LncRNA‐EWSAT1promotes hepatocellular carcinoma metastasis via activation of theSrc‐YAPsignaling axis

The FASEB JournalVolume 36, Issue 12 e22663 RESEARCH ARTICLEOpen Access LncRNA-EWSAT1 promotes hepatocellular carcinoma metastasis via activation of the Src-YAP signaling axis Xigan He, Xigan He orcid.org/0000-0002-5701-1101 Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorJinggui Chen, Jinggui Chen Department of Gastric Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorJiamin Zhou, Jiamin Zhou Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorAnrong Mao, Anrong Mao Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorWeiqi Xu, Weiqi Xu Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorHongxu Zhu, Hongxu Zhu Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorQi Pan, Qi Pan Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorYiming Zhao, Yiming Zhao Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorNing Zhang, Ning Zhang Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorLongrong Wang, Longrong Wang Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorMiao Wang, Miao Wang Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorZeyang Liu, Zeyang Liu Department of General Surgery, Qilu Hospital of Shandong University, Jinan, ChinaSearch for more papers by this authorWeiping Zhu, Corresponding Author Weiping Zhu [email protected] Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, China Correspondence Weiping Zhu and Lu Wang, Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai 200032, China. Email: [email protected] and [email protected]Search for more papers by this authorLu Wang, Corresponding Author Lu Wang [email protected] orcid.org/0000-0002-6856-7084 Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, China Correspondence Weiping Zhu and Lu Wang, Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai 200032, China. Email: [email protected] and [email protected]Search for more papers by this author Xigan He, Xigan He orcid.org/0000-0002-5701-1101 Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorJinggui Chen, Jinggui Chen Department of Gastric Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorJiamin Zhou, Jiamin Zhou Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorAnrong Mao, Anrong Mao Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorWeiqi Xu, Weiqi Xu Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorHongxu Zhu, Hongxu Zhu Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorQi Pan, Qi Pan Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorYiming Zhao, Yiming Zhao Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorNing Zhang, Ning Zhang Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorLongrong Wang, Longrong Wang Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorMiao Wang, Miao Wang Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, ChinaSearch for more papers by this authorZeyang Liu, Zeyang Liu Department of General Surgery, Qilu Hospital of Shandong University, Jinan, ChinaSearch for more papers by this authorWeiping Zhu, Corresponding Author Weiping Zhu [email protected] Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, China Correspondence Weiping Zhu and Lu Wang, Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai 200032, China. Email: [email protected] and [email protected]Search for more papers by this authorLu Wang, Corresponding Author Lu Wang [email protected] orcid.org/0000-0002-6856-7084 Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai, China Correspondence Weiping Zhu and Lu Wang, Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai 200032, China. Email: [email protected] and [email protected]Search for more papers by this author First published: 24 November 2022 https://doi.org/10.1096/fj.202200825R Xigan He, Jinggui Chen, Jiamin Zhou and Anrong Mao contributed equally to this work. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Regardless of the improvements in diagnostic and therapeutic methods, the clinical outcomes of hepatocellular carcinoma (HCC) patients remain poor. Although accumulating evidence indicates that lncRNAs (long noncoding RNAs) are essential within the control of tumorigenesis and the metastasis of cancer, the underlying mechanisms remain largely unknown. This work explored the pattern of expression and functional significance of a newly found lncRNA, Ewing sarcoma-associated transcript 1 (EWSAT1), in HCC metastasis. The results indicated that EWSAT1 was upregulated significantly in HCC relative to that in normal tissues and was correlated with an aggressive phenotype and low patient survival. Functional experiments demonstrated that EWSAT1 could promote proliferation and HCC cell metastasis both in vitro and in vivo. Mechanistically, EWSAT1 binds directly to Yes-associated protein (YAP), promotes Sarcoma gene (Src)-induced phosphorylation of YAP, facilitates nuclear translocation of YAP, and consequently, activates the transcription of Hippo-YAP signaling target genes involved in cancer evolution. This study found that EWSAT1 plays a crucial role in HCC metastasis and that it has the potential to be a prognosis biomarker and a target for therapeutics. 1 INTRODUCTION Hepatocellular carcinoma (HCC) is a widespread cancer type and a foremost contributor to cancer mortality globally.1 Despite improvements in therapeutic and diagnostic methods, the clinical outcomes and prognoses of HCC patients have stayed poor.2 Therefore, there has been an increasing interest in novel strategies that contribute to improving the outcome of HCC patients.3 Integrated studies on genomics have shown that 98% of the transcripts of the human genome consist of noncoding RNAs (ncRNAs) with limited or no capability for coding proteins.4, 5 The long noncoding RNAs (lncRNAs) are novel regulatory ncRNA members with a length exceeding 200 nucleotides. The lncRNAs have recently received considerable attention because of their links to the development of diseases such as cancer.6 To date, hundreds of cancer-related lncRNAs have been identified in multiple malignancies, including HCC, thus allowing novel insights into cancer pathogenesis.7, 8 These lncRNAs play crucial roles in promoting or suppressing cancer cell characteristics, thereby making them attractive therapeutic targets.9-11 Ewing sarcoma-associated transcript 1 (EWSAT1) is a newly identified lncRNA located on chromosome 15q23 and is comprised of 2527 nucleotides. Emerging evidence has demonstrated the aberrant expression and oncogenic role of EWSAT1 in a wide range of cancers, including Ewing sarcoma,12 glioma,13 cervical cancer,14 colorectal cancer,15 osteosarcoma,16-18 ovarian cancer,19 and nasopharyngeal carcinoma.20 However, the potential biological function and clinical importance of EWSAT1 in the metastasis of HCC remains largely unknown. The signaling pathway Hippo-YAP has an important function in regulating the size of organs, tissue regeneration, and cancer progression21 and fundamental parts of this pathway are represented by the Mst1/2-Lats1/2 kinase cascade and transcription coactivators YAP/TAZ located downstream. Direct phosphorylation by the Mst1/2-Lats1/2 kinase cascade impedes YAP/TAZ, causing the retention of cytoplasm and further ubiquitination and degradation. Hippo pathway inhibition results in the translocation to the nucleus of dephosphorylated YAP/TAZ and its binding to transcription factors, such as transcriptional enhanced associate domains (TEADs), resulting in the expression of target genes participating in cell proliferation and survival.22 Notably, while communication between lncRNAs and the Hippo-YAP pathway and the considerable contribution of this network to cancer occurrence and progression have been increasingly recognized,23 the underlying molecular mechanisms remain poorly understood. The current study showed that EWSAT1 was significantly upregulated in HCC tissues, and that elevated expression of EWSAT1 was linked to lowered survival and high recurrence in HCC patients. The results of gain and loss of function assays demonstrated that EWSAT1 could facilitate both in vitro and in vivo HCC cell growth and metastasis. Mechanistic studies revealed that EWSAT1 directly binds to YAP and promotes Src-induced YAP phosphorylation, thus facilitating the nuclear entry of YAP, and consequently activating the transcription of target genes involved in cancer metastasis. 