HIF-1α-regulated Galectin-3 affords the vasculogenic mimicry and poor prognosis of hepatocellular carcinoma

HIF-1α-regulated Galectin-3 affords the vasculogenic mimicry and poor prognosis of hepatocellular carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article HIF-1α-regulated Galectin-3 affords the vasculogenic mimicry and poor prognosis of hepatocellular carcinoma Fei Kong, Meishan Jin, Yu Fu, Zhuoshuai Liang, Jikang Shi, Jing Jiang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5359597/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In present study, we explore the role of HIF-1α-regulated Galectin-3 in VM and poor prognosis of HCC as well as the potential regulatory mechanism. Immunohistochemistry method was used to evaluate HIF-1α and CD31/PAS expression in HCC patients. Vitro experiments were conducted to reveal the molecular mechanisms that regulate Galectin-3 expression under hypoxia. Moreover, the effects of Galectin-3 on the biological function of HCC cells were investigated using MTS kit, wound-healing assay, cell transwell assay, flow cytometry assay and three-dimensional culture under hypoxia. HIF-1α expressions were significantly higher in HCC tissues than those in adjacent hepatic tissues in cirrhosis-free patients with HCC ( P = 0.023). The patients with positive-HIF-1α expression had worse overall survival ( P = 0.002). HIF-1α was positively correlated with Galectin-3 in HCC tumor tissues ( P < 0.05). Higher expression of Galectin-3 linked to poor overall survival ( P = 0.002). HIF-1α and Galectin-3 were correlated with VM of HCC ( P < 0.05). Hypoxia promotes proliferation, migration, invasion and VM formation of HCC cells, and suppresses to apoptosis ( P < 0.05). Galectin-3 is the target genes of HIF-1α transcription factor. Silencing Galectin-3 abolished effect of hypoxia microenvironment on the function of HCC cells. In conclusion, HIF-1α-regulated Galectin-3 contributes to the vasculogenic mimicry and poor prognosis of HCC. Health sciences/Medical research Health sciences/Oncology Hepatocellular carcinoma vasculogenic mimicry HIF-1α Galectin-3 prognosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Hepatocellular carcinoma (HCC) which accounts for 75–85% of primary liver cancer remains the sixth prevalent cancer worldwide and endangers millions of lives [ 1 ]. Globally, about half of newly-diagnosed patients with HCC occur in China[ 2 ]. Despite advances in HCC treatment, the outcome and prognosis of HCC are still depressing: about 70% subjects develop recurrence within five years and the five-year survival rate is only 37% after resection [ 3 , 4 ]. HCC is a one of the highly vascularized tumors, and its microcirculation patterns are complex[ 5 ]. Vasculogenic mimicry (VM) characterized by aggressive tumor cells to form vascular-like structures is a new blood supply system and independent of endothelial cells[ 6 ]. VM is essential for HCC malignancy, correlating with high tumor grade, invasion and metastasis, and short survival in patients with HCC[ 7 , 8 ]. The effect of currently available anti-angiogenic drugs is composed of inhibiting endothelial cell proliferation, inducing endothelial cell apoptosis, and decreasing vascular density. However, vasculogenic networks of VM cannot be restrained by these drugs [ 9 – 11 ]. Of note, anti-angiogenic therapy increases hypoxia, thereby promoting VM formation [ 9 – 11 ]. Therefore, VM is a conundrum of anti-HCC therapy. Hypoxia is one of the most common hallmarks of malignant tumors, conferring uncontrolled growth and proliferation of tumor cells [ 12 ]. As one of the major mediators of hypoxic response, hypoxia-inducible factor-1α (HIF-1α) has been shown to activate hypoxia-responsive genes, which contributes to tumorigenesis and cancer progression[ 13 ]. HIF-1α overexpression correlates with VM and unfavorable prognosis in HCC[ 14 ]. However, HIF-1α-positive expression was not an adverse prognosis factor for HCC patients without cirrhosis in the study of Wang et al . [ 15 ]. Because implications of HIF-1α in HCC leave much to be investigated, we investigated the connectivity between HIF-1α and HCC and crystalized a potential mechanism of HIF-1α involved in VM in this paper. Galectin-3 is a multifunctional member of galectin family. In a previous study, we reported that Galectin-3 was significantly higher expressed in HCC tumor tissues and related to worse overall survival [ 16 ]. Higher Galectin-3 can promote VM in esophageal cancer cells by regulating the EphA2 expression[ 17 ]. However, limited reports focus on the role of Galectin-3 in VM in HCC. Hypoxia is critical for Galectin-3 expression in renal carcinoma cells through HIF-1α[ 18 ]. However, the role of Galectin-3 in HCC under hypoxia microenvironment remain largely unknow, especially regarding molecular regulation mechanisms. In present study, we explore the role of HIF-1α-regulated Galectin-3 in VM and poor prognosis of HCC as well as the potential regulatory mechanism. Methods Study population We enrolled 202 patients (164 men and 38 women) with HCC who underwent surgery at the First Hospital of Jilin University between October 2008 and March 2014. Patients age range from 23 to 83 years, with a mean age of 54.6 years. Of these, 147 patients were from our previous Galectin-3 study population. No patients received chemotherapy or radiotherapy before the surgery. Two independent pathologists confirmed the diagnosis of HCC and evaluated immunohistochemical findings. Paired adjacent normal samples were collected from 140 patients for comparison. All the 202 subjects underwent follow-up by telephone at three months, six months, and every year after the surgery until death or the last scheduled follow-up. The patients who were lost to follow-up at the first time of telephone interview, or died of compliments of the surgery in the perioperative period were excluded from survival analysis. The duration from the date of surgical operation to that of death or that of the last successful interview was defined as survival time. This study protocol was approved by the Ethics Committee of the First Hospital of Jilin University (18YY228-001). Each patient provided a written informed consent. Immunohistochemistry analysis Immunohistochemical staining was done to validate the expression of HIF-1α and CD31 in HCC tumor tissues. After routine dewaxing and hydrating, the tissue sections (thickness, 4 µm) were washed with PBS and then boiled in the EDTA solution for 2.5min in a pressure cooker for epitope retrieval. After blocking endogenous peroxidase activity with endogenous peroxidase blocking buffer (MXB, China), the tissue slides were incubated with anti-human HIF-1α monoclonal antibody (ZA-0552, Zhongshan jinqiao, China) at 4℃ overnight and CD31 monoclonal antibody (MXB, China) at 37℃ for 2 hours. Next, the slides were further treated with horseradish peroxidase-labeled secondary antibody for 30 min at room temperature (MXB, China), further visualized using 3, 3-Diaminobenzidine (DAB), and then counterstained with hematoxylin. Two independent pathologists who were blinded to clinical data and outcome evaluated the stained slides. The expression of HIF-1α were assessed on the basis of H-sore combining staining intensity and percentages of cells stained [ 16 , 19 ]. H-sore > 0 is considered as positive staining and H-sore = 0 is considered as a complete negative staining. CD31/PAS double staining CD31 immunohistochemistry-Periodic Acid-Schiff (PAS) double staining was used to determine the endothelial vessels and vasculogenic mimicry. PAS staining was performed by Glycogen Periodic Acid Schiff stain kit (G1281, Solarbio Life Sciences, China). After the CD31 immunohistochemical staining was performed by the sections as described above. The slides were treated with oxidizer solution for 6.5 min and rinsed with distilled water for 10 min. Then, the slides were submerged in Schiff solution for 15 min in a dark chamber. After washing the slides under running water for 10 min, all of the sections were counterstained with hematoxylin, dehydrated, and mounted. VM channels surrounded by HCC cells were positive for PAS staining, but negative for CD31. Bioinformatic analyses The mRNA expression dataset (RNA-seq data) and related clinical characteristics of HCC patients were downloaded from The Cancer Genome Atlas (TCGA) ( https://cancergenome.nih.gov/ ) database. Cell culture The human HCC cell lines SMMC7721 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Biological Industries, Israel) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) (Biological Industries, Israel) at 37℃ in an incubator supplied with 5% CO 2 . For the hypoxia experiments, the cells were incubated at 37℃ in a tri-gas incubator containing 1% O 2 , 5% CO 2 and 94% N 2 . siRNA transfection SMMC7721 cells were seeded into a 6-well plate with 1.2x10 5 cells/well and transfected with siGalectin-3 and negative control (final concentration 100 nmol/l) respectively, using lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. The culture media were replaced by fresh DMEM media containing 10% FBS and 1% P/S 6 hours after the transfection. The cells were harvested for quantitative real-time PCR analysis after the further culture 24 hours or harvested for Western blot after 48 h. Real-time quantitative PCR analysis Total RNA was extracted from cells using EasyPure RNA Kit (TianGen Biotech, China) and the cDNA was prepared by FastKing gDNA Dispelling RT SuperMix (TianGen Biotech, China) according to the manufacturer’s instructions. β-action was used as endogenous control. The primer sequences used in qPCR was listed in Table 1 . The Real-time qPCR was performed on the basis of Bio-Rad CFX96 Real-Time System using SuperReal PreMix Plus kit (TianGen Biotech, China). The relative expression of the mRNA was calculated using the 2 − ΔΔCt method. Table 1 Sequences of mRNA primers Primers Sequences (5’to 3’) β-action Forward CGCGAGAAGATGACCCAGAT Reverse TCACCGGAGTCCATCACGAT Galectin-3 Forward CAGACAATTTTTCGCTCCATGA Reverse TAGGCCCCAGGATAGGAAG HIF-1α Forward GAACGTCGAAAAGAAAAGTCTC Reverse CCTTATCAAGATGCGAACTCACA Western blot analysis The total protein was extracted using total protein extraction kit (BestBio Science, China) and quantified using BCA Protein Assay Kit (Beyotime Biotechnology, China). The HIF-1α and Galectin-3 protein were by 8% and 10% SDS PAGE gels respectively. The proteins in the gel were transferred to polyvinylidene fluoride membranes (PALL corporation, USA). Next, the membranes were blocked with 5% non-fat milk dissolved in PBS containing 1% Tween-20 (PBST) at room temperature for 1 hour. The membranes were incubated with primary antibodies HIF-1α (1:200, ab51608, Abcam, UK), Galectin-3 (1:1000, ab76245, Abcam, UK), β-action (1:10000, A5316, Sigma, USA) at 4℃ overnight and then incubated with donkey anti-rabbit IgG antibody (1:10000, GE Healthcare, UK) or sheep anti-mouse IgG antibody (1:10000, GE Healthcare, UK) at room temperature for 1 h. The chemiluminescent signal was imaged using a Tanon 5200 Multi Automatic Chemiluminescence/Fluorescence Image Analysis System (China). Cell proliferation analysis Cell proliferation was measured using MTS Kit (Saint-Bio, China). SMMC7721 was seeded in a 96-well plate with 3000 cells/well to investigate the effect of Galectin-3-knockdown under hypoxia. Briefly, 10µl of MTS reagent was added to each well and incubated for 3 hours to test cell proliferation. Optical density (OD) value was measured at 490nm using a multilabel plate reader (Thermo scientific, USA). Cell apoptosis analysis FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, USA) was used to detected cell apoptosis. Cells were harvested and washed twice with phosphate-buffered saline (PBS). Then the cells were stained with PI and FITC for 15 min in the dark at room temperature. Cells apoptosis was analyzed using a FACSCalibur (BD Biosciences, USA) within 1 hour after adding 400µl 1 × Binding Buffer. Cell migration analysis The wound-healing assay was performed to assess the cell migration in response to the mechanical scratch. When cells reached 90% confluence, a straight scratch was made in the center of each well using a micropipette tip. Atter the suspended cells were washed with PBS, the well was replenished with serum-free medium. The cells were observed and images were captured by a microscope at 0, 24, and 48h. The cell migration rate (MR) was calculated using the following equation: MR= (d 0 -d 1 )/d 0 , d 0 means the length of the wound at time 0h, d 1 is the length at time 48h. Cell invasion analysis HCC cells (1x10 5 cells) in 750µl of DMEM without FBS were seeded in the upper chamber of 24-well Growth Factor Reduced Corning Matrigel Invasion chamber (Corning, USA). The lower chamber was filled with medium containing 10% FBS. After 24h, the non-invading cells were removed from the upper surface of the membrane. The cells that invaded to the bottom surface of the membrane were fixed, stained with crystal violet, photographed by a microscope, and counted. Matrigel tube formation assay 3D culture system was used to evaluate VM formation in vitro. A total of 200µl of cold Corning Matrigel Matrix (10 mg/ml) was coated in each well of a 24-well plate and cultured at 37 ℃ for 1 hour. The cells were seeded in the 24-well plate and cultured for 6 hours. The formation of capillary-like structures was observed and photographed with a fluorescence microscope (Leica, Germany). Angio Tool Software was used to calculate percentage area of tubes per area. Chip-qPCR Enzymatic Shearing Kit (Active Motif, USA) was used to prepared sheared chromatin by enzymatic shearing, and ChIP-IT Express Enzymatic Magnetic Chromatin Immunoprecipitation Kit (Active Motif, USA) was used to performed chromatin immunoprecipitation in accordance with the manufacturer’s instructions. Purification of DNA from Chromatin Immunoprecipitation sample was performed using Chromatin IP DNA Purification Kit (Active Motif, USA). Real-time PCR was performed on Bio-Rad CFX96 Real-Time System. The following primers were used for RT-PCR: primer 1: ATCACTTCCCAAGGCCTCCAC (forward), TATCCCGCCAAAGTTCATATGTT (reverse); primer 2: GCCCCTATGACCTAATC ACTTCC (forward), CTCATTTTTACTGTCAGGGTGCTAT (reverse). Statistical analysis The H-score of HIF-1α expression was presented as (interquartile ranges) and compared using the Mann-Whitney test. Categorical variables were expressed as numbers and frequencies (%), and compared using the Pearson Chi-square test or Fisher exact test. The Wilcoxon matched-pairs signed-rank test was used when comparing the expression of HIF-1α in tumor tissue and adjacent non-tumor tissue. Optimal cut-off value of HIF-1α was calculated by time-dependent receiver operating characteristic (ROC) curves using the R language for survival analysis. The overall survival rate was estimated using Kaplan–Meier method and compared using the log-rank test. The Cox proportional hazards model was performed to assess factors potentially associated with prognosis of patients with HCC. The unpaired two-tailed t-test was used to compare differences between two parallel treatment groups in cell experiments. Variables with P values below or equal to 0.10 in univariate analysis were included in the multivariate model. Hazard ratio (HR) and corresponding 95% confidence intervals (95% CI) were used to determine the strength of statistical associations. A two-sided P -value below or equal to 0.05 was considered significant. Statistical analyses were performed using SPSS software package 18.0 (SPSS Inc. USA). Results HIF-1α-positive expressions are implicated in poor overall survival of HCC We evaluated the correlation of HIF-1α expression with clinicopathological characters and prognosis among a cohort of 202 HCC patients. We found that HIF-1α was stained mainly in the cytoplasm or cytoplasm and nucleus (Fig. 1 A); moreover, HIF-1α expressions were higher in tumor tissues than those in adjacent hepatic tissues in the 140 paired samples, but no significant difference was observed ( P = 0.097) (Table 2 ). Of note, HIF-1α expressions were significantly higher in HCC tissues than those in adjacent hepatic tissues in the 53 cirrhosis-free patients with HCC ( P = 0.023) (Table 2 ). HIF-1α expressions were significantly higher in male patients ( P = 0.037) and the patients with HBcAb-positive ( P = 0.040). However, HIF-1α expressions were not significantly associated with tumor histological differentiation, tumor size, number of tumor and lymph-vascular invasion (Table 3 ). ROC curve revealed the cut-off value of HIF-1α in predicting the 3-year survival was H-score = 0. Kaplan–Meier survival analysis indicated that the HIF-1α-positive expression group have poor overall survival compared with HIF-1α-negative expression groups (Log-Rank P = 0.002) (Fig. 1 B). Furthermore, multivariate analysis showed that the prognosis of patients with HCC was associated with number of tumor (HR = 1.83, 95% CI [1.24–2.72]), tumor size ≥ 5 cm (HR = 2.23, 95% CI [1.48–3.37]), and HIF-1α-positive expression (HR = 2.16, 95% CI [1.42–3.30]) significantly (Table 4 ). Table 2 Expressions of HIF-1α in tumor tissue and adjacent non-tumor tissue Group N H-score Median (quartile) P Overall Tumor tissue 140 80(0-150) 0.097 Adjacent non-tumor tissue 140 55(0-120) Cirrhosis Tumor tissue 87 80(0-140) 0.785 Adjacent non-tumor tissue 87 60(0-140) Cirrhosis-free Tumor tissue 53 80(0-160) 0.023 Adjacent non-tumor tissue 53 30(0-100) Table 3 Correlation between clinicopathological characters and expressions of HIF-1α Character N Expression of HIF-1α P H-score Median (quartile) P Negative (%) Positive (%) Gender Male 164 51 (31.1) 113 (68.9) 0.037 80(0-140) 0.077 Female 38 19 (50.0) 19 (50.0) 10(0-120) Age ≤ 55ys 103 35 (34.0) 68 (66.0) 0.883 80(0-150) 0.408 > 55ys 99 35 (35.4) 64 (64.6) 70(0-120) HBV HBsAg positive 136 42 (30.9) 94 (69.1) 0.117 80(0-147.5) 0.118 HBsAg negative 66 28 (42.4) 38 (57.6) 50(0-100) HBeAg positive 44 11 (25.0) 33 (75.0) 0.152 80(5-150) 0.215 HBeAg negative 154 58 (37.7) 96 (62.3) 65(0-120) HBcAb positive 178 57 (32.0) 121 (68.0) 0.040 80(0-140) 0.140 HBcAb negative 24 13 (54.2) 11 (45.8) 0(0-115) HCV Positive 36 14(38.9) 22 (61.1) 0.567 85(0-155) 0.818 Negative 166 56(33.7) 110 (66.3) 70(0-140) AFP <20 ng/ml 97 29(29.9) 68 (70.1) 0.186 90(0-140) 0.250 ≥ 20 ng/ml 105 41 (39.0) 64 (61.0) 60(0-120) Number of tumor single 149 55 (36.9) 94(63.1) 0.314 70(0-120) 0.116 Multiple 53 15 (28.3) 38 (71.7) 80(0-180) Tumor size ≤ 5cm 96 31 (32.3) 65 (67.7) 0.555 90(0-147.5) 0.353 > 5cm 106 39 (36.8) 67 (63.2) 65(0-125) Differentiation Poor 61 20 (32.8) 41 (67.2) 0.750 70(0-120) 0.757 Moderate/well 141 50 (35.5) 91 (64.5) 80(0-145) Lymph-vascular invasion No 107 39 (36.4) 68 (63.6) 0.657 60(0-150) 0.807 Yes 95 31 (32.6) 64 (67.4) 80(0-120) Cirrhosis No 82 34 (41.5) 48 (58.5) 0.100 60(0-150) 0.359 Yes 120 36 (30.0) 84 (70.0) 80(0-140) AJCC clinical stage I 85 32(37.6) 53 (62.4) 0.494 60(0-120) 0.402 II 86 30 (34.9) 56 (65.1) 75(0-125) III 31 8 (25.8) 23(74.2) 90(0-160) VM Negative 131 53 (75.7) 17 (24.3) 0.021 40(0-100) 0.001 Positive 71 78 (59.1) 54 (40.9) 90(20–160) Table 4 Univariate and multivariate analysis of factors associated with prognosis in HCC Character n Univariate analysis Multivariate analysis* HR(95% CI) P HR(95% CI) P HBsAg Negative 66 Reference Reference Positive 136 1.44 (0.97–2.14) 0.072 1.45 (0.97–2.17) 0.073 AFP < 20ng/ml 124 Reference Reference ≥ 20ng/ml 123 1.90 (1.31–2.76) 0.001 1.81 (1.23–2.68) 0.003 Number of tumor Single 183 Reference Reference Multiple 64 2.18 (1.49–3.19) 5cm 110 2.38(1.63–3.47) 0.001 2.23(1.48–3.37) < 0.001 Differentiation Poor 75 Reference Reference Moderate/ well 172 0.60(0.41–0.87) 0.008 0.79(0.52–1.19) 0.255 Lymph-vascular invasion No 138 Reference Reference Yes 109 2.07(1.43–2.99) < 0.001 1.18(0.77–1.82) 0.442 HIF-1α Negative 201 Reference Reference Positive 46 1.89(1.25–2.86) 0.002 2.16(1.42–3.30) < 0.001 *Age, sex, HBsAg, differentiation and lymph-vascular invasion were adjusted in multivariate analysis. Galectin-3 engages in the link between HIF-1α and HCC The expression of Galectin-3 was determined in our previous study. Galectin-3 was significantly higher expressed in HCC tumor tissues and related to worse overall survival [ 16 ]. To understand the clinical relevance of HIF-1α and Galectin-3, we evaluate correlation of HIF-1α and Galectin-3 expression among a cohort of 147 HCC patients. The results showed that HIF-1α expression was positively correlated with Galectin-3 in the HCC tumor tissues (R = 0.167, P = 0.043). To further identify the relationship between HIF-1α and Galectin-3, the mRNA-seq data of 424 HCC patients were recruited from the TCGA database, HIF-1α were found to be positively associated with LGALS3 coding Galectin-3 (R = 0.394, P < 0.001) (Fig. 1 D). To further confirm the relation from HIF-1α, via Galectin-3, to HCC, we investigated expressions of Galectin-3 on survival of patients with HCC on the basis of TCGA database. As expected, compared with low expression of LGALS3, high expression of LGALS3 linked to poor overall survival (Fig. 1 C). HIF-1α and Galectin-3 are involved in VM of HCC Tube cavities derived