Background
Hepatocellular carcinoma (HCC) is the sixth most common malignancy and the fourth leading cause of cancer-related mortality worldwide [
1]. Surgical resection, radiofrequency ablation, liver transplantation, chemotherapy, molecular-targeted therapy, and immunotherapy have been applied in HCC treatment [
2,
3]. However, the survival rate remains unsatisfactory in patients with HCC because typical clinical features and specific indicators in the early stage of HCC are lacking [
4]. Therefore, there is an urgent need to discover and develop more effective biomarkers and targets for better diagnosis, prognosis, and treatment of HCC.
With thousands of non-coding RNAs being identified and annotated, researchers have come to realize the great importance of non-coding RNA, which makes up more than 90% of the human genome [
5]. Non-coding RNAs can be classified as microRNAs and long non-coding RNAs (lncRNAs) according to their size [
6]. LncRNAs are transcripts of more than 200 nucleotides (nt) in length and possess no or limited protein-coding potential [
7]. Recently, the importance of lncRNAs in tumorigenesis has gradually come to light [
8]. LncRNAs have been recognized as key regulators involved in many biological processes rather than byproducts of RNA polymerase II transcription or genomic noise [
9]. Increasing evidence has demonstrated that lncRNAs participate in regulating carcinogenesis through multiple pathways, including transcription modulation, post-transcription modulation, epigenetic modification, and RNA decay [
10]. LncRNAs have also been regarded as potential biomarkers and therapeutic targets for cancers, including HCC [
11]. For instance, lncRNA MCM3AP-AS1, an oncogenic lncRNA that is highly expressed in HCC, promotes the growth of HCC by targeting the miR-194-5p/FOXA1 axis [
12]. LncRNA TUG1 is overexpressed in HCC and promotes proliferation by epigenetically silencing KLF2 [
13]. DILC represses the self-renewal of cancer stem cells by inhibiting the autocrine IL-6/STAT3 axis [
14]. In contrast, our previous findings showed that MIR22HG, a highly conserved lncRNA, was downregulated and predicted poor prognosis in patients with HCC [
15]. These findings indicate that lncRNAs are critically involved in the development and progression of HCC and may serve as biomarkers for HCC diagnosis and prognosis.
UPK1A antisense RNA 1 (UPK1A-AS1) is a newly discovered lncRNA with little information about its functional role and clinical significance in cancers. It has been reported that UPK1A-AS1 is downregulated in esophageal squamous cell carcinoma (ESCC), and suppresses proliferation, migration, and invasion of ESCC cells by sponging microRNA-1248 [
16]. To date, no study has reported the biological role and clinical importance of UPK1A-AS1 in HCC. Here, we determined the functional role of UPK1A-AS1 in HCC progression and uncovered the underlying molecular mechanism. Our results showed that UPK1A-AS1 was overexpressed in HCC, and the upregulation of UPK1A-AS1 predicted poor prognosis in HCC patients. Functionally, UPK1A-AS1 promoted proliferation by accelerating the G1/S transition of HCC cells. UPK1A-AS1 exerted its oncogenic activity by binding with EZH2 to mediate its nuclear translocation and reinforce its binding to SUZ12. Additionally, UPK1A-AS1 promoted HCC cell proliferation in part by sponging miR-138-5p. Our results uncovered the critical role of UPK1A-AS1 in HCC progression, and UPK1A-AS1 might serve as a potential biomarker for HCC diagnosis and prognosis.
Methods
Cell lines and cell culture
The human hepatocyte cell line L02 and HCC cell lines MHCC-97H, SK-Hep-1 Huh7, HCC-LM3, and Hep3B were provided by the Cell Bank of Type Culture Collection (CBTCC, Chinese Academy of Science, Shanghai, China). Cells were maintained in Dulbecco’s modified Eagle medium with 10% fetal bovine serum (Gibco, USA) and cultured in a humidified incubator containing 5% CO2 at 37 °C.
Small Interference RNAs (siRNA) and lentivirus transduction
The siRNAs used in the current study were designed and synthesized by Ribobio Technology (Guangzhou, China). The transfection of siRNAs was performed according to the manufacturer’s instructions using Lipofectamine RNAiMAX Reagent (Invitrogen, USA). For lentivirus UPK1A-AS1 overexpression and knockdown, full-length UPK1A-AS1 was inserted into GV438, and two hairpin precursors specifically targeting UPK1A-AS1 were cloned into GV112. SK-Hep-1 and MHCC-97H cells were infected with lentivirus at a multiplicity of infection of 20 using polybrene (GeneChem, Shanghai, China). The sequences of siRNAs and target sequences of short hairpin RNAs (shRNAs) are listed in Supplementary Table
1.
