Upregulation of UPK1A-AS1 promotes proliferation of HCC cells
Detection of UPK1A-AS1 expression in HCC cell lines suggested that UPK1A-AS1 was highly expressed in HCC cells compared to the human hepatocyte cell line L02 (Supplementary Figure 1A). To investigate the molecular mechanism of UPK1A-AS1, we conducted GSEA of TCGA cohort and found that high UPK1A-AS1-expressing groups were enriched for cell cycle-related gene sets (Figure 1A), suggesting that UPK1A-AS1 may have a role in cell proliferation. To confirm its effect on cell proliferation, lentiviral vectors with full-length UPK1A-AS1 or negative control were introduced into HCC cells, and the proliferation rate of HCC cells was examined. UPK1A-AS1 was successfully overexpressed in HCC cells, and the upregulation of UPK1A-AS1 significantly promoted HCC cell proliferation, as detected by CCK-8 assay (Figure 1B–C). Since the upregulation of UPK1A-AS1 correlated with cell cycle-related gene sets, we further determined whether UPK1A-AS1 could affect HCC cell cycle progression. We then performed EdU dye assays to examine the change in the ratio of cells entering the S phase. The results showed that more UPK1A-AS1-overexpressing cells entered the S phase than the control cells (Figure 1D–G). Collectively, UPK1A-AS1 overexpression promoted HCC proliferation.
UPK1A-AS1 downregulation inhibits HCC cell proliferation
To further confirm the regulatory function of UPK1A-AS1 on cell proliferation, we knocked down UPK1A-AS1 expression in HCC cells using siRNAs and shRNAs. The knockdown efficiency was verified using qRT-PCR (Figure 2A–D). CCK-8 assays showed that the downregulation of UPK1A-AS1 visibly inhibited HCC cell proliferation (Figure 2A–D). Specific locked nucleic acids (LNAs) targeting UPK1A-AS1 were introduced into HCC cells to further verify the effect of UPK1A-AS1 downregulation on HCC proliferation. Consistently, the downregulation of UPK1A-AS1 by LNAs also impaired HCC proliferation (Figure 2E-F). Moreover, cells that entered the S phase in the LNA treatment groups were significantly less than those in the control groups (Figure 2 G–J).
To address the possibility of off-target effects, an overload of UPK1A-AS1 was introduced into HCC cells pre-subjected to UPK1A-AS1 downregulation. Overload of UPK1A-AS1 successfully rescued the expression of UPK1A-AS1 downregulated by siRNAs specifically targeting UPK1A-AS1 (Supplementary Figure 1B–C). Notably, the downregulation of UPK1A-AS1 by specific siRNAs significantly inhibited HCC cell proliferation, and this effect was eliminated by the restored expression of UPK1A-AS1, as detected by CCK-8 and EdU assays (Supplementary Figure 1B–G). In summary, knockdown of UPK1A-AS1 inhibited HCC cell proliferation.
UPK1A-AS1 accelerates the G1/S transition of HCC cells
It is well accepted that rapid cell cycle progression accounts for cancer proliferation. The above results showed that the upregulation of UPK1A-AS1 correlated with cell cycle-related gene sets, and promoted cell proliferation. This led us to hypothesize that UPK1A-AS1 might regulate cell cycle progression. To this end, we carried out flow cytometry analyses to detect the distribution of cell cycle phases in HCC cells following UPK1A-AS1 overexpression or downregulation. The results showed that HCC cells with UPK1A-AS1 overexpression had a decreased rate of G1 phase cells and an increased rate of S phase cells (Figure3 A–D). Consistently, CyclinD1, CDK2, CDK4, and CDK6, important modulators of G1/S transition, were highly expressed in cells overexpressing UPK1A-AS1 (Figure 3E, Supplementary Figure 2B). However, the expression of CyclinE1, p21, and p27 remained unchanged in cells overexpressing UPK1A-AS1 (Supplementary Figure 2A-B). In contrast, si-UPK1A-AS1 resulted in an evident cell cycle arrest at the G1/G0 phase (Supplementary Figure 2C–D), and CyclinD1, CDK2, CDK4, and CDK6 were visibly decreased in cells with UPK1A-AS1 downregulation (Supplementary Figure 2E–F).
We also explored the effect of UPK1A-AS1 on apoptosis and drug resistance. Lower levels of apoptosis were found in UPK1A-AS1-overexpressing cells, indicating that overexpression of UPK1A-AS1 could protect HCC cells from cis-platinum toxicity (Figure 3F–G). Consistently, the expression levels of several well-defined apoptosis markers, including cleaved caspase3 and cleaved PARP, were markedly decreased in UPK1A-AS1-overexpressing cells after cis-platinum exposure (Figure 3H, Supplementary Figure 2G), suggesting that UPK1A-AS1 may boost the resistance to chemotherapy with cis-platinum in HCC cells. In conclusion, the upregulation of UPK1A-AS1 accelerated the G1/S transition of HCC cells.