2 MATERIALS AND METHODS Tissue specimens HCC pairs numbering 146 and matched normal tissues were obtained from the Department of Hepatic Surgery, Fudan University Shanghai Cancer Center (FUSCC), Shanghai, China. The samples were collected through surgical resection, were frozen in dry ice, and were stored at −20°C with RNAlater. The current study was sanctioned and supervised by the Ethics Committee of the hospital, and the written approval of all patients was obtained. Cell culture HCC cell lines (Hep3B, HepG2, Huh7, SMMC-7721, MHCC97L, and HCCLM3) and epithelial cells L02 of normal human liver were obtained from the Chinese Academy of Sciences (Shanghai, China). Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) was used to culture all cell lines at a temperature of 37°C in a humidified incubator with 5% CO2. Production and infection of lentivirus For EWSAT1 knockdown, plasmids encoding short hairpin RNA (shRNA) against EWSAT1 or the control were sourced from Genechem, Shanghai, China. Target sequences were: sh-1: 5′-TTG GGC TCT CAA TGG TAT CAT-3′ and sh-2: 5′-AAG GGA GGG TTA CTA ACT TTA-3′. For the overexpression of EWSAT1, the full length of EWSAT1 was manufactured and subcloned into the pLenti-EF1a plasmids (Obio tech, Shanghai, China). Recombinant lentivirus was produced by transfecting the vectors and viral packaging constructs into 293T cells. The supernatant containing the virus was gathered at 48 h posttransfection, centrifuged, filtered, and concentrated. HepG2 and Huh7 cells were then infected for EWSAT1 knockdown, and Hep3B cells were infected for EWSAT1 overexpression. The infected cells were subsequently chosen by puromycin for 2 weeks to construct stable cells. Quantitative real-time polymerase chain reaction (qRT-PCR) Cell and tissue total RNA were extracted using Trizol reagent (Invitrogen). PrimeScript RT Master Mix (TaKaRa) was used to conduct reverse transcription. Quantitative real-time PCR was implemented using SYBR Green mixture (Takara) with the ABI 7500 Sequence Detection System (Applied Biosystems). The relative level of mRNA expression was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and estimated using the 2‒ΔΔCT method. The primer sequences were: EWSAT1: forward: 5′-TTT GCT GTT GCT GGG CTC ACT G-3′; reverse: 5′-AGG ATG GAG GAG TGG CAG TTC AG-3′, GAPDH: forward: 5′-AGA AGG CTG GGG CTC ATT TG-3′; reverse: 5′-AGG GGC CAT CCA CAG TCT TC -3′, CTGF: forward: 5′-GGC TTA CCG ACT GGA AGA CA-3′; reverse: 5′- AGG AGG CGT TGT CAT TGG TA-3′, CYR61: forward: 5′-TGG AGC CTC GCA TCC TAT AC-3′; reverse: 5′-TGT CAT TGG TAA CTC GTG TGG-3′, and CDX2: forward: 5′-TGT CAT TGG TAA CTC GTG TGG-3′; reverse: 5′-GCT GCT GCA ACT TCT TCT TG-3′. Western blotting The extraction of total cell protein was conducted using radioimmunoprecipitation assay (RIPA) lysis buffer with protease inhibitors (Biyuntian Biotech, China). Protein was detected using the BCA Protein Assay Kit (Biyuntian Biotech). Protein samples of equal amounts were run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, following which they were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). Blocking of the membrane for 1 h with milk (5%) was conducted, following which it was incubated using a primary antibody (Table S1) overnight at a temperature of 4°C. Incubation of the membrane was conducted using a secondary antibody, following which it was detected with enhanced chemiluminescent (ECL) reagents (Millipore) after 30 m of washing with tris-buffered saline (TBS) Tween (TBST) buffer. Immunohistochemistry (IHC) analysis Paraffin-embedded tissue samples of mice were stained as per the standard IHC protocols. Dewaxing and rehydration of samples were conducted in a decreasing gradient series of xylol and ethanol. Antigen recovery was conducted with citrate buffer (pH = 6.0) for 6 min at a temperature of 100°C. After blocking endogenous peroxidase using 3% H2O2, the tissues were incubated using anti-proliferating cell nuclear antigen (anti-PCNA) (Cell Signaling, USA) overnight at a temperature of 4°C, and then by secondary antibody for 1 h room temperature. Subsequently, the signals were developed using 3, 3′-diaminobenzidine (DAB), following which sections were counterstained using hematoxylin. Subcellular fractionation Subcellular fractionation was conducted using the PARIS Kit (Life Technologies, Carlsbad, USA) to determine the cellular distribution of EWSAT1. Briefly, HCC cells were lysed for 10 min, following which they were centrifuged to obtain cytoplasmic RNA from the supernatants and nuclear RNA from the pellets. Cell counting kit-8 (CCK-8) assay The viability