Gene Set Enrichment Analysis (GSEA)
GSEA was carried out using the GSEA program provided by Broad Institute (
http://www.broadinstitute.org/gsea/index.jsp) to examine the gene sets or signatures associated with UPK1A-AS1or EZH2 in HCC samples from The Cancer Genome Atlas (TCGA) dataset. RNA sequencing (RNA-seq) data of HCC samples were downloaded from TCGA project, followed by GSEA analysis. An ordered list of all genes was generated according to their correlation with UPK1A-AS1 or EZH2, and predefined gene sets or signatures received an enrichment score and
P-value.
RNA extraction and Quantitative Polymerase Chain Reaction (qRT-PCR)
Total RNA was isolated from HCC cells and tumor tissues using TRIzol reagent (Invitrogen, USA) following the manufacturer’s instructions, and a total of 500 ng RNA was reverse transcribed into cDNA using a cDNA Reverse Transcription Kit (Takara, Japan). Quantitative PCR was performed using the SYBR Green PCR kit (Takara, Japan). β-actin was used as an internal control. Primer sequences used in the present study are shown in Supplementary Table
1.
Cell proliferation assay
The cell proliferation assay was performed using the Cell Counting Kit (CCK)-8 Kit and Cell-Light EdU Apollo 567 in Vitro Imaging Kit (Ribobio Technology, Guangzhou, China). A total of 2000 cells were seeded in 96-well plates with indication treatments, and the CCK-8 proliferation assay was performed according to the manufacturer’s instructions. The EdU dye assay was performed according to the manufacturer’s recommendations.
Cell cycle analysis and apoptosis assay
Cell cycle distribution was tested by flow cytometry on a FACScan (Beckman Instruments, USA). Cell apoptosis was detected using an Annexin V-FITC kit (Keygen Biotech, China).
Western blotting assay
Proteins were separated on a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (Bio-Rad, USA). The membranes were blocked with 5% bovine serum albumin (BSA) for 50 min at room temperature before incubation with primary antibody at 4 °C overnight. The membranes were incubated with secondary antibody conjugated to horseradish peroxidase, followed by signal detection using an enhanced chemiluminescence western blotting substrate (Bio-Rad, USA). The primary antibodies used here are listed in Supplementary Table
2.
Xenograft assay
Four-week-old male nude mice were subcutaneously injected with 1 × 107 UPK1A-AS1-overexpressing or negative control MHCC-97H cells. Tumor diameters were measured every other day and tumor volumes were calculated as (length × width2)/2. The mice were sacrificed before tumor removal 4 weeks after injection. All animal study procedures were approved by the Animal Use and Care Committee of Nanfang Hospital, Southern Medical University (Guangzhou, China).
Functional enrichment analysis
A total of 500 genes positively correlated with EZH2 in HCC samples from TCGA datasets were subjected to functional enrichment analysis using the online software Metascape (
https://metascape.org/). Only terms with a
P < 0.01, minimum count of 3, and enrichment factor greater than 1.5 were identified as significant.
RNA Immunoprecipitation (RIP)
The RIP assay was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) following the manufacturer’s recommendations. Cells were lysed with lysis buffer, and cell lysates were immunoprecipitated with anti-EZH2 and immunoglobulin (Ig) G antibodies. Immunoprecipitated RNA was extracted, purified, and reverse transcribed to cDNA. The transcribed cDNA was subjected to qRT-PCR using UPK1A-AS1-specific primers. The primer sequences used for UPK1A-AS1 amplification are listed in Supplementary Table
1.
RNA pull-down assay
An RNA pull-down assay was carried out using a Magnetic RNA-Protein Pull-Down Kit (Pierce, USA) following the manufacturer’s instructions. Full-length UPK1A-AS1 and antisense were obtained using RiboMAX Large Scale RNA Production Systems (Promega, USA). Biotin-labeled UPK1A-AS1 and antisense were bound to the beads and incubated with whole-cell protein lysates for immunoprecipitation. The beads were washed before being eluted with SDS-PAGE loading buffer. Samples eluted from beads were subjected to western blotting analysis.
