8 shows Mass Spectrometry of proteins pulled-down by circPDIA4 in HGC-27 cells.</p>
<div>Abstract<p>Circular RNAs (circRNA) are a group of noncoding, covalently uninterrupted loop transcripts, most of which remain to be functionally characterized. Here, we identified circPDIA4 as an oncogenic circRNA in gastric cancer. Clinically, circPDIA4 was significantly upregulated in malignant tissues and was associated with poor survival of patients with gastric cancer. The biogenesis of circPDIA4 was mediated by the RNA-binding protein Quaking, which bound introns 2 and 4 of <i>PDIA4</i> pre-mRNA to promote backsplicing of exons 3 and 4. Elevated expression of circPDIA4 promoted distant metastasis in various mouse xenograft models <i>in vivo</i> and accelerated cancer cell invasion <i>in vitro</i>. CircPDIA4 functioned through distinct oncogenic mechanisms in the cytoplasm and the nucleus. Cytoplasmic circPDIA4 bound to ERK1/2 and sustained hyperactivation of the MAPK pathway by preventing DUSP6-mediated ERK1/2 dephosphorylation. Notably, circPDIA4 depletion enhanced the sensitivity of gastric cancer cells to ERK inhibitors. In the nucleus, circPDIA4 interacted with DHX9 as a decoy and repressed its inhibitory functions on circRNA biogenesis to boost expression of multiple oncogenic circRNAs, which promoted gastric cancer progression. These findings reveal a dual tumor-promoting mechanism for circPDIA4 by regulating oncogenic circRNA biogenesis and increasing MAPK activity. CircPDIA4 should be investigated further as a potential prognostic biomarker and therapeutic target in gastric cancer.</p>Significance:<p>Quaking-regulated circPDIA4 mediates different mechanisms in the nucleus and cytoplasm that coordinate to promote progression and drug resistance in gastric cancer.</p></div>
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