+1 Recommend
1 collections
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Circular RNA circEIF3C promotes intrahepatic cholangiocarcinoma progression and immune evasion via the miR-34a-5p/B7–H4 axis


      Read this article at

          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.


          Circular RNA (circRNA) is a novel type of noncoding RNA that originates from eukaryotic precursor messenger RNA (pre-mRNA). 1 These circRNAs regulate tumor processes through diverse mechanisms, including serving as a miRNA sponge to modulate expression of miRNA-target genes. 2 Eukaryotic translation initiation factor 3 subunit C (eIF3c) is a core subunit of the IF3 complex, involving in the initiation of the translation process of tumor cells. 3 Currently, little is known about the underlying molecular mechanisms of circEIF3C in the pathogenesis of intrahepatic cholangiocarcinoma (ICC). A better understanding of the biological functions of circEIF3C in response to tumor cellular growth and how this affects the tumor signaling pathways would provide important clinical value related to the pathogenesis of ICC. In the current study, we detected expression of 23 circRNAs of the eIF3c gene (predicated by starBase v3.0) in three pairs of ICC compared to adjacent liver tissue. We found that hsa_circ_0005602 (circEIF3C) was significantly upregulated in ICC, and was further identified by sequencing (Fig. S1A; Fig. 1A). Next, we measured circEIF3C expression in 50 ICC and corresponding matched liver samples, and found that circEIF3C was significantly overexpressed in ICC samples (Fig. 1B). The intensity of circEIF3C staining further confirmed that circEIF3C was upregulated in ICC related to matched liver tissues (Fig. S1B, C). Moreover, our results demonstrated that circEIF3C expression was closely related to tumor stage, with higher expression in stage III-IV than in stage I-II (Table S1). Next, we divided patients into circEIF3Chigh and circEIF3Clow groups. Kaplan–Meier analysis suggested that ICC patients in the circEIF3Chigh group had a high recurrence rate and poor prognosis (Fig. S1D, 1C). Thus, these data demonstrate that circEIF3C expression in ICC is higher than that in paratumorous tissues, and increased circEIF3C expression correlate with a worse prognosis in ICC patients. Figure 1 Circular RNA circEIF3C promotes intrahepatic cholangiocarcinoma progression and immune evasion via the miR-34a-5p/B7–H4 axis. (A) Schematic illustration to display the circularization of EIF3Cexons 17–21 to form circEIF3C. (B) A diagram depicting how circEIF3C expression was determined in ICC samples from 50 patients. (C) Kaplan–Meier method was used to analyze the overall survival (OS) rate in 140 patients with ICC. (D) Results of circRIP exhibited a specific enrichment between circEIF3C and miR-34a-5p. (E) Representative images of B7–H4 and CD8 staining in serial TMA (Bar = 100 μm). (F) A diagram showing a positive correlation between circEIF3C and B7–H4 expression, and a negative correlation between CD8 and circEIF3C expression. (G) Work model: High circEIF3C expression promotes the progression of ICC by sponging miR-34a-5p, thereby increasing the translation of B7–H4. Fig. 1 Subsequently, we determined circEIF3C expression in ICC cells. As shown in Figure S2A, circEIF3C expression in RBE and HUCCT1 cells was significantly lower than that in HCCC9810 and QBC939 cells. Next, we overexpressed circEIF3C in RBE cells by transfecting LV-circEIF3C plasmids, and qRT-PCR showed that the circEIF3C was effectively overexpressed (Fig. S2B). Additionally, we effectively inhibited circEIF3 expression in QBC939 cells (Fig. S2C, D). Transwell assays demonstrated that elevated circEIF3C expression increased RBE cell invasion (Fig. S2E, F). CCK-8 assays showed that cell viability was boosted following altered circEIF3C expression in QBC939 and RBE cells (Fig. S2G). Importantly, in vivo tumor assays demonstrated that the tumor volume in RBE-circEIF3C and QBC-939-NC cells was much larger than that of the RBE-Control and QBC-939-shcircEIF3C cells (Fig. S2H, I). Together, these data suggest that circEIF3C promotes the progression of ICC. We used circRNA-RIP to purify circEIF3C-interacting miRNAs, which were specifically against circEIF3C, and used qRT-PCR to analyze the 