A Novel Circular RNA circ-LRIG3 Facilitates the Malignant Progression of Hepatocellular Carcinoma by Modulating the EZH2/STAT3 Signaling

Abstract

Background: Circular RNA (circRNA) is emerging as an important player in human diseases, especially cancer. In our previous study, we identified a series of deregulated circRNAs in hepatocellular carcinoma (HCC) by performing circRNA microarray expression profile. Here, we aimed to explore the role of circ-LRIG3 (hsa_circ_0027345) in HCC.Methods: qRT-PCR and western blot were used to asses gene and protein expression, respectively. CCK-8, EdU and Transwell assays were used to detect cell proliferation, migration and invasion. GSEA software was applied to analyze the pathway related to circ-LRIG3. Co-IP, RIP and ChIP assays were used to identify the positive feedback axis of circ-LRIG3/EZH2/STAT3. Animal study was carried to test the role of circ-LRIG3 in vivo.Results: Circ-LRIG3 was notably upregulated in HCC and promoted HCC cell proliferation, migration, invasion and reduced apoptosis. Circ-LRIG3 formed a ternary complex with EZH2 and STAT3, facilitating EZH2-induced STAT3 methylation and subsequent phosphorylation, resulting in the activation of STAT3 signaling. In turn, activated STAT3 could directly bind to circ-LRIG3 promoter to increase circ-LRIG3 transcription activity, thus forming a positive feedback loop. The animal models showed that exogenous expression of circ-LRIG3 enhanced tumorigenicity and metastasis in vivo, whereas these effects were blocked after treatment with C188-9, a specific STAT3 small-molecule inhibitor. Clinically, high circ-LRIG3 was closely linked with aggressive clinicopathological features and was identified as an independent risk prognostic factor of overall survival. Importantly, plasma circ-LRIG3 was found to be a highly sensitive and specific non-invasive diagnostic indicator for HCC. Conclusions: Our study reveals the carcinogenic role of circ-LRIG3 in HCC, which may provide a new therapeutic target for HCC patients.

CircPLCE1 facilitates the malignant progression of colorectal cancer by repressing the SRSF2‐dependent PLCE1 pre‐RNA splicing

“…23,24 Many molecular functions are performed by cir-cRNAs to implement their cellular effects, such as interacting with different proteins to form specific RNPs and influence their intrinsic function. [25][26][27] In our study, we present evidence that PLCE1 mRNA production is negatively controlled by its circRNA via suppression of pre-PLCE1 splicing. One of our main findings is that circPLCE1 directly binds to the SRSF2 protein and suppresses the spliceosome assembly on nascent pre-PLCE1 to catalyse intron removal before the completion of transcription (Figure 6F).…”

“…Recently, evidence has accumulated that circRNAs are frequently dysregulated in human cancer and affect tumorigenesis, invasion and metastasis 23,24 . Many molecular functions are performed by circRNAs to implement their cellular effects, such as interacting with different proteins to form specific RNPs and influence their intrinsic function 25‐27 . In our study, we present evidence that PLCE1 mRNA production is negatively controlled by its circRNA via suppression of pre‐PLCE1 splicing.…”

CircARID1A binds to IGF2BP3 in gastric cancer and promotes cancer proliferation by forming a circARID1A-IGF2BP3-SLC7A5 RNA–protein ternary complex

“…In addition to dominant localization in the cytoplasm, some circRNAs are located in the nucleus. For example, circLRIG3 preferentially located in the nucleus and interacts with both EZH2 and STAT3 in hepatocellular carcinoma to form a circRNA-protein complex, facilitating EZH2-induced activation of STAT3 signaling and promoted HCC cell proliferation and metastasis [57]. CircURI1 was reported to preferentially locate in the nucleus in both AGS and SGC7901 cells.…”

CircRNAs and their regulatory roles in cancers

“…For positive regulation, RNA binding proteins (RBPs) promote circularization by reducing spatial proximity as most of them bind to their binding sites in flanking introns (Ashwal-Fluss et al 2014;Conn et al 2015;Dai et al 2018). Besides, the upstream promoter of the circRNA is activated under some circumstances, which also contributes to the formation of the circRNA (Zhao et al 2020b;Wang et al 2018;Sun et al 2020a). However, it's reported that circularization can be promoted by most RBPs except DHX9, which inhibits the production of circDLC1 as it can bind to the flanking reverse complementary sequence of circDLC1 and inhibit the pairing of these sequences .…”

“…For example, circLRIG3 physically interacts with EZH2 and STAT3 and acts as a scaffold to increase EZH2-induced STAT3 methylation and subsequent phosphorylation. In turn, activated STAT3 directly binds to the circLRIG3 promoter region, which enhances the circLRIG3 transcription, and at last, forms a positive regulatory circuit that improves hepatocellular carcinoma (HCC) processes (Sun et al 2020a). Another example is circNDUFB2 that binds to the IGF2BPs KH domain and acts as a stent to enhance the interaction between TRIM25 and IGF2BPS, eventually expediting TRIM25-mediated IGF2BPS ubiquitination and degradation (Li et al 2021a).…”