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PVT1, which maps to chromosome 8q24, is a copy number amplification-associated long non-coding RNA. Overexpression of PVT1 is a powerful predictor of tumor progression and patient survival in a diverse range of cancer types. However, the association between PVT1 and hepatocellular carcinoma (HCC) remains unclear. The aim of the present study was to examine the expression pattern of PVT1, and its clinical significance in HCC. Between 2003 and 2012, reverse transcription-quantitative polymerase chain reaction was used to determine the expression levels of PVT1 in two independent cohorts: Cohort one, 58 HCC resection samples; and cohort 2, 214 HCC transplant samples. Additionally, the correlation between PVT1 expression levels and clinical parameters and outcomes was analyzed. The relative expression levels of PVT1 were significantly higher in cancerous tissues compared with the corresponding non-cancerous tissues (cohort one, P=0.0016; cohort two, P=0.0274). Furthermore, overexpression of PVT1 was associated with a higher serum α-fetoprotein expression level (P=0.011) and a higher recurrence rate (P=0.004). Kaplan-Meier analysis indicated that the patients with high PVT1 expression exhibited poor recurrence-free survival (P=0.021), and multivariate analysis demonstrated that high levels of PVT1 expression are an independent predictor for HCC recurrence (P=0.042; hazard ratio, 1.653). Thus, the high expression levels of PVT1 in HCC may serve as a novel biomarker for predicting tumor recurrence in HCC patients, and as a potential therapeutic target.
Hepatocellular carcinoma (HCC) represents the fifth most prevalent malignancy and the second most common cause of cancer-related mortality worldwide, with ~695,900 mortalities per year (
Previously, RNA sequencing revealed a novel class of transcripts termed long non-coding RNAs (lncRNAs). LncRNAs are >200 nucleotides long and lack protein-coding potential, thus, they were previously regarded as random transcriptional noises. However, increasing evidence has implicated lncRNAs in critical regulatory roles in cancer biology (
PVT1, which maps to chromosome 8q24, is a copy number amplification-associated lncRNA. Overexpression of PVT1 is a powerful predictor of tumor progression and patient survival in colorectal (
The aim of the present study was to examine the expression pattern of PVT1 and its clinical significance in HCC.
Fifty-eight snap-frozen HCC tissues and the corresponding non-cancerous tissues were obtained from patients undergoing liver resection at the First Affiliated Hospital of Zhejiang University (Hangzhou, China) between 2009 and 2012 (cohort one). An additional 214 HCC tissues were collected from patients undergoing liver transplantation at the First Affiliated Hospital of Zhejiang University between 2003 and 2012 (cohort two), and were used for survival analysis and validation. The liver tissue specimens were immediately frozen in liquid nitrogen following surgical resection and stored at −80°C prior to the extraction of total RNA. A postoperative histopathological examination by experienced pathologists was used to establish a diagnosis of HCC in these patients. The histological grade of differentiation was evaluated on hematoxylin and eosin-stained sections according to the Edmondson-Steiner grading method (
The present study was conducted with the approval of the Institutional Review Board and Ethics Committee of the First Affiliated Hospital, Zhejiang University (Hangzhou, China) and in accordance with the Declaration of Helsinki. Thus, the study conformed to international and national regulations. Informed consent was obtained from all of the patients.
Patient follow-up was conducted every 2–3 months during the first two years following surgery and 3–6 months thereafter. The endpoint of study was September 3, 2013. During the follow-up period, all patients were monitored using abdomen ultrasonography, chest X-ray, emission computed tomography and serum AFP tests. Following a suspected recurrence, computed tomography, magnetic resonance imaging or positron emission tomography-computed tomography, were immediately performed. These imaging techniques were required for a new lesion to be detected and recurrence to be diagnosed; an increase in serum AFP levels alone was not regarded as recurrence. Once recurrent tumors were diagnosed, treatment was implemented based on the tumor size, number, location and vascular invasion, as well as the liver function. The recurrence-free survival (RFS) period was calculated from the date of surgery to the date of detection of tumor recurrence, mortality or the most recent observation. All follow-up examinations were performed by two physicians who were unaware of the study. The mean follow-up time was 27.58 months (range, 2–124 months).