UPK1A-AS1 promotes tumor growth in vivo
Based on our in vitro UPK1A-AS1 findings, we speculated that it might play an important role in tumor growth in vivo. HCC cells with stable UPK1A-AS1-overexpressing or negative controls were then subcutaneously injected into nude mice. The tumors formed in the UPK1A-AS1-overexpressing group (n = 6) grew faster than those in the negative control group (n = 6). The tumor weight and volume were significantly higher in the UPK1A-AS1-overexpressing group than in the negative control group (Figure 4A–C, Supplementary Figure 3A). UPK1A-AS1 was remarkably overexpressed in the UPK1A-AS1-overexpressing group, as detected by qRT-PCR (Figure 4D). In addition, the positive rate of the proliferation marker Ki-67 was obviously increased in tumors with UPK1A-AS1-overexpressing cells (Figure 4E). Collectively, UPK1A-AS1 boosted tumor growth in vivo.
UPK1A-AS1 correlates with EZH2-mediated cell cycle progression
To dissect the molecular mechanism involved in UPK1A-AS1-associated HCC progression, GSEA was carried out with HCC tumor samples in TCGA datasets. GSEA results suggested that high expression of UPK1A-AS1 was correlated with EZH2 targets (Figure 5A). Interestingly, the GSEA results showed that high EZH2 expression was positively correlated with cell cycle gene sets (Figure 5B). Additionally, the functions of EZH2 and its correlated genes in HCC were predicted by analyzing Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathways in Metascape. The top 20 GO enrichment items also suggested that EZH2 was associated with the cell cycle (Figure 5C). It has been long recognized that EZH2 plays a crucial role in regulating cancer cell proliferation[17]. Consistent with previous studies, EZH2 downregulation with siRNA significantly inhibited HCC cell proliferation (Figure 5D–E, Supplementary Figure 2B). CCND1, CDK2, and CDK4, which accelerate cell cycle progression, were reported to be downstream targets of EZH2[18]. Not surprisingly, these genes were significantly downregulated after EZH2 silencing in HCC cells (Figure 5F, Supplementary Figure 3C–D). We also found that CCNB1 and CCNB2 were significantly decreased after EZH2 silencing in HCC cells (Figure 5F, Supplementary Figure 3C-D). These results suggested that CCND1, CDK2, CDK4, CCNB1, and CCNB2 were direct targets of EZH2 in HCC. We further investigated the correlation between EZH2 and its targets from TCGA datasets. Strong positive correlations between EZH2 and CCND1, CDK2, CDK4, CCNB1, and CCNB2 were found in HCC samples (Supplementary Figure 4A), suggesting that EZH2 promoted HCC proliferation by regulating cell cycle-related genes.
Given that UPK1A-AS1 was correlated with the EZH2 target, and both regulated HCC proliferation, we speculated that UPK1A-AS1 boosted HCC cell progression by regulating these cell cycle-related EZH2 targets. As expected, the upregulation of UPK1A-AS1 significantly increased the expression of EZH2 targets, including CCND1, CDK2, CDK4, CCNB1, and CCNB2 (Figure 5G, Supplementary Figure 4C–D). Furthermore, positive correlations between UPK1A-AS1 and CDK2, CDK4, CCNB1, and CCNB2, except for CCND1, were found in HCC samples (Supplementary Figure 4B), indicating that UPK1A-AS1 regulated CCND1 in a more complicated manner. In short, these results indicated that UPK1A-AS1 regulated EZH2-mediated cell cycle progression.
UPK1A-AS1 interacts with EZH2
To further investigate the molecular mechanisms by which UPK1A-AS1 contributes to the progression of HCC, we examined the subcellular distribution of UPK1A-AS1 in HCC cells by fractionation and FISH assays. UPK1A-AS1 was localized to both the nucleus and cytosol of HCC cells, indicating that it could function as a modulator of gene transcription (Figure 6A, Supplementary Figure 5A). It has been reported that one-fifth of human lncRNAs physically interact with polycomb repressive complex 2 (PRC2), consisting of EZH2, SUZ12, and EED, among which EZH2 is highlighted as a crucial component of PRC2[19]. We showed that UPK1A-AS1 regulated EZH2-mediated cell cycle progression, suggesting that UPK1A-AS1 may interact with and bind to EZH2. To test our hypothesis, an RIP assay against EZH2 was performed. The RIP assay showed that UPK1A-AS1 was significantly enriched with the EZH2 antibody compared with that of the negative control (IgG) in HCC cells (Figure 6B–C). To further confirm our assumption, the interaction of UPK1A-AS1 with EZH2 was determined using an RNA pull-down assay. The results showed that biotin-labeled UPK1A-AS1, but not antisense, exhibited the ability to harbor EZH2 protein (Figure 6D). These results demonstrated that UPK1A-AS1 could physically interact with EZH2.
