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 in comparison with 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 UPK1A-AS1 may hold a function on cell proliferation. To confirm its function on cell proliferation, lentivirus with full length of 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 upregulation of UPK1A-AS1 significantly promoted HCC cell proliferation, as detected by CCK-8 assay (Figure 1B-C). Since upregulation of UPK1A-AS1 correlated with cell cycle-related gene sets, we further determine whether UPK1A-AS1 could affect HCC cell cycle progression. We then performed EdU dye assay to examine the ratio change of cells entering the S phase. The results showed that more UPK1A-AS1-overexpressing cells entered S phase than its control cells (Figure 1D-G). Taken together, overexpression of UPK1A-AS1 could promote 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 knocked down efficiency was verified using qRT-PCR (Figure 2A-D). CCK-8 assays showed that downregulation of UPK1A-AS1 visibly inhibited HCC cell proliferation (Figure 2A-D). Locked nucleic acids (LNAs) specific targeting UPK1A-AS1 were introduced into HCC cells to further verified the effect of UPK1A-AS1 downregulation on HCC proliferation. Consistently, downregulation of UPK1A-AS1 by LNAs also impaired HCC proliferation (Figure 2E-F). Moreover, cells that entered S phase in LNAs treatment groups were significantly less than the control groups (Figure 2 G-J).
To rule out possible off-target effects, overload of UPK1A-AS1 were 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, downregulation of UPK1A-AS1 by specific siRNAs significantly inhibited HCC cell proliferation, and this effect was eliminated by restored expression of UPK1A-AS1, as detected by CCK-8 and EdU assays (Supplementary Figure 1B-G). In summary, knocked down of UPK1A-AS1 inhibits HCC cell proliferation.
UPK1A-AS1 accelerates G1/S transition of HCC cells
It is well accepted that rapid cell cycle progression accounts for cancer proliferation. Above results showed that upregulation of UPK1A-AS1 correlated with cell cycle-related gene sets and UPK1A-AS1 promotes cell proliferation. This led us to hypothesize that UPK1A-AS1 might regulate cell cycle progression. To this end, we carried out flow cytometry analysis to detect cell cycle distributions in HCC cells after UPK1A-AS1 overexpression or downregulation. The results showed that HCC cells with UPK1A-AS1 overexpression had a decreased rate of G1 phase cells and increased rate of S phase cells (Figure3 A-D). Consistently, CyclinD1, CDK2, CDK4, and CDK6, important modulators in G1/S transition, were highly expressed in cells with UPK1A-AS1 overexpression (Figure 3E, Supplementary Figure 2B). However, the expression of CyclinE1, p21 and p27 remained unchanged in cells with UPK1A-AS1 overexpression (Supplementary Figure 2A-B). On the contrary, 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 level of several well-defined apoptosis markers, including cleaved caspase3 and cleaved PARP, were obviously decreased in UPK1A-AS1-overexpressing cells after cis-platinum exposure (Figure 3H, Supplementary Figure 2G). This suggests that UPK1A-AS1 may boost the resistance to chemotherapy with cis-platinum in HCC cells. In conclusion, upregulation of UPK1A-AS1 accelerates G1/S transition of HCC cells.