The healthy liver cell line HL7702, the liver cancer cell lines Huh7, SK-hep1, SMMC-7721, HepG2, Hep3B, PLC/PRF/5 and Bel-7402, as well as the metastasis-capable human HCC cell lines MHCC97L, MHCC97H and HCCLM3, were purchased from the American Type Culture Collection (Manassas, VA, USA), the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China), and the Liver Cancer Institute of Fudan University (Shanghai, China), respectively. All of the cell lines were maintained in Dulbecco’s modified Eagle’s medium with high glucose or RPMI-1640, containing 10% fetal bovine serum, and cultured in a humidified 5% CO2 incubator at 37°C.
Total RNAs from the specimens and cell lines were extracted using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA); and complementary DNA was synthesized using Moloney murine leukemia virus reverse transcriptase (Promega Corporation, Madison, WI, USA) according to the manufacturer’s instructions. The expression of PVT1 was determined using qPCR, which was performed on an Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems Life Technologies, Foster City, CA, USA) and SYBR® Green dye (Takara Biotechnology Co., Inc., Dalian, China). All PCRs were performed in triplicate and GAPDH was used to normalize mRNA expression levels. Relative quantification was performed using the comparative threshold cycles (2−ΔΔCt method) as described in the manufacturer’s instructions. The primer sequences were as follows: Sense, 3′-CATCCGGCGCTCAGCT-5′ and antisense, 3′-TCATGATGGCTGTATGTGCCA-5′ for PVT1; and sense, 3′-ATGGGGAAGGTGAAGGTCG-5′ and antisense, 3′-GGGGTCATTGATGGCAACAATA-5′ for GAPDH.
Comparisons of continuous data were analyzed using the independent t-test between the two groups, whereas categorical data were analyzed by the χ2 test. Comparisons of continuous data among multiple groups were calculated using one-way analysis of variance. A receiver operating characteristic (ROC) curve was used to determine the cut-off value of PVT1 expression that yielded the highest combined sensitivity and specificity for discriminating between patients exhibiting HCC recurrence and those not exhibiting recurrence. Recurrence-free survival was analyzed using the Kaplan-Meier method and compared by performing the log-rank test. Independent prognostic factors were assessed in the univariate and multivariate analysis using the Cox proportional-hazards regression model. All statistical analyses were performed using SPSS for Windows (version 16.0; SPSS, Inc., Chicago, IL, USA) and GraphPad Prism (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA) software. P<0.05 was considered to indicate a statistically significant difference.
Snap-frozen HCC tissues were obtained from 272 HCC patients who had undergone liver surgery at the First Affiliated Hospital of Zhejiang University between 2003 and 2012. The present study involved two independent cohorts of HCC patients: Cohort one included 58 HCC patients who had undergone radical resection between 2009 and 2012; while cohort two included 214 HCC patients who had received a liver transplant between 2003 and 2012.
The majority of the HCC patients in the two cohorts were male (91.18%) with a tumor size of >5 cm at the time of surgery (45.96%), an elevated serum AFP level (47.79%) and with tumors exceeding the Milan and UCSF criteria (63.60 and 54.41%, respectively;
In the present study, the expression levels of PVT1 in the 272 HCC patients from the two independent cohorts were determined using RT-qPCR. Compared with the corresponding non-tumor liver tissues of the HCC cohorts, PVT1 expression was significantly increased in the cancerous tissues of the patients in cohort one (P=0.0016;
As PVT1 expression was significantly increased in HCC, the association between PVT1 expression in HCC and disease progression was evaluated. First, the expression level of PVT1 in different TNM-stage patients was assessed, as TNM staging is a widely accepted system for HCC stratification. In HCC cohort two, advanced-stage patients (stages III and IV; n=115) exhibited increased expression levels of PVT1 compared with early-stage patients (stages I and II; n=99) (P=0.0466;
To investigate the clinicopathological correlation of PVT1 expression in HCC tissues, the patients were divided into high and low expression groups according to the cut-off value obtained from the ROC curve analysis. No significant correlations with any of the clinicopathological parameters tested were observed in HCC cohort one (
To determine whether PVT1 could be employed as a prognostic biomarker for HCC, clinical data of HCC cohort two were analyzed in detail. Using the cut-off value, the 214 patients were divided into two groups: A low-expression group (n=57) and a high-expression group (n=157). Kaplan-Meier analysis indicated that the patients with high PVT1 expression levels had a poor RFS period (P=0.021;