from HCC cells were positive for PAS staining, but negative for CD31 staining, indicating that VM, rather than endothelium-dependency, is characteristic of these tube cavities (Fig. 1 E, green arrow). The channels positive for CD31 and PAS were considered as endothelium-dependent vessels (Fig. 1 E, red arrow). VM was observed in 35.1% (71/202) patients with HCC. HIF-1α was implicated in VM formation, higher HIF-1α expression was observed in the patients with VM ( P = 0.021) (Table 3 ). Furthermore, the relationship between expression of Galectin-3 and tumor VM formation was explored among 147 HCC patients. The results showed that the presence of VM related to Galectin-3, higher Galectin-3 expression was observed in VM-positive patients ( P = 0.026). Effect of hypoxia microenvironment on the function of HCC cells Hypoxia microenvironment has been characterized in HCC tumor tissues. After SMMC7721 cells were cultured under hypoxia for 24 hours, we found that hypoxia triggered proliferation of SMMC7721 cells significantly ( P < 0.001). As expected, hypoxia decreased apoptosis of SMMC7721 cells ( P = 0.006). Cell scratch assay showed that hypoxia accelerated cell migration remarkably ( P = 0.166). Similarly, cell transwell assay also revealed that hypoxia induced invasion of SMMC7721 cells significantly ( P = 0.001). Three-dimensional cultures of SMMC7721 cells showed that hypoxia promoted the formation of tube-like strictures area significantly ( P = 0.003) (Fig. 2 ). HIF-1α modulates Galectin-3 expression in HCC cells under hypoxia We investigated the effects of hypoxia on expressions of HIF-1α and Galectin-3 in SMMC7721 cells, showing that 48 hours of hypoxia enhanced the transcription and translation of HIF-1α and Galectin-3 significantly ( P < 0.05) (Fig. 3 A, Fig. 3 C). After HIF-1α knockdown under hypoxia, we found the expression of Galectin-3 decreased significantly ( P < 0.05) (Fig. 3 B, Fig. 3 C). To further confirm the relation between Galectin-3 and HIF-1α, we performed ChIP-qPCR assay, corroborating that LGALS3 coding Galectin-3 was a target gene of HIF-1α (Fig. 3 D). Galectin-3 is required for the function of HCC cells under hypoxia microenvironment Hypoxic microenvironment increased Galectin-3 expression in HCC cells. To explore connectivity of Galectin-3 with the effect of hypoxia microenvironment on the function of HCC cells, we utilized small interfering RNA (siRNA) to silence Galectin-3, parsing that the silencing reduced proliferation ( P < 0.001), increased apoptosis ( P = 0.008), and decreased migration and invasion of SMMC7721 cells (both P < 0.001). Indeed, the formation of tube-like strictures area was reduced by silencing Galectin-3 under hypoxia significantly ( P < 0.001). As shown in Fig. 4 . Discussion In this paper, we reveal that HIF-1α-regulated Galectin-3 is required for the pathogenesis of HCC, establishing connection from HIF-1α, via Galectin-3, to the VM and poor prognosis of HCC. Hypoxia and the activation of hypoxia inducible factors are essential for the pathogenesis of HCC. Although the expression of HIF-1α in HCC tissues was significantly higher than that in the paracancerous tissues [ 20 ], we showed that there was no difference of HIF-1α expression between HCC tumor tissues and adjacent non-tumor tissues among the patients with cirrhosis. While we found that higher positive frequency of HIF-1α in HCC tissues than that in adjacent non-tumor tissues in cirrhosis-free patients with HCC. Similar to the discovery of Wang et al.[ 15 ]. Hypoxia causes the accumulation and activation of HIF-1α[ 21 ]. HIF-1α, as a transcription factor, regulates expressions of target genes to facilitate cellular adaptation to tumor hypoxia by binding to hypoxic response elements[ 15 , 22 ]. Hypoxia in HCC is dynamic[ 21 ]. HIF-1α expression appears to depends on intensity of hypoxia. The results of present study found that HIF-1α is mainly expressed in cirrhosis tissues, rather than in normal liver tissues. Similar findings were also reported by Li et al . in serum levels[ 23 ]. During the progression of chronic liver diseases, due to persisting inflammatory response, parenchymal injury, liver architecture changes, and vascular reorganization, can significantly result in tissue hypoxia and a hypoxic cellular response[ 24 ]. Hypoxic represent a very common and major feature of fibrotic and cirrhotic liver [ 25 ]. The connection between HIF-1α expressions and HCC risks we found suggested that HIF-1α may serve as a diagnostic marker for HCC in non-cirrhotic patients, but cannot be used as an effective marker to distinguish liver cirrhosis from hepatocellular carcinoma. VM gives rise to the poor prognosis in HCC via forming vasculogenic networks[ 14 ]. Our present study showed that HIF-1α were implicated in VM formation and patients with positive HIF-1α expression had poor survival rate. VM formation correlates with the expression of vimentin or α-SMA in HCC tissues significantly[ 26 ]. HIF-1α affords EMT and VM formation via up-regulating LOXL2 in hypoxic microenvironment of HCC[ 27 ]. In this paper, the positive correlation between HIF-1α and Galectin-3 expression in HCC tissues triggered us to investigate the connection between HIF-1α and Galectin-3 expression. We, further, unveiled that LGALS3 coding Galectin-3 was a target gene of HIF-1α in HCC. Similar findings were also reported in renal cell carcinoma and thyroid cancer[ 18 , 28 ]. HCC is one of the most hypoxic malignancies[ 21 ]. In a previous study, we concluded that Galectin-3 was highly expressed in tumor tissue [ 16 ]. These data provided molecular mechanisms underlying the accumulation of Galectin-3 in HCC. Conceivably, Galectin-3 has been found to regulate VM formation in esophageal cancer cell [ 17 ]. In this paper, we revealed that increased Galectin-3 expressions were significantly associated with VM formation. In our previous study, we found that increased Galectin-3 expressions in tumor tissue correlate with poor prognosis in HCC[ 16 ]. Further vitro experiments showed that Galectin-3 was required for the function of HCC cells under hypoxia microenvironment. Therefore, Galectin-3 is critical for linking HIF-1α to VM formation and poor prognosis in HCC. Conclusions In conclusion, HIF-1α-regulated Galectin-3 contributes to the vasculogenic mimicry and poor prognosis of HCC. This study highlights the potential therapeutic value of targeting Galectin-3 for suppression of HCC metastasis and progression. Declarations Authors’ contributions Fei Kong and Mei-shan Jin contributed equally to this work; Yi Cheng and Yawen Liu designed and conducted most of the study; Fei Kong wrote the first draft of the manuscript; Meishan Jin performed immunohistochemistry testing and evaluation; Yu Fu and Jing Jiang collected the epidemiological and clinical data; Fei Kong and Jikang Shi performed the cells experiments. Fei Kong and zhuoshuai Liang performed the statistical analyses; All the authors reviewed the manuscript. Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Funding The study was sponsored by the National Natural Science Foundation of China (grants No. 82304216), the Natural Science Foundation of Jilin Province (YDZJ202401197ZYTS), the Natural Science Foundation of First Hospital of Jilin University (JDYY72016012). Conflict of Interest The authors declare no conflicts of interest. Ethics approval and consent to participate This study protocol was approved by the Ethics Committee of the National Natural Science Foundation of Jilin Province (YDZJ202401197ZYTS). Each patient provided a written informed consent. We confirm that all methods were performed in accordance with the Declaration of Helsinki. Consent for publication Not applicable. References Sung, H. et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 71 (3), 209–249 (2021). Chen, M. et al. Design and rationale of the HCC BRIDGE study in China: a longitudinal, multicenter cohort trial in hepatocellular carcinoma. BMC Gastroenterol. 11 , 53 (2011). Tabrizian, P. et al. Recurrence of hepatocellular cancer after resection: patterns, treatments, and prognosis. Ann. Surg. 261 (5), 947–955 (2015). Yang, X. D. et al. 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Galectin-3 not Galectin-9 as a candidate prognosis marker for hepatocellular carcinoma. PeerJ . 8 , e9949 (2020). Zhang, J. et al. Effect of galectin-3 on vasculogenic mimicry in esophageal cancer cells. Oncol. Lett. 15 (4), 4907–4911 (2018). Gu, X. et al. Hypoxia contributes to galectin-3 expression in renal carcinoma cells. Eur. J. Pharmacol. 890 , 173637 (2021). McCarty, K. S. Jr. et al. Estrogen receptor analyses. Correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies. Arch. Pathol. Lab. Med. 109 (8), 716–721 (1985). Liang, H. et al. Expression of caspase-3 and hypoxia inducible factor 1alpha in hepatocellular carcinoma complicated by hemorrhage and necrosis. World J. Clin. Cases . 9 (23), 6725–6733 (2021). Bao, M. H. & Wong, C. C. Hypoxia, Metabolic Reprogramming, and Drug Resistance in Liver Cancer . Cells , 10 (7). (2021). Le, Q. T. & Courter, D. Clinical biomarkers for hypoxia targeting. Cancer Metastasis Rev. 27 (3), 351–362 (2008). Li, N. et al. [Clinical significance of circulating hypoxia inducible factor-1alpha and hemeoxygenase-1 in patients with liver cirrhosis]. Zhonghua Yi Xue Za Zhi . 94 (48), 3842–3844 (2014). Foglia, B. et al. Hypoxia, Hypoxia-Inducible Factors and Liver Fibrosis . Cells , 10 (7). (2021). Cannito, S. et al. Hypoxia, hypoxia-inducible factors and fibrogenesis in chronic liver diseases. Histol. Histopathol . 29 (1), 33–44 (2014). She, Q. et al. The effect of hepatocellular carcinoma-associated fibroblasts on hepatoma vasculogenic mimicry. Am. J. Cancer Res. 10 (12), 4198–4210 (2020). Wang, M. et al. HIF-1alpha promoted vasculogenic mimicry formation in hepatocellular carcinoma through LOXL2 up-regulation in hypoxic tumor microenvironment. J. Exp. Clin. Cancer Res. 36 (1), 60 (2017). Zheng, J. et al. Galectin-3 induced by hypoxia promotes cell migration in thyroid cancer cells. Oncotarget . 8 (60), 101475–101488 (2017). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5359597","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":387900100,"identity":"be6b5005-2176-4eff-b419-33492c5ed853","order_by":0,"name":"Fei Kong","email":"","orcid":"","institution":"the First Hospital of Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Kong","suffix":""},{"id":387900102,"identity":"184e4fde-ef0f-4300-83a8-b6120b225315","order_by":1,"name":"Meishan Jin","email":"","orcid":"","institution":"the First Hospital of Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Meishan","middleName":"","lastName":"Jin","suffix":""},{"id":387900103,"identity":"f865ad9d-9022-4908-af93-5687a2f49819","order_by":2,"name":"Yu Fu","email":"","orcid":"","institution":"the First Hospital of Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Fu","suffix":""},{"id":387900106,"identity":"85057923-2515-4df3-9ca3-bf7a8e72ae73","order_by":3,"name":"Zhuoshuai Liang","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Zhuoshuai","middleName":"","lastName":"Liang","suffix":""},{"id":387900107,"identity":"6f0403a6-97f1-4e42-a78f-811eb6e7e5d8","order_by":4,"name":"Jikang Shi","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Jikang","middleName":"","lastName":"Shi","suffix":""},{"id":387900108,"identity":"2507810c-40d7-48d4-a5c0-bc02839a2dd3","order_by":5,"name":"Jing Jiang","email":"","orcid":"","institution":"the First Hospital of Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Jiang","suffix":""},{"id":387900110,"identity":"ba54b430-1139-4213-8c0f-b35392a9d25e","order_by":6,"name":"Yawen Liu","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Yawen","middleName":"","lastName":"Liu","suffix":""},{"id":387900112,"identity":"6e53aba8-705b-42b8-948e-1280a989bfef","order_by":7,"name":"Yi Cheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYDACCQgpx9gOotlI0GLM2EyiFobEBmZitfDPbj728GubRXpzM48Bw4eyw0CRBgKW3DmWbizbJpHbCNTCOOPcYaDIAfxaDCRyzKQloVqYedsOA0USCGnJ/wbSks4I0vKXOC05bJIf2yQSwFoYidEicSPNTJrhnIRhYzNbwcGec+k8EjcIaOGfkfxM8kdZnbxhe/PGBz/KrOX4ZxDQAgLMvMDoMGxgYDgA5PAQVg8EjD/+MDDIE6V0FIyCUTAKRiQAACaCO8xoH7HUAAAAAElFTkSuQmCC","orcid":"","institution":"the First Hospital of Jilin University","correspondingAuthor":true,"prefix":"","firstName":"Yi","middleName":"","lastName":"Cheng","suffix":""}],"badges":[],"createdAt":"2024-10-30 08:23:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5359597/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5359597/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71688514,"identity":"bd063763-aa9d-4bb5-9b40-5c869f6f5aed","added_by":"auto","created_at":"2024-12-17 17:41:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1006152,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of HIF-1α and VM in liver tissues of tumor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Expression of HIF-1α in liver tissues of tumor (SP, ×200); (B) Association of HIF-1α expression with overall survival in HCC patients; (C) Association of \u003cem\u003eLGALS3 \u003c/em\u003eexpression with overall survival in HCC patients in TCGA database; (D) Correlation analysis of \u003cem\u003eHIF1A \u003c/em\u003eand \u003cem\u003eLGALS3 \u003c/em\u003ein HCC patients in TCGA database; (E) Expressions of VM in liver tissues of tumor (SP, ×200): VM Channels made of HCC cells were positive for PAS staining, but negative for CD31 (green arrow); Endothelium-dependent vessels (red arrow) exhibited CD31-positive on their luminal surface, and positive reaction to PAS in their wall.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5359597/v1/2233e3fb955956d0d67a21ff.png"},{"id":71688511,"identity":"84c2a781-55f3-4c1d-b3df-5974bc1612fd","added_by":"auto","created_at":"2024-12-17 17:41:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2207762,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of hypoxia microenvironment on the function of HCC cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Hypoxia promotes the proliferation of SMMC7721 cells; (B) Hypoxia inhibits apoptosis of HCC cells; (C) Hypoxia promotes migration of SMMC7721 cells; (D) Hypoxia promotes invasion of SMMC7721 cells; (E) Hypoxia promotes VM of SMMC7721. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5359597/v1/16c905b98a948388e6b733c0.png"},{"id":71688512,"identity":"a04f10e4-e306-41d8-b79f-368ba0a906d9","added_by":"auto","created_at":"2024-12-17 17:41:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":417386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHIF-1α modulates Galectin-3 expression in HCC cell lines under hypoxia microenvironment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Effect of hypoxia on \u003cem\u003eHIF-1α and Galectin-3 \u003c/em\u003eexpression; (B) mRNA levels of \u003cem\u003eHIF-1α\u003c/em\u003e and \u003cem\u003eGalectin-3\u003c/em\u003e in SMMC7721 cells treated with siHIF-1α\u003cem\u003e \u003c/em\u003eunder hypoxia; (C) Western blot analysis for the expression of HIF-1α and Galectin-3 in SMMC7721 cells treated with siHIF-1α and hypoxia; (D) ChIP-qPCR assay confirms the recruitment of HIF-1α to \u003cem\u003eGalectin-3.\u003c/em\u003e *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5359597/v1/be0d982b0d72f824caa75742.png"},{"id":71689018,"identity":"a5085e2d-548f-4b8e-b4d3-14e79b054d1f","added_by":"auto","created_at":"2024-12-17 17:49:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":864466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGalectin-3 is required for the function of HCC cells under hypoxia microenvironment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) \u003cem\u003eGalectin-3 \u003c/em\u003eknockdown inhibits proliferation of SMMC7721cells under hypoxia; (B) \u003cem\u003eGalectin-3\u003c/em\u003e knockdown promotes apoptosis of HCC cells under hypoxia; (C) \u003cem\u003eGalectin-3 \u003c/em\u003eknockdown inhibits the migration of SMMC7721 cells under hypoxia; (D) \u003cem\u003eGalectin-3 \u003c/em\u003eknockdown inhibits the invasion of SMMC7721 cells under hypoxia; (E) \u003cem\u003eGalectin-3 \u003c/em\u003eknockdown inhibits the VM of SMMC7721 cells under hypoxia. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5359597/v1/6163932aa0ab57f8cf26135e.png"},{"id":106413983,"identity":"2ebd4bbc-f265-47db-8fd6-8273ac0b32f7","added_by":"auto","created_at":"2026-04-08 10:06:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5672285,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5359597/v1/ee6d0897-0b5d-45a7-86ca-2d19c1a0ef5a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"HIF-1α-regulated Galectin-3 affords the vasculogenic mimicry and poor prognosis of hepatocellular carcinoma","fulltext":[{"header":"Background","content":"\u003cp\u003eHepatocellular carcinoma (HCC) which accounts for 75\u0026ndash;85% of primary liver cancer remains the sixth prevalent cancer worldwide and endangers millions of lives [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Globally, about half of newly-diagnosed patients with HCC occur in China[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite advances in HCC treatment, the outcome and prognosis of HCC are still depressing: about 70% subjects develop recurrence within five years and the five-year survival rate is only 37% after resection [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. HCC is a one of the highly vascularized tumors, and its microcirculation patterns are complex[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Vasculogenic mimicry (VM) characterized by aggressive tumor cells to form vascular-like structures is a new blood supply system and independent of endothelial cells[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. VM is essential for HCC malignancy, correlating with high tumor grade, invasion and metastasis, and short survival in patients with HCC[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The effect of currently available anti-angiogenic drugs is composed of inhibiting endothelial cell proliferation, inducing endothelial cell apoptosis, and decreasing vascular density. However, vasculogenic networks of VM cannot be restrained by these drugs [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Of note, anti-angiogenic therapy increases hypoxia, thereby promoting VM formation [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, VM is a conundrum of anti-HCC therapy.\u003c/p\u003e \u003cp\u003eHypoxia is one of the most common hallmarks of malignant tumors, conferring uncontrolled growth and proliferation of tumor cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. As one of the major mediators of hypoxic response, hypoxia-inducible factor-1α (HIF-1α) has been shown to activate hypoxia-responsive genes, which contributes to tumorigenesis and cancer progression[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. HIF-1α overexpression correlates with VM and unfavorable prognosis in HCC[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, HIF-1α-positive expression was not an adverse prognosis factor for HCC patients without cirrhosis in the study of Wang \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Because implications of HIF-1α in HCC leave much to be investigated, we investigated the connectivity between HIF-1α and HCC and crystalized a potential mechanism of HIF-1α involved in VM in this paper.\u003c/p\u003e \u003cp\u003eGalectin-3 is a multifunctional member of galectin family. In a previous study, we reported that Galectin-3 was significantly higher expressed in HCC tumor tissues and related to worse overall survival [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Higher Galectin-3 can promote VM in esophageal cancer cells by regulating the EphA2 expression[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, limited reports focus on the role of Galectin-3 in VM in HCC. Hypoxia is critical for Galectin-3 expression in renal carcinoma cells through HIF-1α[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, the role of Galectin-3 in HCC under hypoxia microenvironment remain largely unknow, especially regarding molecular regulation mechanisms.\u003c/p\u003e \u003cp\u003eIn present study, we explore the role of HIF-1α-regulated Galectin-3 in VM and poor prognosis of HCC as well as the potential regulatory mechanism.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy population\u003c/h2\u003e \u003cp\u003eWe enrolled 202 patients (164 men and 38 women) with HCC who underwent surgery at the First Hospital of Jilin University between October 2008 and March 2014. Patients age range from 23 to 83 years, with a mean age of 54.6 years. Of these, 147 patients were from our previous Galectin-3 study population. No patients received chemotherapy or radiotherapy before the surgery. Two independent pathologists confirmed the diagnosis of HCC and evaluated immunohistochemical findings. Paired adjacent normal samples were collected from 140 patients for comparison. All the 202 subjects underwent follow-up by telephone at three months, six months, and every year after the surgery until death or the last scheduled follow-up. The patients who were lost to follow-up at the first time of telephone interview, or died of compliments of the surgery in the perioperative period were excluded from survival analysis. The duration from the date of surgical operation to that of death or that of the last successful interview was defined as survival time. This study protocol was approved by the Ethics Committee of the First Hospital of Jilin University (18YY228-001). Each patient provided a written informed consent.