52 candidate miRNAs (predicated by starBase v3.0). Interestingly, we uncovered a specific enrichment of circEIF3C and miR-34a-5p compared to the negative control. However, other miRNAs showed little to no enrichment (Fig. 1D), indicating that miR-34a-5p functions as a circEIF3C-interacting miRNA in QBC939 cells. Moreover, circEIF3C and miR-34a-5p were pulled down using AGO2 antibodies (Fig. S3A). To further validate the role of miR-34a-5p as a circEIF3C RNA sponge, a dual-luciferase reporter assay was performed in HEK293 T cells. Wild type (wt) circEIF3C and mutant circEIF3C were cloned into a luciferase expression vector. The results demonstrated that circEIF3C (wt) significantly decreased the luciferase activity of miR-34a-5p, but not mutant circEIF3C (Fig. S3B). Here, the luciferase activity was decreased more than 35%, which indicated a direct interaction between circEIF3C and miR-34a-5p. Furthermore, miR-34a-5p showed obvious fluctuations following overexpression or knockdown of circEIF3C in RBE or QBC939 cells (Fig. S3D, E). These data confirm that circEIF3C may act as a sponge for miR-34a-5p. Initially, B7–H4 is present on the surface of antigen-presenting cells including tumor cells and T cells, which contributes to inhibition of T cell function and tumor immune escape. 4 As a result, B7–H4 dysregulation is closely correlated with tumor progression. 5 We demonstrated that B7–H4 might be a target of miR-34a-5p and identified a binding site for miR-34a-5p in the 3′-UTR of B7–H4 through bioinformatics analysis (Fig. S4A). To verify the relationship among circEIF3C, miR-34a-5p, and B7–H4, we constructed miR-34a-5p and B7–H4 pLG3 luciferase reporter plasmids with mutated and wild type potential binding sites. As expected, the dual-luciferase reporter assay further revealed obviously reduced levels of luciferase activity with wt-B7-H4 compared to mu-B7-H4 (Fig. S4B). Moreover, we found that B7–H4 was elevated in RBE-shmiR-34a-5p (Fig. S4C, D) but was markedly reduced in GBC939-shcircEIF3C (Fig. S4E, F). We also showed that circEIF3C-upregulated cells secreted more B7–H4 compared to circEIF3C-downregulated cells (Fig. S4G). Finally, to test specific effects of B7–H4 on pro-apoptosis of CD8+ T cells, we performed an in vitro functional assay. Supernatants that were collected from the circEIF3C-overexpressing cells induced high apoptosis rate of CD8+ T cells compared to control cells (Fig. S4H–K), suggesting a positive correlation between circEIF3C and B7–H4 expression, but a negative correlation between circEIF3C expression and CD8+ T cell infiltration in ICC tissues (Fig. 1E, F). Together, these data further suggest that circEIF3C induces ICC progression and immunosuppression via the miR-34a-5p/B7–H4 axis in ICC cells. We aimed to address whether the oncogenic function of circEIF3C could be reversed by interfering with B7–H4 expression. Thus, RBE-circEIF3C cells were transfected with a B7–H4 shRNA plasmid, followed by quantification of shRNA knockdown efficacy via Western blotting and qRT-PCR. As expected, B7–H4 expression was reduced in plasmid-transfected cells compared to the control group (Fig. S5A, B). Importantly, B7–H4 knockdown greatly suppressed cell migration and proliferation induced by circEIF3C overexpression in vitro (Fig. S5C, D). Together, these data demonstrate that circEIF3C promotes ICC development via the miR-34a-5p/B7–H4 axis (Fig. 1G). In this study, we found that overexpression of circEIF3C was associated with the low overall survival and high recurrence in ICC patients. Intriguingly, circRNA-RIP, pull down and luciferase reporter assays with wt/mutant circEIF3C or knockdown of circEIF3C showed the specific interaction between circEIF3C and miR-34a-5p. In addition, our data showed that 3′-UTR of B7–H4 directly binds and recognizes miR-34a-5p, serving as an RNA binding protein, which induced apoptosis of CD8+ T cells. Collectively, these data indicate that high levels of circEIF3C act reliably to promote ICC progression via sponging miR-34a-5p to up-regulate B7–H4. Author contributions Conceived and designed the experiments: XM Zhong. Revised the manuscript: CX Ji. Analyzed the data: DB Ren. Performed the experiments: AW Ke. Wrote the paper: ZW Yang. Conflict of interests The authors declare no conflict of interest.

