CircUBAP2 was upregulated in HCC tissues and indicated poor prognosis
To evaluate the relationship between circUBAP2 and outcomes among patients with HCC, we leveraged the StarBase database to perform bioinformatic analyses of the expression of circUBAP2 in HCC tissues. We found that circUBAP2 expression was significantly higher in HCC tissues relative to healthy liver tissues (Fig. 1B). In order to confirm the findings from the public database, we compared the relative expression of circUBAP2 in 20 paired formalin-fixed, paraffin-embedded tissue samples of liver cancer and adjacent healthy liver tissues via qRT-PCR (Fig. 1C). To investigate the diagnostic and prognostic value of circUBAP2 in HCC, we next measured the expression of circUBAP2 in 125 HCC tissues. Unexpectedly, we found that circUBAP2 was associated with markedly worse OS (p < 0.001) and TTR (p < 0.001) after surgical resection (Fig. 1D-E).
We investigated the association between the expression of circUBAP2 in HCC tissues and clinicopathological parameters through univariate and multivariate Cox proportional regression analyses. Multivariate subgroup analyses showed that high circUBAP2 expression was an independent factor for predicting both TTR [HR 2.83 (1.69-4.74), p < 0.001] and OS [HR 4.68 (2.45-8.89), p < 0.001, Tables 2-3]. Indeed, patients with higher circUBAP2 expression were more likely to have microvascular invasion (MVI) and worse differentiation (Tables 2-3).
We measured the relative expression of circUBAP2 in seven different characteristic HCC cell lines (Fig. 1F). In good agreement with our findings in patient samples, we found that circUBAP2 expression was higher in the high-grade malignancy cell lines HCCLM3 and MHCC97H relative to MHCC97L, a less malignant cell line, suggesting the cicrUBAP2 may facilitate the progression of HCC. Thus, we considered HCCLM3 and MHCC97H cells to be suitable for further experiments. These results collectively suggested that circUBAP2 is of significant importance in the pathogenesis of HCC.
CircUBAP2 promotes HCC cell proliferation and induces cell apoptosis
To investigate the biological effects of circUBAP2 in HCC cells, we generated two circUBAP2-specific shRNAs to target the back-splice sequence. As expected, these shRNAs led to downregulation of circUBAP2 expression in HCCLM3 and MHCC97H cell lines, as validated by qRT-PCR (Fig. 2A). We next performed a series of functional experiments. Here, we found that reduced circUBAP2 expression markedly suppressed cell proliferation (CCK8 assay, Fig. 2B-C). Consistently, flow cytometry assays showed that downregulation of circUBAP2 led to increased apoptosis in circUBAP2high (Fig. 2D-E).
To further evaluate the function of circUBAP2 in vivo, we generated a xenograft tumor model using NOD/SCID/γc(null) (NOG) mice. We found that tumor volumes and weights were decreased in mice with down-regulated circUBAP2 relative to the control group (Fig. 2F). Further, the rate of tumor growth was significantly decreased (Fig. 2F).
CircUBAP2 promotes HCC cell migration and invasion
Pathological epithelial-mesenchymal transition (EMT) is a significant stage in cancer progression, in which cancer cells gain invasive potential[16]. Many important drivers of EMT, such as SNAIL1, bind to and suppress the activity of E-cadherin promoters and are correlated with increased chance of relapse and decreased survival in patients with HCC[17]. Notably, we found that, after knockdown of circUBAP2, HCCLM3 and MHCC97H had decreased migration in both transwell and wound healing assays (Fig. 3A-D). Moreover, in vitro transwell assays found decreased invasion of cells in the circUBAP2-KD group compared to the control group. Collectively, the above experiments confirmed that circUBAP2 promotes migration and invasion of HCC cells in vitro. To identify whether EMT was at the root of these malignant phenotypic changes, qRT-PCR and WB assays were performed to analyze changes in epithelial markers such as E-cadherin and mesenchymal markers such as N-cadherin and SNAIL1 (Fig. 3E-G). We found that circUBAP2-KD cell lines had decreased expression of N-cadherin and SNAIL1 compared with the mock-transfected cell lines. In accordance with the results from functional and molecular experiments, we found that circUBAP2 was associated with EMT and promoted migration and invasion of HCC cells.