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry analysis\u003c/h3\u003e\n\u003cp\u003eImmunohistochemical staining was done to validate the expression of HIF-1α and CD31 in HCC tumor tissues. After routine dewaxing and hydrating, the tissue sections (thickness, 4 \u0026micro;m) were washed with PBS and then boiled in the EDTA solution for 2.5min in a pressure cooker for epitope retrieval. After blocking endogenous peroxidase activity with endogenous peroxidase blocking buffer (MXB, China), the tissue slides were incubated with anti-human HIF-1α monoclonal antibody (ZA-0552, Zhongshan jinqiao, China) at 4℃ overnight and CD31 monoclonal antibody (MXB, China) at 37℃ for 2 hours. Next, the slides were further treated with horseradish peroxidase-labeled secondary antibody for 30 min at room temperature (MXB, China), further visualized using 3, 3-Diaminobenzidine (DAB), and then counterstained with hematoxylin. Two independent pathologists who were blinded to clinical data and outcome evaluated the stained slides. The expression of HIF-1α were assessed on the basis of H-sore combining staining intensity and percentages of cells stained [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. H-sore\u0026thinsp;\u0026gt;\u0026thinsp;0 is considered as positive staining and H-sore\u0026thinsp;=\u0026thinsp;0 is considered as a complete negative staining.\u003c/p\u003e\n\u003ch3\u003eCD31/PAS double staining\u003c/h3\u003e\n\u003cp\u003eCD31 immunohistochemistry-Periodic Acid-Schiff (PAS) double staining was used to determine the endothelial vessels and vasculogenic mimicry. PAS staining was performed by Glycogen Periodic Acid Schiff stain kit (G1281, Solarbio Life Sciences, China). After the CD31 immunohistochemical staining was performed by the sections as described above. The slides were treated with oxidizer solution for 6.5 min and rinsed with distilled water for 10 min. Then, the slides were submerged in Schiff solution for 15 min in a dark chamber. After washing the slides under running water for 10 min, all of the sections were counterstained with hematoxylin, dehydrated, and mounted. VM channels surrounded by HCC cells were positive for PAS staining, but negative for CD31.\u003c/p\u003e\n\u003ch3\u003eBioinformatic analyses\u003c/h3\u003e\n\u003cp\u003eThe mRNA expression dataset (RNA-seq data) and related clinical characteristics of HCC patients were downloaded from The Cancer Genome Atlas (TCGA) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cancergenome.nih.gov/\u003c/span\u003e\u003cspan address=\"https://cancergenome.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database.\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eThe human HCC cell lines SMMC7721 cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Biological Industries, Israel) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) (Biological Industries, Israel) at 37℃ in an incubator supplied with 5% CO\u003csub\u003e2\u003c/sub\u003e. For the hypoxia experiments, the cells were incubated at 37℃ in a tri-gas incubator containing 1% O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e and 94% N\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003esiRNA transfection\u003c/h2\u003e \u003cp\u003eSMMC7721 cells were seeded into a 6-well plate with 1.2x10\u003csup\u003e5\u003c/sup\u003e cells/well and transfected with siGalectin-3 and negative control (final concentration 100 nmol/l) respectively, using lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, USA) according to the manufacturer\u0026rsquo;s instructions. The culture media were replaced by fresh DMEM media containing 10% FBS and 1% P/S 6 hours after the transfection. The cells were harvested for quantitative real-time PCR analysis after the further culture 24 hours or harvested for Western blot after 48 h.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReal-time quantitative PCR analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from cells using EasyPure RNA Kit (TianGen Biotech, China) and the cDNA was prepared by FastKing gDNA Dispelling RT SuperMix (TianGen Biotech, China) according to the manufacturer\u0026rsquo;s instructions. β-action was used as endogenous control. The primer sequences used in qPCR was listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The Real-time qPCR was performed on the basis of Bio-Rad CFX96 Real-Time System using SuperReal PreMix Plus kit (TianGen Biotech, China). The relative expression of the mRNA was calculated using the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt method.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSequences of mRNA primers\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimers\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequences (5\u0026rsquo;to 3\u0026rsquo;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-action\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCGCGAGAAGATGACCCAGAT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTCACCGGAGTCCATCACGAT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGalectin-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCAGACAATTTTTCGCTCCATGA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTAGGCCCCAGGATAGGAAG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHIF-1α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAACGTCGAAAAGAAAAGTCTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCCTTATCAAGATGCGAACTCACA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eThe total protein was extracted using total protein extraction kit (BestBio Science, China) and quantified using BCA Protein Assay Kit (Beyotime Biotechnology, China). The HIF-1α and Galectin-3 protein were by 8% and 10% SDS PAGE gels respectively. The proteins in the gel were transferred to polyvinylidene fluoride membranes (PALL corporation, USA). Next, the membranes were blocked with 5% non-fat milk dissolved in PBS containing 1% Tween-20 (PBST) at room temperature for 1 hour. The membranes were incubated with primary antibodies HIF-1α (1:200, ab51608, Abcam, UK), Galectin-3 (1:1000, ab76245, Abcam, UK), β-action (1:10000, A5316, Sigma, USA) at 4℃ overnight and then incubated with donkey anti-rabbit IgG antibody (1:10000, GE Healthcare, UK) or sheep anti-mouse IgG antibody (1:10000, GE Healthcare, UK) at room temperature for 1 h. The chemiluminescent signal was imaged using a Tanon 5200 Multi Automatic Chemiluminescence/Fluorescence Image Analysis System (China).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation analysis\u003c/h2\u003e \u003cp\u003eCell proliferation was measured using MTS Kit (Saint-Bio, China). SMMC7721 was seeded in a 96-well plate with 3000 cells/well to investigate the effect of Galectin-3-knockdown under hypoxia. Briefly, 10\u0026micro;l of MTS reagent was added to each well and incubated for 3 hours to test cell proliferation. Optical density (OD) value was measured at 490nm using a multilabel plate reader (Thermo scientific, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell apoptosis analysis\u003c/h2\u003e \u003cp\u003eFITC Annexin V Apoptosis Detection Kit I (BD Biosciences, USA) was used to detected cell apoptosis. Cells were harvested and washed twice with phosphate-buffered saline (PBS). Then the cells were stained with PI and FITC for 15 min in the dark at room temperature. Cells apoptosis was analyzed using a FACSCalibur (BD Biosciences, USA) within 1 hour after adding 400\u0026micro;l 1 \u0026times; Binding Buffer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell migration analysis\u003c/h2\u003e \u003cp\u003eThe wound-healing assay was performed to assess the cell migration in response to the mechanical scratch. When cells reached 90% confluence, a straight scratch was made in the center of each well using a micropipette tip. Atter the suspended cells were washed with PBS, the well was replenished with serum-free medium. The cells were observed and images were captured by a microscope at 0, 24, and 48h. The cell migration rate (MR) was calculated using the following equation: MR= (d\u003csub\u003e0\u003c/sub\u003e-d\u003csub\u003e1\u003c/sub\u003e)/d\u003csub\u003e0\u003c/sub\u003e, d\u003csub\u003e0\u003c/sub\u003e means the length of the wound at time 0h, d\u003csub\u003e1\u003c/sub\u003e is the length at time 48h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCell invasion analysis\u003c/h2\u003e \u003cp\u003eHCC cells (1x10\u003csup\u003e5\u003c/sup\u003e cells) in 750\u0026micro;l of DMEM without FBS were seeded in the upper chamber of 24-well Growth Factor Reduced Corning Matrigel Invasion chamber (Corning, USA). The lower chamber was filled with medium containing 10% FBS. After 24h, the non-invading cells were removed from the upper surface of the membrane. The cells that invaded to the bottom surface of the membrane were fixed, stained with crystal violet, photographed by a microscope, and counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMatrigel tube formation assay\u003c/h2\u003e \u003cp\u003e3D culture system was used to evaluate VM formation in vitro. A total of 200\u0026micro;l of cold Corning Matrigel Matrix (10 mg/ml) was coated in each well of a 24-well plate and cultured at 37 ℃ for 1 hour. The cells were seeded in the 24-well plate and cultured for 6 hours. The formation of capillary-like structures was observed and photographed with a fluorescence microscope (Leica, Germany). Angio Tool Software was used to calculate percentage area of tubes per area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eChip-qPCR\u003c/h2\u003e \u003cp\u003e Enzymatic Shearing Kit (Active Motif, USA) was used to prepared sheared chromatin by enzymatic shearing, and ChIP-IT Express Enzymatic Magnetic Chromatin Immunoprecipitation Kit (Active Motif, USA) was used to performed chromatin immunoprecipitation in accordance with the manufacturer\u0026rsquo;s instructions. Purification of DNA from Chromatin Immunoprecipitation sample was performed using Chromatin IP DNA Purification Kit (Active Motif, USA). Real-time PCR was performed on Bio-Rad CFX96 Real-Time System. The following primers were used for RT-PCR: primer 1: ATCACTTCCCAAGGCCTCCAC (forward), TATCCCGCCAAAGTTCATATGTT (reverse); primer 2: GCCCCTATGACCTAATC\u003c/p\u003e \u003cp\u003eACTTCC (forward), CTCATTTTTACTGTCAGGGTGCTAT (reverse).