Immunofluorescence (IF)
MHCC-97H cells overexpressing UPK1A-AS1 were seeded on coverslips for IF staining. Briefly, cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton-X-100, and blocked with 5% BSA at room temperature. Cells on the coverslips were incubated with anti-EZH2 (1:100) at 4 °C overnight, followed by incubation with Cy3-conjugated CA goat antibodies against rabbit IgG (Santa Cruz Biotechnology, USA) for 1 h at room temperature. The cells were further counterstained with 4, 6-diamidino-2-phenylindole (DAPI, Invitrogen, USA) before imaging with a microscope (Carl Zeiss LSM880, Germany).
Fluorescence in Situ Hybridization Analysis (FISH)
The UPK1A-AS1 RNA FISH probe was designed and synthesized by Yeshan Bio (Guangzhou, China). In brief, cells on the coverslips were fixed with 4% paraformaldehyde for 20 min, washed with PBS twice, treated with pepsin, and pre-hybridized with hybridization reaction solution at 55 °C for 2 h. After pre-hybridization, 20 μL hybridization reaction solution containing 3 μM probes was added to the coverslips. Cells on the coverslips were hybridized at 37 °C overnight and then washed with 2 × saline sodium citrate at 37 °C, followed by washing with PBS twice at room temperature. Finally, cells on the coverslips were stained with DAPI before imaging with a microscope (Carl Zeiss LSM880).
Luciferase reporter assay
The sequence of the full-length UPK1A-AS1 (UPK1A-AS1-wt) and miR-138-5p binding site mutation of UPK1A-AS1 (UPK1A-AS1-mut) were cloned and inserted into the psiCHEK-2.0 vector (Promega, USA). For the dual-luciferase reporter assay, MHCC-97H cells were co-transfected with miR-138-5p and UPK1A-AS1-wt or UPK1A-AS1-mut plasmids. Luciferase activity was measured 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.
Statistical methods
All statistical analyses were carried out using SPSS statistical software version 22 (Abbott Laboratories, USA). Student’s t-test and one-way ANOVAs were performed for statistical analysis when appropriate. Kaplan-Meier and log-rank tests were used for survival analysis. A P-value < 0.05 (two-tailed) was considered statistically significant.
Discussion
Despite the profound advances made in HCC therapeutic strategies, the long-term prognosis of HCC patients remains poor due to limited understanding of the underlying mechanisms of tumor initiation and development [
21]. Dysregulation of lncRNAs is involved in the onset and progression of malignancies, suggesting their clinical potential as biomarkers for diagnosis and prognosis, as well as therapeutic targets. Here, we demonstrated that UPK1A-AS1 was highly expressed in HCC, and high expression of UPK1A-AS1 predicted poor prognosis in patients with HCC. Biological experiments showed that UPK1A-AS1 promoted proliferation and tumor growth by accelerating the G1/S transition of HCC cells. Furthermore, we also found that overexpression of UPK1A-AS1 could protect HCC cells against cis-platinum toxicity, suggesting that UPK1A-AS1 may promote resistance to chemotherapy in HCC cells. Our findings suggest that UPK1A-AS1 may serve as a novel prognostic biomarker and a potential therapeutic target for HCC.
Little is known about the functional role and clinical significance of UPK1A-AS1 in cancers. UPK1A-AS1, downregulated in ESCC, inhibites the proliferation, migration, and invasion of ESCC cells by serving as a miRNA decoy [
16]. In contrast, our findings showed that UPK1A-AS1 was upregulated in HCC, and the overexpression of UPK1A-AS1 promoted proliferation by regulating cell cycle progression. It is well accepted that lncRNAs consistently act in a tissue- or disease-specific manner [
22]. RNA-seq data from GTEx revealed that UPK1A-AS1 was highly expressed in the bladder and modestly expressed in the esophagus, cervix, and vagina but hardly expressed in other tissues (Supplementary Figure
7A), indicating that the expression of UPK1A-AS1 was tissue-specific. Thus, the biological role of UPK1A-AS1 may vary depending on the organic context. The tissue- or disease-specific context of UPK1A-AS1 may account for the distinct roles of UPK1A-AS1 in ESCC and HCC.