circUBAP2 upregulated c-MYC expression by sponging miR-1294
Fluorescence in situ hybridization (FISH) and nuclear-plasma extraction assays suggested that circUBAP2 was primarily localized in the cytoplasm of the two HCC cell lines (Fig. 4A-B). A number of prior studies have reported that some circRNAs act as microRNA sponges. Accordingly, we conducted dual-luciferase reporter assays to explore this possibility for circUBAP2. We constructed a luciferase report vector of wild-type (WT) and mutant (Mut) circUBAP2 and transfected that vector into both HCCLM3 and MHCC97H cell lines (Fig. 4C). After transfection, we found that overexpression of the miR-1294 mimic significantly reduced luciferase activity relative to the negative control (Fig. 4F). Furthermore, we performed RNA immunoprecipitation (RIP) studies with anti-Ago2 antibodies in the same two HCC cells and found that endogenous circUBAP2 was enriched (Fig. 4D). The results of a microRNA pull-down assay using biotin-labeled miR-1294 mimics showed significant enrichment of circUBAP2 compared with the control group (Fig. 4E). Meanwhile, a circRNA pull-down assay using a probe specific for circUBAP2 detected a clear increase in miR-1294 (Fig. 4D-E). Taken together, these results suggested that circUBAP2 functions as a sponge for miR-1294.
Next, we sought to determine the regulatory relationship and binding properties between miR-1294 and c-MYC through qRT-PCR (Fig. 4G), dual-luciferase reporter assays (Fig. 4H), and WB (Fig. 4I). We found that circUBAP2-KD cells had increased miR-1294 levels (Fig. 4G). Conversely, overexpression of miR-1294 reduced the expression of c-MYC protein (Fig. 4I). This result was confirmed by the reduced activity of a WT LUC-c-MYC reporter gene (Fig. 4H), however overexpression of miR-1294 had no effect on the activity of a LUC-c-MYC-mutant reporter gene (Fig. 4H). Collectively, these studies suggested that circUBAP2 may function as a sponge of miR-1294 to upregulate c-MYC expression.
miRNA inhibitor and overexpression of mRNA prevented the effects of circUBAP2 in liver cancer cells.
To further illustrate the regulatory interactions between circUBAP2 and miR-1294 that affect the c-MYC signaling pathway, we used a miR-1294 inhibitor to attenuate the induced expression in the initially circUBAP2 knockdown cells. qRT-PCR results confirmed that, as expected, addition of the miR-1294 inhibitor decreased the expression of miR-1294 in both MHCC97H and HCCLM3 cells (Fig. 5B). Of interest, we found that treatment with the miR-1294 inhibitor partially rescued the expression of c-MYC mRNA and protein relative to the control group (Fig. 5C-D). However, qRT-PCR analysis found that neither the addition of miR-1294 inhibitor nor the overexpression of c-MYC affected the expression of circUBAP2 (Fig. 5A).
Functional experiments revealed that the addition of miR-1294 inhibitor reversed the circUBAP2-knockdown-mediated increase in apoptosis and decrease in the proliferation, migration, and invasion in HCCLM3 and MHCC97H cells (Fig. 5E-H). Of note, the overexpression of c-MYC led to a reversal of the suppressive effects caused by circUBAP2 knockdown. circUBAP2-KD cells overexpressing c-MYC had significantly increased rates of proliferation and apoptosis, as determined by CCK8 and flow cytometry assays (Fig. 5E-F). Transwell migration and invasion assays showed attenuated motility and invasive potential in circUBAP2-KD cells, however these effects were partially reversed by overexpression of c-MYC (Fig. 5G-H). In summary, we determined that the addition of miR-1294 inhibitor and the overexpression of c-MYC reversed the oncogenic effects of circUBAP2 in HCC cells.
mRNA overexpression abolished the effects of miRNA on liver cancer cells.
In order to understand the exact interactions between miR-1294 and c-MYC, we transfected a miR-1294 mimic into MHCC97H and HCCLM3 cell lines. The effects of the miR-1294 mimic were confirmed by qRT-PCR in those two cell lines compared with a scrambled control miRNA mimic. Next, qRT-PCR was combined with WB assays to show that the overexpression of miR-1294 in MHCC97H and HCCLM3 cells led to decreased c-MYC expression (Fig. 6B-C). However, c-MYC overexpression did not alter miR-1294 expression in the same two cell lines (Fig. 5B). Therefore, we next tested whether c-MYC overexpression would attenuate the effects of miR-1294 in HCC cells. Indeed, overexpression of miR-1294 inhibited proliferation, migration, and invasion in MHCC97H and HCCLM3 cell lines (Fig. 6E-G). Conversely, when we also introduced c-MYC into the HCC cells, the tumor suppressive role of miR-1294 was significantly weakened. These findings matched with the finding of decreased cell viability, migration, and invasion (Fig. 6E-G). In summary, overexpression of c-MYC reversed the tumor suppressive role of miR-1294.