Circ-LRIG3 is highly expressed in HCC and preferentially located in the nucleus
We collected 130 pairs of HCC and paracancerous normal tissues to conduct qRT-PCR. The results showed that circ-LRIG3 was significantly upregulated in HCC (Fig. 1A). Similarly, increased expression of circ-LRIG3 was observed in HCC cells in comparison to human normal LO2 hepatocytes (Fig. 1B). Sanger sequencing verified that circ-LRIG3 is produced by the back splicing of pre-LRIG3 mRNA exon 2 ~ 11, and its mature full-length is 1080 bp (Fig. 1C). Then, we designed divergent and convergent primers to amplify circ-LRIG3 and linear LRIG3, respectively (Fig. 1D). As shown in Fig. 1E, circ-LRIG3 was only amplified in cDNA, but not in gDNA. Further, we treated LO2 and SMMC-7721 cells with 5 µg/ml actinomycin D, and found that circ-LRIG3 had a half-life of more than 24 h (Fig. 1F). Besides, circ-LRIG3, not linear LRIG3, was highly resistance to RNase R (Fig. 1G). These suggest that circ-LRIG3 is a bona fide circRNA. Clinically, HCC patients with high circ-LRIG3 expression had larger tumor size, more vascular invasion, higher edmondson’s grade and later TNM stage than patients with low circ-LRIG3 expression (Table S1). Moreover, high circLRIG3 was positively correlated with short overall and disease-free survival time (Fig. 1H), and it was identified as an independent risk prognostic factor (Table S2). Of note, the qRT-PCR results showed that circLRIG3 was also increased in HCC plasma (Fig. 1I), and the area under the ROC curve (AUC) was 0.8681 (95%CI: 0.7843 ~ 0.9519) (Fig. 1J), implying that circLRIG3 is an excellent indicator for HCC diagnosis. The FISH assay showed that circLRIG3 was mainly localized in the nucleus (Fig. 1K).
Circ-LRIG3 promotes HCC cell malignant phenotype
To investigate the biological function of circ-LRIG3 in HCC cells, we stably overexpressed circ-LRIG3 in HepG2 cells relatively lowly expressing circ-LRIG3 by using pLC5-ciR lentiviral vector (Fig. 2A, B). And we knocked down circ-LRIG3 in SMMC-7721 cells relatively highly expressing circ-LRIG3 by using antisense oligonucleotide (ASO) targeting the junction site of circ-LRIG3 (Fig. 2C, D). Neither pLC5-circ-LRIG3 nor ASO-LRIG3 affected linear LRIG3 expression (Fig. 2B, D). The CCK-8 results showed that overexpression of circ-LRIG3 increased HepG2 cell viability, while knockdown of circ-LRIG3 decreased SMMC-7721 cell viability (Fig. 2E). Likewise, more DNA synthesis was observed in circ-LRIG3-overexpressing HepG2 cells (Fig. 2F), while less DNA synthesis was observed circ-LRIG3-silenced SMMC-7721 cells (Fig. 2G) as compared with their respective control cells. The migration and invasion of cells were enhanced after ectopic expression of circ-LRIG3 (Fig. 2H), and circ-LRIG3 depletion resulted in an opposite effect (Fig. 2I). In addition, the flow cytometry results showed that circ-LRIG3 overexpression reduced the number of apoptotic cells, whereas circ-LRIG3 depletion increased the number of apoptotic cells (Fig. 2J). These above functional data indicate that circ-LRIG3 is a carcinogenic circRNA in HCC.
Circ-LRIG3 activates STAT3 signaling pathway in HCC
To determine how circ-LRIG3 promotes HCC progression, we performed RNA sequencing in control and circ-LRIG3-silenced SMMC-7721 cells, and found that circ-LRIG3 significantly affected the downstream genes of STAT3 signaling (Fig. 3A). The gene-set enrichment analysis (GSEA) results confirmed the the close connection between circ-LRIG3 and STAT3 signaling (Fig. 3B). Then, we performed qRT-PCR assay to verify RNA sequencing results, as shown in Fig. 3C, the levels of FAS, SOCS1, DUSP5, CXCL1 and STAM2 were notably increased in circ-LRIG3-overexpressing HepG2 cells in comparison to control cells, and depletion of circ-LRIG3 in SMMC-7721 cells led to an opposite trend (Fig. 3D). Further, overexpression of circ-LRIG3 markedly increased, while knockdown of circ-LRIG3 reduced STAT3 transcriptional activity (Fig. 3E). Consistently, the phosphorylation level of STAT3 (p-STAT3) significantly increased after circ-LRIG3 overexpression, and decreased after circ-LRIG3 silencing, but the total STAT3 level remained unchanged (Fig. 3F). Besides, we performed p-STAT3 immunohistochemistry (IHC) staining in HCC tissues, and found that its level was strongly positively associated with circ-LRIG3 level (r = 0.716) (Fig. 3G). Functionally, when HepG2 cells were treated with C188-9, a specific STAT3 small-molecule inhibitor, the enhanced cell viability and invasion caused by circ-LRIG3 overexpression were effectively abolished (Fig. 3H, I). These findings demonstrate that circ-LRIG3 functions via activating STAT3 signaling in HCC.