Given the subcellular localization of lncRNAs in both the cytoplasm and nucleus, they utilize multiple molecular mechanisms to modulate protein function and gene activity [
23]. Cytoplasmic lncRNAs can act as competing endogenous RNAs to interact directly with and sponge miRNA [
24]. For example, lncRNA-HGBC regulates gallbladder cancer progression by interacting with miR-502-3p, thus sequestering miR-502-3p and downregulating SET expression [
25]. SNHG1 specifically interacts with miR-154-5p, resulting in the induced expression of CCND2 [
26]. In concert with these findings, our study demonstrated that lncRNA UPK1A-AS1 interactd with miR-138-5p, which is reported to be a tumor suppressor in HCC. Notably, miR-138-5p was downregulated in HCC and acted as a tumor suppressor to inhibit several cell cycle-related genes such as
CDK6 [
27‐
29]. Indeed, UPK1A-AS1-induced CDK6 overexpression was partially dependent on the activity of miR-138-5p, as overexpression of miR-138-5p ablated UPK1A-AS1 activity in HCC. Given that UPK1A-AS1 also interacts with and sequesters other miRNAs (e.g., miR-1248) not limited to miR-138-5p, it is worthwhile to explore other miRNAs that potentially accelerate the growth of HCC during progression.
EZH2 serves as the core enzymatic subunit of PRC2, a complex that can methylate lysine 27 of histone H3 and facilitates chromatin remodeling and transcriptional silencing [
30]. A growing body of evidence has implicated the oncogenic role of EZH2 in the progression of a variety of human malignancies [
31]. Consistently, our findings also confirmed that EZH2 was highly expressed in various cancer types, including HCC [
32]. Here, we found that high EZH2 levels correlated with the development and progression of HCC. The upregulation of EZH2 predicts poor prognosis in patients with HCC. Moreover, in patients undergoing sorafenib treatment, EZH2 was a factor affecting their survival, indicating that the expression level of EZH2 may distinguish patients who would benefit from sorafenib treatment. Notably, patients with simultaneous high UPK1A-AS1 and EZH2 expression exhibited shorter OS than those with low expression of UPK1A-AS1 and EZH2, suggesting that the combined detection of UPK1A-AS1 and EZH2 expression can better predict the prognosis of patients with HCC.
Piling studies have highlighted that EZH2 participates in cancer cell proliferation by regulating several cell cycle-related genes [
33]. GSEA analysis showed that high expression of UPK1A-AS1 was correlated with EZH2 targets that were cell cycle-related, suggesting that UPK1A-AS1 may contribute to HCC progression by regulating EZH2-correlated signaling. Indeed, our current study showed that UPK1A-AS1 plays an oncogenic role by modulating EZH2 activity because the downregulation of EZH2 abrogated the tumor-promoting activity of UPK1A-AS1 in HCC. It has been reported that the subcellular localization of EZH2 correlates with the mechanism of EZH2 oncogenic activity. EZH2 present in the cytoplasm may participate in actin polymerization to influence tumor dissemination [
34]. Conversely, EZH2, SUZ12, and EED form a complex in the nucleus and transcriptionally regulate gene expression [
35]. The association of EZH2, SUZ12, and EED is responsible for PRC2 activation. Here, we found that UPK1A-AS1 induced the translocation of EZH2 from the cytoplasm to the nucleus. Furthermore, UPK1A-AS1 increased the interaction between EZH2 and SUZ12 that promoted the methylation of lysine 27 in histone H3, indicating that UPK1A-AS1 contributes to the formation and activation of the PRC complex. EZH2-mediated PRC2 activation contributes to the transcriptional silencing of tumor suppressor genes, leading to the activation of NOTCH [
36], JAK-STAT [
37], or β-catenin signaling pathways [
38] and upregulation of cell cycle genes, such as CDK2, CDK4, and CCND1. However, UPK1A-AS1 overexpression did not change the expression of p-STAT3 (data not shown) or β-catenin (Fig.
6g). In contrast, si-EZH2 abolished the upregulation of CDK2, CDK4, and CCND1 caused by UPK1A-AS1, suggesting that UPK1A-AS1 upregulated the aforementioned genes via EZH2, but not EZH2-mediated JAK-STAT and β-catenin signaling activation. Growing evidence has shown that EZH2 can function independently of PRC2 to facilitate transcriptional activation rather than repression [
39‐
41]. EZH2 directly binds to the promoter regions of CCND1 and promotes its transcriptional activation [
42]. Whether the UPK1A-AS1-mediated upregulation of CDK2, CDK4, and CCND1 is EZH2-dependent transcriptional activation still requires further investigation.
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