UPK1A-AS1 promotes tumor growth in vivo
Based on the results of UPK1A-AS1 in vitro assay, we speculated that UPK1A-AS1 may take an important part in tumor growth in vivo. HCC cells with stable UPK1A-AS1-overexpressing or negative control were then subcutaneously injected into nude mice. The tumors formed in UPK1A-AS1-overexpressing group (n = 6) grew faster than those in negative control group (n = 6). The tumor weight and volume were significantly higher in UPK1A-AS1-overexpressing group than those in negative control group (Figure 4A-C, Supplementary Figure 3A). UPK1A-AS1 was remarkedly overexpressed in UPK1A-AS1-overexpressing group, as detected by qRT-PCR (Figure 4D). In addition, the positive rate of proliferation marker Ki-67, was obviously increased in tumor with UPK1A-AS1-overexpressing (Figure 4E). Collectively, UPK1A-AS1 boosts 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 the HCC tumor samples in TCGA dataset. GSEA results suggested that high expression of UPK1A-AS1 was correlated with EZH2 targets (Figure 5A). Interestingly, GSEA result showed that high expression of EZH2 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 GO and KEGG in Metascape. The top 20 Go enrichment items also suggested that EZH2 was associated with cell cycle (Figure 5C). It has been long recognized that EZH2 has a crucial role in regulating cancer cell proliferation. Consistently with previous studies, downregulation of EZH2 with siRNA significantly inhibited HCC cell proliferation (Figure 5D-E, Supplementary Figure 2B). CyclinD1, CDK2, CDK4, which accelerates cell cycle progression, were reported to be downstream targets of EZH2. Not surprisingly, these genes were significantly downregulated after EZH2 silencing in HCC cells (Figure 5F, Supplementary Figure 3C-D). We also founded that CCNB1 and CCNB2 were significantly decreased after EZH2 silencing in HCC cells (Figure 5F, Supplementary Figure 3C-D). These results suggested that CyclinD1, CDK2, CDK4, CCNB1, and CCNB2 were direct targets of EZH2 in HCC. We further investigated the correlation between EZH2 and its targets from TCGA dataset. Strong positive correlations between EZH2 and CyclinD1, CDK2, CDK4, CCNB1, and CCNB2 were found in HCC samples (Supplementary Figure 4A). These results suggested that EZH2 promoted HCC proliferation by regulating cell cycle-related genes.
Given that UPK1A-AS1 correlated with EZH2 target, and both of them regulated HCC proliferation, we speculated that UPK1A-AS1 boosted HCC cell progression by regulating these cell cycle-related EZH2 targets. As expected, upregulation of UPK1A-AS1 significantly increased the expression of EZH2 targets, including CyclinD1, CDK2, CDK4, CCNB1 and CCNB2 (Figure 5G, Supplementary Figure 4C-D). Furthermore, positive correlations between UPK1A-AS1 and CDK2, CDK4, CCNB1, and CCNB2, but not CyclinD1 were found in HCC samples (Supplementary Figure 4B), indicating that UPK1A-AS1 regulated CyclinD1 in a more complicated way. In short, these results indicated that UPK1A-AS1 regulates 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 assay and FISH assay. UPK1A-AS1 located in both nucleus and cytosol in HCC cells, indicating that UPK1A-AS1 could function as a modulator of gene transcription (Figure 6A, Supplementary Figure 5A). It is reported that one-fifth of human lncRNAs identified physically interacted with polycomb repressive complex 2 (PRC2), consisting of EZH2, SUZ12, and EED, among which EZH2 is highlighted as a crucial component of PRC2. Our results that UPK1A-AS1 regulates EZH2-mediated cell cycle progression enlightened us to assume that UPK1A-AS1 may interact with and bind to EZH2. To test our hypothesis, RNA immunoprecipitation assay (RIP) against EZH2 was performed. RIP assay showed that UPK1A-AS1 was significantly enriched with the EZH2 antibody compared with negative control (IgG) in HCC cells (Figure 6B-C). To further confirm our assumption, the interaction of UPK1A-AS1 with EZH2 was determined using 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 next wondered if UPK1A-AS1 had an impact on the expression level of EZH2. Western blot 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 level (Figure 6F). These results demonstrated that UPK1A-AS1 interacted with EZH2 without changing the expression of EZH2. Surprisingly, overexpression of UPK1A-AS1 increased the trimethylation of H27K3 which was caused by PRC2 activation. On the contrary, silencing UPK1A-AS1 led to obvious reduction of trimethylation on H27K3 (Figure 6E, Supplementary Figure 5B), suggesting that interaction between UPK1A-AS1and EZH2 led to PRC2 activation.