of cells was observed using the CCK-8 assay. Here, cells were plated in a 96-well plate with a 100 μl DMEM medium at a density of 1 × 103/well. The cells contained in each well were then incubated with 10 μl CCK-8 reagent (Dojindo, Japan) at a temperature of 37°C for 2 h. Measurement of absorbance occurred for 5 days at an optical density of 450 nm. Colony formation assay Cells numbering ~1 × 103 were individually plated on 6-well plates and incubated using a complete medium for 2 weeks. After fixation for 15 min with 4% paraformaldehyde, Giemsa solution was for cell staining for 20 min, following which the colonies were photographed, counted. Ethynyldeoxyuridine (EdU) incorporation assay An EdU incorporation assay was conducted to examine cell proliferation. Briefly, cells were incubated with a complete medium supplemented with EdU (RiboBio, China) for 2 h, fixed using paraformaldehyde (4%) for 20 min, permeabilized using 0.5% Triton X-100 for 15 min, conjugated for 30 min to Apollo solution, dyed with Hoechst for 3 min, and finally visualized using a confocal microscope. Transwell migration and invasion assays During the migration assay, ~1 × 105 cells were plated into the upper chamber for each insert (BD Biosciences, USA) with pores of 8 μm diameter, whereas the lower chamber was filled with a DMEM medium (500 μl) supplemented with 10% FBS. The cells located on the upper surface of the filter were scratched after incubation for 48 h at 37°C, whereas 4% paraformaldehyde and crystal violet were used to fix and stain cells located on the lower surface, respectively. For the invasion assay, a layer of 20% Matrigel was used to coat the upper chamber (Corning, USA), and the procedures conducted subsequently were as described above. In vivo study The in vivo animal studies conducted in the current study were as per the FUSCC Animal Care Guidelines. For the tumor xenograft model, 2 × 106 stable cells in phosphate-buffered saline (PBS) solution (200 μl) were injected subcutaneously into BALB/C nude mice (4-week-old; n = 5) sourced from the Institute of Zoology, Chinese Academy of Sciences, Shanghai, China, and the sizes of tumors were monitored every 4 days. The mice were euthanized 31 days after the injection and the tumors were removed. The volumes of tumors (V) were calculated by V (mm3) = 4π/3 × (width/2)2 × (length/2). For the orthotopic tumor model, 2 × 106 stable HCC cells in phosphate-buffered saline solution (200 μl) were directly injected into the liver of BALB/C nude mice. After 30 days, the mice were euthanized, and liver tissue was isolated and captured. A model of lung metastasis was established to validate the impact of EWSAT1 on in vivo metastasis of HCC. Briefly, 5 × 106 stable cells in PBS (200 μl) were injected into nude mice tail veins. The mice were euthanized after 6 weeks and luciferase imaged. Lung tissues were then dissected, fixed using 4% formalin, and analyzed using hematoxylin & eosin (HE) staining. RNA-sequencing analysis RNA preparation, library construction, and sequencing were performed on the Hiseq3000 (Illumina, USA) platform at Sangon Biotech, Shanghai, China. Raw sequencing reads were aligned to the human reference genome (hg19) for the processing of data. Read counts for each gene were normalized into Reads Per Kilobase per Million mapped reads (RPKM) values. Luciferase reporter assay Cotransfection of cells was conducted with EWSAT1 overexpression or knockdown plasmids and YAP/TEAD luciferase reporter plasmids using Lipofectamine 2000 (Invitrogen). Triplicates of each group were run in 96-well plates. Luciferase activity was detected by a Synergy 2 Multidetection Microplate Reader (BioTek Instruments, Inc.) subsequent to 48 h of transfection. The activity of renilla luciferase was normalized relative to that of Firefly luciferase. RNA pull-down RNA pull-down was conducted to detect the interaction between lnc-EWSAT1 and YAP protein. Biotin-labeled EWSAT1 was manufactured with Biotin RNA Labeling Mix using T7 RNA polymerase, following which it was incubated for 4 h with the cell lysates. The protein with biotin-labeled EWSAT1 was pulled down with streptavidin magnetic beads (Thermo, USA), following which it was exposed to electrophoresis and western blotting of anti-YAP. RNA immunoprecipitation (RIP) The RIP assays were conducted using the