We then wondered whether UPK1A-AS1 had an impact on the expression level of EZH2. Western blotting assay showed that neither overexpression nor downregulation of UPK1A-AS1 altered the expression of EZH2 (Figure 6E, Supplementary Figure 5B). Moreover, no significant correlation was found between UPK1A-AS1 and EZH2 expression levels (Figure 6F). These results demonstrated that UPK1A-AS1 interacted with EZH2 without changing the expression of EZH2. Surprisingly, the overexpression of UPK1A-AS1 increased the trimethylation of H27K3, which was caused by PRC2 activation. In contrast, silencing UPK1A-AS1 led to an obvious reduction of trimethylation on H27K3 (Figure 6E, Supplementary Figure 5B), suggesting that the interaction between UPK1A-AS1and EZH2 led to PRC2 activation.
It has been reported that lncRNAs physically interact with and bind to proteins to alter their subcellular distribution[20]. Fractionation assays showed that the overexpression of UPK1A-AS1 decreased the cytoplasmic expression of EZH2 but increased the expression level of EZH2 in the nucleus (Figure 6G, Supplementary Figure 5C). IF experiments also confirmed that overexpression of UPK1A-AS1 induced the translocation of EZH2 from the cytoplasm to the nucleus (Figure 6H). EZH2, SUZ12, and EED form a complex in the nucleus for PRC2 activation. An increased interaction between EZH2 and SUZ12 was found after UPK1A-AS1 overexpression (Figure 6I). In brief, UPK1A-AS1 interacted with EZH2, mediated its nuclear translocation, and reinforced its binding to SUZ12, leading to increased trimethylation of H27K3.
UPK1A-AS1 functions through EZH2
To explore whether EZH2 mediated the regulative effect of UPK1A-AS1 on HCC cell proliferation, we co-transfected EZH2 siRNA and UPK1A-AS1 vectors into HCC cells and analyzed the expression of EZH2 targets related to the cell cycle. Overexpression of UPK1A-AS1 increased the expression of CCND1, CDK2, CDK4, CCNB1, and CCNB2, which was eliminated by downregulation of EZH2 (Figure 7A–C, Supplementary Figure 5D). Consistent with the results of qRT-PCR, the EdU assay showed that more UPK1A-AS1-overexpressing cells entered the S phase than the control cells. The increase in the S phase ratio by UPK1A-AS1 overexpression was partly reversed by silencing EZH2 (Figure 7D–G). Taken together, targeting EZH2 with specific siRNA impaired the UPK1A-AS1-mediated upregulation of proliferation and cell cycle progression related genes.
UPK1A-AS1 promotes HCC cell proliferation partially by sponging miR-138-5p
It is well recognized that cytoplasmic lncRNAs can bind to miRNAs and function as sponges to modulate the availability of miRNAs for binding with their target mRNAs. Given that UPK1A-AS1 was located in both the nucleus and cytosol of HCC cells (Figure 6A, Supplementary Figure 5A), we speculated that cytoplasmic UPK1A-AS1 might directly interact with miRNAs. A putative miR-138-5p binding site was identified within the UPK1A-AS1 sequence, as predicted by miRcode (http://mircode.org/index.php) online software (Supplementary Figure 6A). To test whether miR-138-5p recognized the predicted binding site within UPK1A-AS1, we constructed luciferase activity reporter vectors of wild type and mutant UPK1A-AS1 (the binding motif for miR-138-5p was mutated, Supplementary Figure 6A). Dual-luciferase assays showed that overexpression of miR-138-5p significantly decreased the relative luciferase activity of reporter vectors containing UPK1A-AS1-wt but that with UPK1A-AS1-mut remained unchanged (Supplementary Figure 6B).
Accumulating evidence indicates that miRNAs bind to their target mRNAs and cause RNA degradation, translation suppression, or both by forming an RNA-induced silencing complex (RISC) with Argonaute 2 (AGO2). To test whether endogenous UPK1A-AS1 existed in miRNA-containing RISC, an RIP assay against the AGO2 antibody was employed in HCC cells. Both UPK1A-AS1 and miR-138-5p were enriched in the AGO2-associated complex but not in the control IgG (Supplementary Figure 6 C–D), indicating that miR-138-5p is a bona fide UPK1A-AS1-targeting miRNA. Collectively, these results suggested that UPK1A-AS1 physically bound to miR-138-5p and might act as a sponge for miR-138-5p.
miR-138-5p targets multiple cell cycle-related genes, including CDK6, which play an important role in the G1/S transition. We wondered whether miR-138-5p regulated CDK6 expression in HCC cells. Overexpression of miR-138-5p markedly decreased both mRNA and protein expression of CDK6, as detected by qRT-PCR and western blotting assays (Supplementary Figure 6 E–G). Given that the overexpression of UPK1A-AS1 considerably increased the expression of CDK6, we hypothesized that UPK1A-AS1 regulated CDK6 expression by sponging miR-138-5p. As expected, the upregulation of UPK1A-AS1 induced CDK6 expression. However, overexpression of miR-138-5p downregulated the expression of CDK6, while UPK1A-AS1 induction partly reversed this result (Supplementary Figure 6 H–I).