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe H-score of HIF-1α expression was presented as (interquartile ranges) and compared using the Mann-Whitney test. Categorical variables were expressed as numbers and frequencies (%), and compared using the Pearson Chi-square test or Fisher exact test. The Wilcoxon matched-pairs signed-rank test was used when comparing the expression of HIF-1α in tumor tissue and adjacent non-tumor tissue. Optimal cut-off value of HIF-1α was calculated by time-dependent receiver operating characteristic (ROC) curves using the R language for survival analysis. The overall survival rate was estimated using Kaplan\u0026ndash;Meier method and compared using the log-rank test. The Cox proportional hazards model was performed to assess factors potentially associated with prognosis of patients with HCC. The unpaired two-tailed t-test was used to compare differences between two parallel treatment groups in cell experiments. Variables with \u003cem\u003eP\u003c/em\u003e values below or equal to 0.10 in univariate analysis were included in the multivariate model. Hazard ratio (HR) and corresponding 95% confidence intervals (95% CI) were used to determine the strength of statistical associations. A two-sided \u003cem\u003eP\u003c/em\u003e-value below or equal to 0.05 was considered significant. Statistical analyses were performed using SPSS software package 18.0 (SPSS Inc. USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHIF-1α-positive expressions are implicated in poor overall survival of HCC\u003c/h2\u003e \u003cp\u003eWe evaluated the correlation of HIF-1α expression with clinicopathological characters and prognosis among a cohort of 202 HCC patients. We found that HIF-1α was stained mainly in the cytoplasm or cytoplasm and nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA); moreover, HIF-1α expressions were higher in tumor tissues than those in adjacent hepatic tissues in the 140 paired samples, but no significant difference was observed (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.097) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Of note, HIF-1α expressions were significantly higher in HCC tissues than those in adjacent hepatic tissues in the 53 cirrhosis-free patients with HCC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.023) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). HIF-1α expressions were significantly higher in male patients (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037) and the patients with HBcAb-positive (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.040). However, HIF-1α expressions were not significantly associated with tumor histological differentiation, tumor size, number of tumor and lymph-vascular invasion (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). ROC curve revealed the cut-off value of HIF-1α in predicting the 3-year survival was H-score\u0026thinsp;=\u0026thinsp;0. Kaplan\u0026ndash;Meier survival analysis indicated that the HIF-1α-positive expression group have poor overall survival compared with HIF-1α-negative expression groups (Log-Rank \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Furthermore, multivariate analysis showed that the prognosis of patients with HCC was associated with number of tumor (HR\u0026thinsp;=\u0026thinsp;1.83, 95% CI [1.24\u0026ndash;2.72]), tumor size\u0026thinsp;\u0026ge;\u0026thinsp;5 cm (HR\u0026thinsp;=\u0026thinsp;2.23, 95% CI [1.48\u0026ndash;3.37]), and HIF-1α-positive expression (HR\u0026thinsp;=\u0026thinsp;2.16, 95% CI [1.42\u0026ndash;3.30]) significantly (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExpressions of HIF-1α in tumor tissue and adjacent non-tumor tissue\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eH-score\u003c/p\u003e \u003cp\u003eMedian (quartile)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOverall\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTumor tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80(0-150)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.097\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdjacent non-tumor tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e55(0-120)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCirrhosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTumor tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80(0-140)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.785\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdjacent non-tumor tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60(0-140)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCirrhosis-free\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTumor tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80(0-160)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.023\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdjacent non-tumor tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30(0-100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation between clinicopathological characters and expressions of HIF-1α\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eCharacter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c4\" namest=\"c3\" rowspan=\"2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eExpression of HIF-1α\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" morerows=\"1\" nameend=\"c10\" namest=\"c8\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c12\" namest=\"c11\" rowspan=\"2\"\u003e \u003cp\u003eH-score\u003c/p\u003e \u003cp\u003eMedian (quartile)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNegative (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ePositive (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e164\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51 (31.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e113 (68.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.037\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e80(0-140)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.077\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19 (50.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e19 (50.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e10(0-120)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;55ys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35 (34.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e68 (66.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e0.883\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e80(0-150)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.408\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;55ys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35 (35.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e64 (64.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e70(0-120)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eHBV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eHBsAg positive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42 (30.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e94 (69.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e0.117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e80(0-147.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.118\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eHBsAg negative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28 (42.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e38 (57.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e50(0-100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eHBeAg positive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11 (25.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e33 (75.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e0.152\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e80(5-150)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.215\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eHBeAg negative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e154\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e58 (37.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e96 (62.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e65(0-120)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eHBcAb positive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e178\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e57 (32.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e121 (68.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.040\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e80(0-140)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.140\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eHBcAb negative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13 (54.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e11 (45.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e0(0-115)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eHCV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14(38.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e22 (61.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e0.567\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e85(0-155)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.818\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e166\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e56(33.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e110 (66.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e70(0-140)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eAFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u0026lt;20 ng/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29(29.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e68 (70.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e0.186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e90(0-140)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.250\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;20 ng/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41 (39.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e64 (61.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e60(0-120)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eNumber of tumor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003esingle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e55 (36.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e94(63.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e0.314\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e70(0-120)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.116\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eMultiple\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15 (28.