Magna RIP Kit (Millipore) as per manufacturer instructions. RIP lysis buffer with protease and RNase inhibitors (Thermo) was used to lyse cells. Next, 3 μg of antibody was incubated with 30 μl of magnetic protein G beads (Invitrogen, USA), followed by the addition of the cell lysate and full reaction. The coprecipitated RNAs were then isolated by TRIzol and subjected to qRT-PCR analysis. Co-immunoprecipitation (IP) Co-IP experiments were executed to examine the effect of EWSAT1 on the physical association between Src and YAP protein. The total protein was obtained by I the lysing of the indicated cells, following which the cells were incubated overnight on ice with 2 μg primary antibody. On the following day, 30 μl A/G PLUS-Agarose beads (Santa Cruz Biotechnology, USA) were added to the cell lysate and primary antibody mixture, and the solution was gently incubated for 3 h at a temperature of 4°C under rotation. The beads were then washed with lysate buffer on ice at least five times, following which they were subjected to standard western blotting analysis. Statistical analyses All statistical analyses were conducted in the software GraphPad Prism 6 (GradPad Software, Inc). All data are shown in the current study as means ± standard deviation (SD). The Student's t-test was used to detect significant differences between the two groups. Kaplan–Meier analysis was used to perform survival analysis. p < .05 on a two-sided test was regarded as statistically significant. One-way ANOVA in more than two group circumstances to detect the significant differences. After conducting a homogeneity of variance test to confirm equal variances among subgroups, p values were obtained from a post hoc test using the least significant difference (LSD) method. 3 RESULTS EWSAT1 is significantly upregulated in HCC and linked to poor prognosis To determine its clinical relevance, the present study first investigated EWSAT1 expression using qRT-PCR analysis and normalized relative to the endogenous control (GAPDH). The results suggested that the level of EWSAT1 in HCC samples markedly exceeded that in adjacent normal tissues (p < .001) (Figure 1A). Meanwhile, ~75.3% (110/146) of HCC cases showed elevated EWSAT1 expression, among which ~56.2% (82/146) of HCC samples showed a significant increase (fold change > 2) (Figure 1B). Most importantly, EWSAT1 expression gradually increased with the progression of the tumor stage and differentiation (Figure 1C,D), indicating the potential role of EWSAT1 in the evolution of HCC. Therefore, the results of the current study clearly confirm the significantly elevated expression of EWSAT1 in HCC tissues. FIGURE 1Open in figure viewer EWSAT1 is significantly upregulated in HCC and can be linked to a poor prognosis. (A) The levels of Ewing sarcoma-associated transcript 1 (EWSAT1) expression in 146 hepatocellular carcinoma (HCC) and paired normal tissues were identified through quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was applied as an internal control. (B) Fold-changes of EWSAT1 expression in 146 paired tissues (HCC/non-tumor). (C) The levels of expression of EWSAT1 in 146 HCC tissues were analyzed according to tumor stage. (D) The levels of expression of EWSAT1 in 146 HCC tissues were analyzed according to tumor differentiation. (E and F) The influence of EWSAT1 patterns of expression on disease-free survival (DFS) and overall survival (OS) as determined by Kaplan–Meier analyses (log-rank test). One hundred and forty-six HCC patients were assigned to groups of high and low EWSAT1 expression according to the median value (Data are shown as mean ± standard deviation (SD) in figures A, C, and D. **p < .01, ***p < .001). The current study examined the relationship between EWSAT1 expression and clinicopathologic parameters by dividing all patient groups of high and low EWSAT1 expression by the median expression value. Furthermore, the curves of disease-free survival (DFS) and overall survival (OS) were constructed using the Kaplan–Meier method and compared with the log-rank test to evaluate the prognostic influence of EWSAT1 (Figure 1E,F). Patients with higher expression of EWSAT1 showed reduced OS relative to those with lower expression (log-rank test, p = .0011). In addition, a significant difference in the DFS curve existed between the high and the low