          Related collections

          Most cited references5

          • Record: found
          • Abstract: found
          • Article: not found

          The biogenesis, biology and characterization of circular RNAs

          Circular RNAs (circRNAs) are covalently closed, endogenous biomolecules in eukaryotes with tissue-specific and cell-specific expression patterns, whose biogenesis is regulated by specific cis-acting elements and trans-acting factors. Some circRNAs are abundant and evolutionarily conserved, and many circRNAs exert important biological functions by acting as microRNA or protein inhibitors ('sponges'), by regulating protein function or by being translated themselves. Furthermore, circRNAs have been implicated in diseases such as diabetes mellitus, neurological disorders, cardiovascular diseases and cancer. Although the circular nature of these transcripts makes their detection, quantification and functional characterization challenging, recent advances in high-throughput RNA sequencing and circRNA-specific computational tools have driven the development of state-of-the-art approaches for their identification, and novel approaches to functional characterization are emerging.
            • Record: found
            • Abstract: found
            • Article: found
            Is Open Access

            Circular RNA: metabolism, functions and interactions with proteins

            Circular RNAs (CircRNAs) are single-stranded, covalently closed RNA molecules that are ubiquitous across species ranging from viruses to mammals. Important advances have been made in the biogenesis, regulation, localization, degradation and modification of circRNAs. CircRNAs exert biological functions by acting as transcriptional regulators, microRNA (miR) sponges and protein templates. Moreover, emerging evidence has revealed that a group of circRNAs can serve as protein decoys, scaffolds and recruiters. However, the existing research on circRNA-protein interactions is quite limited. Hence, in this review, we briefly summarize recent progress in the metabolism and functions of circRNAs and elaborately discuss the patterns of circRNA-protein interactions, including altering interactions between proteins, tethering or sequestering proteins, recruiting proteins to chromatin, forming circRNA-protein-mRNA ternary complexes and translocating or redistributing proteins. Many discoveries have revealed that circRNAs have unique expression signatures and play crucial roles in a variety of diseases, enabling them to potentially act as diagnostic biomarkers and therapeutic targets. This review systematically evaluates the roles and mechanisms of circRNAs, with the hope of advancing translational medicine involving circRNAs.
              • Record: found
              • Abstract: not found
              • Article: not found

              A T-cell–engaging B7-H4/CD3-bispecific Fab-scFv Antibody Targets Human Breast Cancer


                Author and article information

                Genes Dis
                Genes Dis
                Genes & Diseases
                Chongqing Medical University
                19 May 2022
                March 2023
                19 May 2022
                : 10
                : 2
                : 370-372
                [a ]Department of Oncology Radiotherapy, Jiangxi Cancer Hospital, Nanchang, Jiangxi 330000, China
                [b ]Department of Interventional Treatment, Songjiang District Central Hospital, Shanghai 201600, China
                [c ]Department of Gastroenterology, Songjiang District Central Hospital, Shanghai 201600, China
                [d ]Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai 200032, China
                [e ]Department of Pharmacy, Songjiang District Central Hospital, Shanghai 201600, China
                Author notes
                []Corresponding author. Songjiang District Central Hospital, Shanghai 201600, China. yangzhiwen2009@ 123456sina.com

                Xiaoming Zhong, Changxue Ji, and Dabin Ren contributed equally to this work.

                © 2022 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                : 29 January 2022
                : 28 April 2022
                : 1 May 2022
                Rapid Communication


                Comment on this article