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e38 (71.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e80(0-180)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTumor size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;5cm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31 (32.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e65 (67.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e0.555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e90(0-147.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.353\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;5cm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e39 (36.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e67 (63.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e65(0-125)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eDifferentiation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003ePoor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20 (32.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e41 (67.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e0.750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e70(0-120)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.757\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eModerate/well\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50 (35.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e91 (64.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e80(0-145)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eLymph-vascular invasion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e39 (36.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e68 (63.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e0.657\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e60(0-150)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.807\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31 (32.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e64 (67.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e80(0-120)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eCirrhosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34 (41.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e48 (58.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e0.100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e60(0-150)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.359\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36 (30.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e84 (70.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e80(0-140)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eAJCC clinical stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32(37.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e53 (62.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e0.494\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e60(0-120)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.402\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30 (34.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e56 (65.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e75(0-125)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (25.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e23(74.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e90(0-160)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eVM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e53 (75.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e17 (24.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e \u003cp\u003e\u003cb\u003e0.021\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e40(0-100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e78 (59.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003e54 (40.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e90(20\u0026ndash;160)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eUnivariate and multivariate analysis of factors associated with prognosis in HCC\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCharacter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eUnivariate analysis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eMultivariate analysis*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHR(95% CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHR(95% CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHBsAg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.44 (0.97\u0026ndash;2.14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.072\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.45 (0.97\u0026ndash;2.17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.073\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt; 20ng/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e124\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge; 20ng/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e123\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.90 (1.31\u0026ndash;2.76)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.81 (1.23\u0026ndash;2.68)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.003\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of tumor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSingle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e183\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMultiple\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.18 (1.49\u0026ndash;3.19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.83 (1.24\u0026ndash;2.72)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.003\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;5cm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;5cm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.38(1.63\u0026ndash;3.47)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.23(1.48\u0026ndash;3.37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDifferentiation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePoor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModerate/ well\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.60(0.41\u0026ndash;0.87)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.79(0.52\u0026ndash;1.19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.255\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymph-vascular invasion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.07(1.43\u0026ndash;2.99)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.18(0.77\u0026ndash;1.82)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.442\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHIF-1α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.89(1.25\u0026ndash;2.86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.16(1.42\u0026ndash;3.30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e*Age, sex, HBsAg, differentiation and lymph-vascular invasion were adjusted in multivariate analysis.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eGalectin-3 engages in the link between HIF-1α and HCC\u003c/h2\u003e \u003cp\u003eThe expression of Galectin-3 was determined in our previous study. Galectin-3 was significantly higher expressed in HCC tumor tissues and related to worse overall survival [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To understand the clinical relevance of HIF-1α and Galectin-3, we evaluate correlation of HIF-1α and Galectin-3 expression among a cohort of 147 HCC patients. The results showed that HIF-1α expression was positively correlated with Galectin-3 in the HCC tumor tissues (R\u0026thinsp;=\u0026thinsp;0.167, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.043). To further identify the relationship between HIF-1α and Galectin-3, the mRNA-seq data of 424 HCC patients were recruited from the TCGA database, HIF-1α were found to be positively associated with LGALS3 coding Galectin-3 (R\u0026thinsp;=\u0026thinsp;0.394, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). To further confirm the relation from HIF-1α, via Galectin-3, to HCC, we investigated expressions of Galectin-3 on survival of patients with HCC on the basis of TCGA database. As expected, compared with low expression of LGALS3, high expression of LGALS3 linked to poor overall survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eHIF-1α and Galectin-3 are involved in VM of HCC\u003c/h2\u003e \u003cp\u003eTube cavities derived from HCC cells were positive for PAS staining, but negative for CD31 staining, indicating that VM, rather than endothelium-dependency, is characteristic of these tube cavities (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, green arrow). The channels positive for CD31 and PAS were considered as endothelium-dependent vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, red arrow). VM was observed in 35.1% (71/202) patients with HCC. HIF-1α was implicated in VM formation, higher HIF-1α expression was observed in the patients with VM (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.021) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Furthermore, the relationship between expression of Galectin-3 and tumor VM formation was explored among 147 HCC patients. The results showed that the presence of VM related to Galectin-3, higher Galectin-3 expression was observed in VM-positive patients (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.026).