To determine whether the proliferation promotion effect of UPK1A-AS1 was mediated by miR-138-5p, HCC cells with UPK1A-AS1-overexpressing or negative control were co-transfected with miR-138-5p mimic or miR-NC. EdU experiments demonstrated that miR-138-5p overexpression increased cell proliferation induced by UPK1A-AS1 overexpression (Supplementary Figure 6 J–M). In summary, these results suggested that UPK1A-AS1 promoted HCC cell proliferation partially by sponging miR-138-5p.
High expression of UPK1A-AS1 predicts poor prognosis for patients with HCC
UPK1A-AS1 is a newly identified lncRNA, and little is known about its clinical implication in cancers. Genotype-Tissue Expression (GTEx) benign tissue RNA-seq revealed that UPK1A-AS1 was highly expressed in the bladder but scarcely in other tissues (Supplementary Figure 7A). However, data from TCGA datasets showed that UPK1A-AS1 was relatively induced in some types of cancers, including HCC (Supplementary Figure 7B), indicating its important role in the development and progression of malignancies.
To elucidate the clinical implication of UPK1A-AS1 in HCC, UPK1A-AS1 expression levels in HCC were analyzed using RNA-seq data from TCGA datasets. UPK1A-AS1 was highly expressed in HCC cells (Figure 8A). To eliminate the possible contribution of the imbalanced sample size to the statistical significance, we continued to compare the expression of UPK1A-AS1 in paired HCC and corresponding non-tumor samples. The results confirmed that UPK1A-AS1 was significantly overexpressed in HCC (Figure 8B). Moreover, the high expression of UPK1A-AS1 was positively correlated with the tumor stage of HCC (Figure 8C). Survival analysis showed that patients with high expression of UPK1A-AS1 exhibited worse overall survival (OS) than those with low UPK1A-AS1 expression (Figure 8D). As UPK1A-AS1 expression correlated with the HCC stage, we reanalyzed the data from subgroups. Patients with high levels of UPK1A-AS1 presented shorter OS than those with low UPK1A-AS1 expression, although the difference did not reach statistical significance (Figure 8E). Vascular invasion is a sign of poor prognosis in patients with HCC. Survival analysis showed that in the vascular invasion group, patients with high levels of UPK1A-AS1 suffered poorer OS. Due to limitations in sample size, the difference did not reach statistical significance (Figure 8F). Since infection with hepatitis virus and alcohol abuse were risk factors for HCC, we also clarified the correlation between UPK1A-AS1 expression level and prognosis in patients with HCC risk factor exposure. It is shown that patients with high UPK1A-AS1 expression suffered shortened OS in patients with HCC risk factors (Figure 8G). Furthermore, univariate Cox regression analysis identified UPK1A-AS1 as a risk factor for OS in patients with HCC (Table 1).
We also explored the clinical significance of EZH2 in cancer. Data from TCGA datasets showed that EZH2 was highly expressed in various cancers, including HCC (Supplementary Figure 8A). EZH2 overexpression predicted poor prognosis in various cancers, suggesting its oncogenic role in tumorigenesis (Supplementary Figure 8B). A series of HCC datasets from the Gene Expression Omnibus confirmed that EZH2 was highly expressed in HCC (Supplementary Figure 8C). Moreover, high EZH2 expression correlated with the development and progression of HCC (Supplementary Figure 8 D–G). Survival analysis showed that EZH2 predicted poor prognosis for patients with HCC (Supplementary Figure 9A, 9C). Nonetheless, in patients undergoing sorafenib treatment, EZH2 was a factor affecting their survival (Supplementary Figure 9B). Furthermore, high expression of EZH2 was associated with poor prognosis in patients with vascular invasion (Supplementary Figure 9D). EZH2 was also potent in clarifying prognosis in patients with hepatitis virus and alcohol consumption (Supplementary Figure 9 E–F). Our results showed that UPK1A-AS1 functioned through EZH2, at least in part. Consistently, patients with simultaneous high UPK1A-AS1 and EZH2 expression also exhibited shorter OS (Figure 8H). Collectively, UPK1A-AS1 was significantly upregulated in HCC, and the upregulation of UPK1A-AS1 predicted poor prognosis in patients with HCC.