It has been reported that lncRNA physically interacts with and binds to proteins to alter their subcellular distribution. Fractionation assays showed that 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). Immunofluorescence experiment also confirmed that overexpression of UPK1A-AS1 induced the translocation of EZH2 from cytoplasm to 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 nucleus translocation, and reinforced its binding to SUZ12, leading to the 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 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 results of qRT-PCR, EdU assay showed that more UPK1A-AS1-overexpressing cells entered S phase than its control cells. The increase of S phase ratio by UPK1A-AS1 overexpression was partly reversed by silencing EZH2 (Figure 7D-G). Taken together, targeting EZH2 with specific siRNA impaired 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 could bind to miRNAs and function as sponges to modulate the availability of miRNAs for binding with their target mRNAs. Given that UPK1A-AS1 located in both nucleus and cytosol in 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 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 wild type UPK1A-AS1 (UPK1A-AS1-wt), but that with mutant UPK1A-AS1 (UPK1A-AS1-mut) remained unchanged (Supplementary Figure 6B).
Accumulating evidence prove that miRNAs bind to their target mRNAs and cause RNA degradation and/or translation suppression by forming an RNA-induced silencing complex (RISC) with Argonaute 2 (AGO2). To test whether endogenous UPK1A-AS1 exited in miRNA-containing RISC, RIP assay against AGO2 antibody was employed in HCC cells. Both UPK1A-AS1 and miR-138-5p were enriched in 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 suggest that UPK1A-AS1 physically binding to miR-138-5p and may act as a sponge for miR-138-5p.
miR-138-5p is appreciated to target multiple cell cycle-related genes including CDK6, which play an important role in G1/S transition. We wondered whether miR-138-5p regulates expression of CDK6 in HCC cells. Overexpression of miR-138-5p obviously decreased both mRNA and protein expression of CDK6, as detected by qRT-PCR and western blot assays (Supplementary Figure 6 E-G). Given that overexpression of UPK1A-AS1 considerably increased the expression of CDK6, we thus supposed that UPK1A-AS1 regulated the expression of CDK6 through sponging miR-138-5p. As expected, upregulation of UPK1A-AS1 induced CDK6 expression. However, overexpression of miR-138-5p downregulated the expression of CDK6, while UPK1A-AS1 induction partly reversed the 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 ablated the increased cell proliferation induced by UPK1A-AS1 overexpression (Supplementary Figure 6 J-M). In summary, these results suggest that UPK1A-AS1 promotes 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. Analysis from 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 UPK1A-AS1 was relatively induced in some kinds of cancers, including HCC (Supplementary Figure 7B), indicating its important role in development and progression of malignancies.
To explicit the clinical implication of UPK1A-AS1 in HCC, UPK1A-AS1 expression level in HCC was analyzed with RNA-seq data from TCGA dataset. UPK1A-AS1 was highly expressed in HCC (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 convinced that UPK1A-AS1 was significantly overexpressed in HCC (Figure 8B). Moreover, high expression of UPK1A-AS1 positively correlated with tumor stage of HCC (Figure 8C). Survival analysis showed that patients with high expression of UPK1A-AS1 exhibited worse overall survival (OS) as compared with those with low UPK1A-AS1 expression group (Figure 8D). Because UPK1A-AS1 expression correlated with HCC stage, we reanalyzed the data from subgroups. Patients with high UPK1A-AS1 level of UPK1A-AS1 presented shorter OS than those with low UPK1A-AS1 expression, though the difference did not reach statistical significance (Figure 8E). Vascular invasion is a sign of poor prognosis for patients with HCC. Survival analysis showed that in vascular invasion group, patients with high level of UPK1A-AS1 suffered poorer OS. Due to limitation in sample size, the difference did not reach statistical significance (Figure 8F). Since infection of hepatitis virus and alcohol abuse were risk factors for HCC, we also clarified 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 for the OS of 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). Overexpression of EZH2 predicted poor prognosis in various cancer, suggesting its oncogenic role in tumorigenesis (Supplementary Figure 8B). A series of HCC datasets from Gene Expression Omnibus (GEO) confirmed that EZH2 was highly expressed in HCC (Supplementary Figure 8C). Moreover, high expression of EZH2 correlated with 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 obviously effecting their survival (Supplementary Figure 9B). Furthermore, high expression of EZH2 indicated 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 upregulation of UPK1A-AS1 predicted poor prognosis for patients with HCC.