Circ-LRIG3 enhances EZH2-mediated STAT3 activation in HCC
Given that EZH2 can directly bind STAT3 to increase STAT3 methylation and subsequent phosphorylation[19], we wondered whether circ-LRIG3 activates STAT3 through EZH2. The RPISeq online tool result showed that circ-LRIG3 and EZH2 are highly likely to interact (RF classifier = 0.91, SVM classifier = 0.92) (http://pridb.gdcb.iastate.edu/) (Fig. 4A). We then designed biotinylated circ-LRIG3 probe to perform RNA pull-down assay, the circ-LRIG3 probe was verified to effectively enrich circ-LRIG3, but not linear LRIG3 (Fig. 4B). As shown in Fig. 4C, EZH2 was abundantly enriched by circ-LRIG3 probe in HepG2 cells, and this enrichment was significantly increased by circ-LRIG3 overexpression. Likewise, the interaction between circ-LRIG3 and EZH2 was also observed in SMMC-7721 cells, and circ-LRIG3 depletion evidently decreased this phenomenon (Fig. 4D). To confirm above results, we performed RNA immunoprecipitation (RIP) assay, and found that circ-LRIG3 was significantly immunoprecipitated by anti-EZH2 antibody as compared with negative anti-IgG antibody (Fig. 4E). Importantly, circ-LRIG3 overexpression increased STAT3 methylation and phosphorylation, whereas these effects were completely blocked after treatment with EZH2 siRNA or GSK-126, a highly selective EZH2 inhibitor (Fig. 4F). Similarly, EZH2 siRNA or GSK-126 effectively abrogated the enhanced malignant phenotype induced by circ-LRIG3 overexpression in HepG2 cells (Fig. 4G). These indicate that EZH2 is required for circ-LRIG3-induced STAT3 activation. In additions, the RPISeq online tool also showed that circ-LRIG3 and STAT3 are highly likely to interact (RF classifier = 0.81, SVM classifier = 0.88) (Fig. 4H), and this prediction was confirmed by RNA pull-down assay using biotin-labeled circ-RIG3 probe (Fig. 4I) and RIP assay using anti-STAT3 antibody (Fig. 4J) in both HepG2 and SMMC-7721 cells. More importantly, the Co-IP results showed that endogenous EZH2 and STAT3 were directly bound, whereas this interaction was almost disappeared after circ-LRIG3 knockdown (Fig. 4K), suggesting that circ-LRIG3 is critical for the binding between EZH2 and STAT3. And the immunofluorescence (IF) results showed that circ-LRIG3 was co-located with EZH2 and p-STAT3 in the nucleus (Fig. 4L), and this phenomenon was decreased by circ-LRIG3 depletion (Fig. 4L).
Circ-LRIG3 was transcriptionally regulated by activated STAT3
Given that STAT3 is a critical transcription factor, we then test whether circ-LRIG3 is also regulated by STAT3. After treatment of HepG2 and SMMC-7721 cells with colivelin, a specific STAT3 activator, the expression levels of circ-LRIG3 were remarkably increased, while treatment with C188–9 resulted in an opposite effect (Fig. 5A). Through analyzing the JASPAR database, two STAT3 binding motifs were found on circ-LRIG3 promoter (Fig. 5B, C). We then performed chromatin immunoprecipitation (ChIP) coupled PCR assay, the results showed that p-STAT3 was abundantly enriched on site 2 adjacent to the transcription initiation site (TSS), but not on site 1 and negative control region (Fig. 5D, E). Further, we performed luciferase reporter assay using pGL3-basic vector with wild-type or mutant STAT3 binding site (Fig. 5F), the results showed that colivelin and C188–9 significantly increased and decreased wild-type circ-LRIG3 transcriptional activity, respectively, whereas mutant of site 2, not site 1, completely blocked above effects (Fig. 5G). These data reveal that p-STAT3 directly binds to circ-LRIG3 promoter and activates its transcription.
Exogenous expression of circ-LRIG3 enhances tumorigenicity and metastasis in vivo, which is effectively counteracted by C188-9
Lastly, we tested whether circ-LRIG3 was functional in vivo via subcutaneous injection of HepG2 cells into nude mice. As shown in Fig. 6A, B, mice bearing tumors of circ-LRIG3-overexpressing cells had larger tumor volume and weight than mice bearing tumors of control cells. Moreover, more Ki-67 and p-STAT3 positive cells, while less apoptotic cells were observed in circ-LRIG3-overexpressing group as compared with control group (Fig. 6C). Further, we established lung metastasis model by caudal vein injection of circ-LRIG3-overexpressing HepG2 cells into nude mice, the results showed that circ-LRIG3 overexpression notably increased the number of lung metastasis nodules (Fig. 6D). Importantly, when nude mice were treated with C188-9, the increased tumor size and lung metastasis nodules caused by circ-LRIG3 overexpression were restored to control levels (Fig. 6A-D).