EWSAT1 groups (log-rank test, p = .0054), indicating that HCC patients with elevated expression of EWSAT1 had a higher likelihood of developing local recurrence or metastasis. Furthermore, univariate Cox regression showed that EWSAT1 expression, tumor size, differentiation, and American Joint Committee on Cancer (AJCC) stage were significantly correlated with both DFS and OS (Tables 1 and 2). In addition, multivariate Cox regression suggested that the expression of EWSAT1 remained an independent factor for prognosing increased occurrence of disease and reduced survival (Tables 1 and 2). TABLE 1. The results of univariate and multivariate analyses of overall patient survival of HCC (Bold values indicate a significant difference) Variable Univariate analysis Multivariate analysis HR 95% CI p value HR 95% CI p value Age 1.210 0.652–2.017 .527 Gender 1.035 0.723–1.216 .613 AJCC stage 3.507 2.254–4.923 .001 2.245 1.528–3.272 .000 HBsAg 1.625 1.071–2.872 .026 Tumor size 1.529 1.336–2.994 .003 Differentiation 1.602 1.104–2.316 .032 1.502 1.183–2.249 .016 Vascular invasion 1.726 0.649–2.526 .098 EWSAT1 3.454 2.028–6.004 .000 2.516 1.187–4.301 .000 Abbreviations: AJCC, American Joint Committee on Cancer; CI, confidence interval; EWSAT1, Ewing sarcoma-associated transcript 1; HCC, hepatocellular carcinoma; HR, hazard ratio. TABLE 2. The results of univariate and multivariate analyses of disease-free patient survival of HCC (Bold values indicate a significant difference) Variable Univariate analysis Multivariate analysis HR 95% CI p value HR 95% CI p value Age 1.143 0.649–2.074 .572 Gender 1.061 0.517–2.925 .289 AJCC stage 3.861 1.947–6.031 .007 3.601 1.592–5.416 .000 HBsAg 1.362 0.674–1.890 .571 Tumor size 3.085 1.893–5.862 .002 3.019 1.681–5.318 .000 Differentiation 1.417 1.055–3.144 .016 1.627 1.152–2.963 .013 Vascular invasion 1.772 0.573–2.738 .427 EWSAT1 2.267 1.338–4.002 .001 2.163 1.410–3.711 .002 Abbreviations: AJCC, American Joint Committee on Cancer; CI, confidence interval; EWSAT1, Ewing sarcoma-associated transcript 1; HCC, hepatocellular carcinoma; HR, hazard ratio. EWSAT1 promotes the in vitro proliferation of HCC cells A nuclear fraction assay was conducted to identify the cellular distribution of EWSAT1. The results suggested that EWSAT1 was predominately situated in the HCC cell cytoplasm (Figure 2A). The influence of EWSAT1 on the proliferation of HCC cells was investigated using lentiviral vectors to stably silence endogenous EWSAT1 expression in HepG2 and Huh7 cells, whereas Hep3B cells were selected for EWSAT1 overexpression (Figure 2B,C). The CCK-8 assay showed that depletion of EWSAT1 in both cell types significantly compromised cell viability (Figure 2D). Moreover, the colony formation assay demonstrated that depletion of EWSAT1 resulted in a considerably reduced colony number relative to that of the control cells (Figure 2E). Conversely, EWSAT1 overexpression significantly facilitated the growth of HCC cells (Figure 2D,E). Similarly, the results of the EdU incorporation assay showed that depletion and overexpression of EWSAT1 markedly inhibited and promoted the percentage of EdU-positive cells, respectively (Figure 2F). Accordingly, it can be concluded that EWSAT1 promotes the in vitro proliferation of HCC cells. FIGURE 2Open in figure viewer EWSAT1 increases the in vitro proliferation of HCC cells. (A) Fractionation of HepG2 and Hep3B cell lysates demonstrated cytoplasmic expression of Ewing sarcoma-associated transcript 1 (EWSAT1). U6 RNA acted as the internal control for the expression of nuclear genes, and the internal control used for cytoplasmic gene expression was glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (B) EWSAT1 levels of expression in normal liver epithelial cell L02 and 7 hepatocellular carcinoma (HCC) cell lines were identified through quantitative real-time polymerase chain reaction (qRT-PCR) analysis. (C) The effects of EWSAT1 overexpression and knockdown were validated by qRT-PCR analysis. The internal control used was GAPDH. (D) Reduced EWSAT1 lowered HCC cell proliferation, whereas ectopic expression of EWSAT1 increased HCC cell proliferation as shown by the CCK-8 assay. (E) The effect of modified EWSAT1 expression on the colony formation of HCC cells. (F) Representative micrographs and quantification of EdU labeling indicat