To identify the risk factors associated with post-transplant RFS, 11 clinicopathological factors were evaluated by performing Cox univariate and multivariate analyses. Univariate analysis demonstrated that the significant prognostic factors for HCC recurrence were tumor size, tumor number, histopathological grade, PVTT, preoperative AFP level, TNM stage and PVT1 expression (all P<0.05). Only tumor size (HR, 2.462; 95% CI, 1.652–3.671; P<0.001), tumor number (HR, 1.802; 95% CI, 1.194–2.719; P=0.005), PVTT (HR, 2.075, 95% CI, 1.418–3.037; P<0.001), preoperative AFP level (HR, 1.539;95% CI, 1.027–2.305; P=0.037) and PVT1 expression (HR, 1.653; 95% CI, 1.019–2.681; P=0.042) were identified as independent prognostic factors associated with tumor recurrence following liver transplantation, as determined by the Cox multivariate analysis (
The complexity of the human transcriptome has been highlighted by various high-throughput studies (
Although the majority of previous studies have focused on short RNAs in cancer research, such as miRNAs, lncRNAs are gaining prominence. A number of classic lncRNAs have been implicated in human hepatocarcinogenesis, exhibiting oncogenic or tumor suppressive roles. One such example of oncogenic lncRNA is HOTAIR, which was initially identified in foreskin fibroblasts. HOTAIR resides in the HOX C locus, acting as a modular scaffold to recruit the polycomb repressive complex 2 to specific target sequences that ultimately results in the suppression of numerous genes (
Furthermore, tumor suppressive lncRNAs may affect various tumor suppressor pathways. For example, MEG3 was identified to be frequently downregulated in HCC by miR29a-mediated promoter methylation, and MEG3 inhibited cell growth by functionally interacting with the p53 signaling pathway (
Various reports have presented evidence that PVT1 contributes to cancer pathophysiology. For example, PVT1 was markedly overexpressed in colorectal (
Furthermore, the present study identified that PVT1 was more likely to be overexpressed in advanced-stage and recurrence patients. Correlation analysis indicated that increased expression of PVT1 was associated with a higher AFP level and a higher recurrence rate. These data support the hypothesis that PVT1 is associated with disease progression. In addition, survival analysis demonstrated that the patients with high PVT1 expression exhibited poor RFS. Multivariate analysis identified that PVT1 was an independent prognostic factor for RFS. Therefore, data from the present study indicate that PVT1 may be a novel biomarker for risk surveillance and adjuvant therapy screening of HCC patients following liver transplantation. Furthermore, overexpression of PVT1 may be used by surgeons to identify high-risk patients who may benefit from preventive strategies as opposed to surgery.
The effects and precise molecular mechanisms underlying the altered expression of PVT1 in HCC are unclear. Guan
In conclusion, the present study demonstrated that PVT1 was overexpressed in two independent human HCC cohorts and 10 liver cancer cell lines. Increased expression levels of PVT1 were associated with a higher AFP level and a higher recurrence rate. Furthermore, PVT1 served as an independent prognostic factor for RFS. Thus, the findings of the present study indicate that PVT1 may act as a novel biomarker for predicting tumor recurrence in HCC patients and may be a potential therapeutic target.
This abstract was presented at the American College of Surgeons Clinical Congress (October 26–October 30, 2014), San Francisco, CA, USA, and was published as abstract S23 in the Journal of the American College of Surgeons 219: S3, 2014. The present study was supported by grants from the National High Technology Research and Development Program of China (863 Program), the Special Fund for Health Research in the Public Welfare, the Zhejiang Provincial Natural Science Foundation for Young Distinguished Scholars, the National Science and Technology Major Project and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (grant nos. 2012AA021002, 201302009, R2110125, 2012ZX10002017 and 81121002, respectively). The authors of the present study would also like to thank all of the patients enrolled in the study for their support.
PVT1 expression levels in hepatocellular carcinoma tumor (HCC) tissue and the corresponding non-tumorous tissue. PVT1 expression levels were normalized to an internal control and log2-transformed. (A) Cohort one consists of 58 paired HCC resection samples and (B) cohort two consists of 128 paired HCC transplant samples. Horizontal lines in the plots represent the mean values. P-values between samples were obtained by performing a paired t-test.
Relative PVT1 expression levels in 10 liver cancer cell lines. HL-7702 is a healthy liver cell line. Data are the mean ± standard error of the mean (n=3). *P<0.05, vs. the control (HL-7702 cells), according to the paired t-test.