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eEffect of hypoxia microenvironment on the function of HCC cells\u003c/h2\u003e \u003cp\u003eHypoxia microenvironment has been characterized in HCC tumor tissues. After SMMC7721 cells were cultured under hypoxia for 24 hours, we found that hypoxia triggered proliferation of SMMC7721 cells significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). As expected, hypoxia decreased apoptosis of SMMC7721 cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006). Cell scratch assay showed that hypoxia accelerated cell migration remarkably (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.166). Similarly, cell transwell assay also revealed that hypoxia induced invasion of SMMC7721 cells significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001). Three-dimensional cultures of SMMC7721 cells showed that hypoxia promoted the formation of tube-like strictures area significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eHIF-1α modulates Galectin-3 expression in HCC cells under hypoxia\u003c/h2\u003e \u003cp\u003eWe investigated the effects of hypoxia on expressions of HIF-1α and Galectin-3 in SMMC7721 cells, showing that 48 hours of hypoxia enhanced the transcription and translation of HIF-1α and Galectin-3 significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). After HIF-1α knockdown under hypoxia, we found the expression of Galectin-3 decreased significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). To further confirm the relation between Galectin-3 and HIF-1α, we performed ChIP-qPCR assay, corroborating that \u003cem\u003eLGALS3\u003c/em\u003e coding Galectin-3 was a target gene of HIF-1α (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eGalectin-3 is required for the function of HCC cells under hypoxia microenvironment\u003c/h2\u003e \u003cp\u003eHypoxic microenvironment increased Galectin-3 expression in HCC cells. To explore connectivity of Galectin-3 with the effect of hypoxia microenvironment on the function of HCC cells, we utilized small interfering RNA (siRNA) to silence Galectin-3, parsing that the silencing reduced proliferation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), increased apoptosis (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008), and decreased migration and invasion of SMMC7721 cells (both \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Indeed, the formation of tube-like strictures area was reduced by silencing Galectin-3 under hypoxia significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this paper, we reveal that HIF-1α-regulated Galectin-3 is required for the pathogenesis of HCC, establishing connection from HIF-1α, via Galectin-3, to the VM and poor prognosis of HCC.\u003c/p\u003e \u003cp\u003eHypoxia and the activation of hypoxia inducible factors are essential for the pathogenesis of HCC. Although the expression of HIF-1α in HCC tissues was significantly higher than that in the paracancerous tissues [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], we showed that there was no difference of HIF-1α expression between HCC tumor tissues and adjacent non-tumor tissues among the patients with cirrhosis. While we found that higher positive frequency of HIF-1α in HCC tissues than that in adjacent non-tumor tissues in cirrhosis-free patients with HCC. Similar to the discovery of Wang et al.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Hypoxia causes the accumulation and activation of HIF-1α[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. HIF-1α, as a transcription factor, regulates expressions of target genes to facilitate cellular adaptation to tumor hypoxia by binding to hypoxic response elements[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Hypoxia in HCC is dynamic[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. HIF-1α expression appears to depends on intensity of hypoxia. The results of present study found that HIF-1α is mainly expressed in cirrhosis tissues, rather than in normal liver tissues. Similar findings were also reported by Li \u003cem\u003eet al\u003c/em\u003e. in serum levels[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. During the progression of chronic liver diseases, due to persisting inflammatory response, parenchymal injury, liver architecture changes, and vascular reorganization, can significantly result in tissue hypoxia and a hypoxic cellular response[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Hypoxic represent a very common and major feature of fibrotic and cirrhotic liver [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The connection between HIF-1α expressions and HCC risks we found suggested that HIF-1α may serve as a diagnostic marker for HCC in non-cirrhotic patients, but cannot be used as an effective marker to distinguish liver cirrhosis from hepatocellular carcinoma.\u003c/p\u003e \u003cp\u003eVM gives rise to the poor prognosis in HCC via forming vasculogenic networks[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our present study showed that HIF-1α were implicated in VM formation and patients with positive HIF-1α expression had poor survival rate. VM formation correlates with the expression of vimentin or α-SMA in HCC tissues significantly[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. HIF-1α affords EMT and VM formation via up-regulating LOXL2 in hypoxic microenvironment of HCC[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this paper, the positive correlation between HIF-1α and Galectin-3 expression in HCC tissues triggered us to investigate the connection between HIF-1α and Galectin-3 expression. We, further, unveiled that \u003cem\u003eLGALS3\u003c/em\u003e coding Galectin-3 was a target gene of HIF-1α in HCC. Similar findings were also reported in renal cell carcinoma and thyroid cancer[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. HCC is one of the most hypoxic malignancies[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In a previous study, we concluded that Galectin-3 was highly expressed in tumor tissue [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These data provided molecular mechanisms underlying the accumulation of Galectin-3 in HCC. Conceivably, Galectin-3 has been found to regulate VM formation in esophageal cancer cell [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In this paper, we revealed that increased Galectin-3 expressions were significantly associated with VM formation. In our previous study, we found that increased Galectin-3 expressions in tumor tissue correlate with poor prognosis in HCC[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Further vitro experiments showed that Galectin-3 was required for the function of HCC cells under hypoxia microenvironment. Therefore, Galectin-3 is critical for linking HIF-1α to VM formation and poor prognosis in HCC.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, HIF-1α-regulated Galectin-3 contributes to the vasculogenic mimicry and poor prognosis of HCC. This study highlights the potential therapeutic value of targeting Galectin-3 for suppression of HCC metastasis and progression.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFei Kong and Mei-shan Jin contributed equally to this work; Yi Cheng and Yawen Liu designed and conducted most of the study; Fei Kong wrote the first draft of the manuscript; Meishan Jin performed immunohistochemistry testing and evaluation; Yu Fu and Jing Jiang collected the epidemiological and clinical data; Fei Kong and Jikang Shi performed the cells experiments. Fei Kong and zhuoshuai Liang performed the statistical analyses; All the authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was sponsored by the National Natural Science Foundation of China (grants No. 82304216), the Natural Science Foundation of Jilin Province (YDZJ202401197ZYTS), the Natural Science Foundation of First Hospital of Jilin University (JDYY72016012).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study protocol was approved by the Ethics Committee of the National Natural Science Foundation of Jilin Province (YDZJ202401197ZYTS). Each patient provided a written informed consent. We confirm that all methods were performed in accordance with the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung, H. et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. \u003cem\u003eCA Cancer J. Clin.\u003c/em\u003e \u003cb\u003e71\u003c/b\u003e (3), 209\u0026ndash;249 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, M. et al. Design and rationale of the HCC BRIDGE study in China: a longitudinal, multicenter cohort trial in hepatocellular carcinoma. \u003cem\u003eBMC Gastroenterol.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 53 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTabrizian, P. et al. 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Galectin-3 induced by hypoxia promotes cell migration in thyroid cancer cells. \u003cem\u003eOncotarget\u003c/em\u003e. \u003cb\u003e8\u003c/b\u003e (60), 101475\u0026ndash;101488 (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hepatocellular carcinoma, vasculogenic mimicry, HIF-1α, Galectin-3, prognosis","lastPublishedDoi":"10.21203/rs.3.rs-5359597/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5359597/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn present study, we explore the role of HIF-1α-regulated Galectin-3 in VM and poor prognosis of HCC as well as the potential regulatory mechanism. Immunohistochemistry method was used to evaluate HIF-1α and CD31/PAS expression in HCC patients. Vitro experiments were conducted to reveal the molecular mechanisms that regulate Galectin-3 expression under hypoxia. Moreover, the effects of Galectin-3 on the biological function of HCC cells were investigated using MTS kit, wound-healing assay, cell transwell assay, flow cytometry assay and three-dimensional culture under hypoxia. HIF-1α expressions were significantly higher in HCC tissues than those in adjacent hepatic tissues in cirrhosis-free patients with HCC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.023). The patients with positive-HIF-1α expression had worse overall survival (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002). HIF-1α was positively correlated with Galectin-3 in HCC tumor tissues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Higher expression of Galectin-3 linked to poor overall survival (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002). HIF-1α and Galectin-3 were correlated with VM of HCC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Hypoxia promotes proliferation, migration, invasion and VM formation of HCC cells, and suppresses to apoptosis (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eGalectin-3\u003c/em\u003e is the target genes of HIF-1α transcription factor. Silencing Galectin-3 abolished effect of hypoxia microenvironment on the function of HCC cells. In conclusion, HIF-1α-regulated Galectin-3 contributes to the vasculogenic mimicry and poor prognosis of HCC.\u003c/p\u003e","manuscriptTitle":"HIF-1α-regulated Galectin-3 affords the vasculogenic mimicry and poor prognosis of hepatocellular carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-17 17:41:21","doi":"10.21203/rs.3.rs-5359597/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ab4fffd2-3111-46f2-b244-9be273824fa8","owner":[],"postedDate":"December 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":41332104,"name":"Health sciences/Medical research"},{"id":41332105,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-04-08T09:51:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-17 17:41:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5359597","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5359597","identity":"rs-5359597","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}