PVT1 expression levels defined according to the tumor-node-metastasis stage and recurrence status. (A) Cohort two: Early (stages I + II) vs. advanced stage (stages III + IV). (B) Cohort two: Non-recurrence vs. recurrence. (C) Cohort one: Three different stages (I vs. II vs. III). PVT1 expression levels were normalized to an internal control and log2-transformed. Horizontal lines in the plots represent the mean values. P-values were analyzed using (A and B) the independent t-test and (C) one-way analysis of variance.
Kaplan-Meier survival curves according to PVT1 level. Patients with high PVT1 expression had poor (A) recurrence free survival and (B) overall survival rates. P-values were calculated using the log-rank test.
Clinical characteristics of HCC patients.
Cohort one (n=58) | Cohort two (n=214) | P-value | |
---|---|---|---|
Age, years | 0.000 | ||
Median | 60 | 49 | |
Range | 26–86 | 20–71 | |
Gender, n (%) | 0.951 | ||
Female | 5 (8.62) | 19 (8.88) | |
Male | 53 (91.38) | 195 (91.12) | |
HBV, n (%) | 0.000 | ||
Negative | 11 (18.97) | 6 (2.34) | |
Positive | 47 (81.03) | 209 (97.66) | |
Cirrhosis, n (%) | 0.000 | ||
No | 22 (37.93) | 2 (0.93) | |
Yes | 36 (62.07) | 212 (99.07) | |
Tumor size, n (%) | 0.059 | ||
≤5 cm | 25 (43.10) | 122 (57.01) | |
>5 cm | 33 (56.90) | 92 (42.99) | |
Tumor number, n (%) | 0.000 | ||
=1 | 52 (89.66) | 89 (41.59) | |
>1 | 6 (10.34) | 125 (58.41) | |
PVTT, n (%) | 0.000 | ||
Negative | 54 (93.10) | 142 (66.36) | |
Positive | 4 (6.90) | 72 (33.64) | |
AFP, n (%) | 0.270 | ||
≤400 ng/ml | 34 (58.62) | 108 (50.47) | |
>400 ng/ml | 24 (41.38) | 106 (49.53) | |
Histopathological grade, n (%) | 0.512 | ||
Well + moderately | 27 (46.55) | 110 (51.40) | |
Poorly | 31 (53.45) | 104 (48.60) | |
TNM stage, n (%) | 0.000 | ||
I + II | 52 (89.66) | 99 (46.26) | |
III + IV | 6 (10.34) | 115 (53.74) | |
Milan criteria ( |
0.374 | ||
Within criteria | 24 (41.38) | 75 (35.05) | |
Beyond criteria | 34 (58.62) | 139 (64.95) | |
UCSF criteria ( |
0.175 | ||
Within criteria | 31 (53.45) | 93 (43.46) | |
Beyond criteria | 27 (46.55) | 121 (56.54) | |
Hangzhou criteria ( |
0.000 | ||
Within criteria | 46 (79.31) | 105 (49.07) | |
Beyond criteria | 12 (20.69) | 109 (50.93) |
P<0.05 was considered to indicate a statistically significant difference, using the independent samples t-test for age and the χ2 test for all other data.
HCC, hepatocellular carcinoma; HBV, hepatitis B virus; PVTT, portal vein tumor thrombosis; AFP, serum α-fetoprotein; TNM, tumor-node-metastasis; UCSF, University of California, San Francisco.
Clinicopathological correlation of PVT1 expression in human HCC (cohort one).
Low PVT1 expression | High PVT1 expression | |||||
---|---|---|---|---|---|---|
|
|
|||||
Factors | Cases, n | n | % | n | % | P-value |
Age, years | ||||||
≤60 | 33 | 5 | 55.56 | 28 | 57.14 | 0.930 |
>60 | 25 | 4 | 44.44 | 21 | 42.86 | |
Gender | ||||||
Female | 5 | 0 | 0.00 | 5 | 10.20 | 0.316 |
Male | 53 | 9 | 100.00 | 44 | 89.80 | |
HBV | ||||||
Negative | 11 | 1 | 11.11 | 10 | 20.41 | 0.513 |
Positive | 47 | 8 | 88.89 | 39 | 79.59 | |
Cirrhosis | ||||||
No | 22 | 4 | 44.44 | 18 | 36.73 | 0.661 |
Yes | 36 | 5 | 55.56 | 31 | 63.27 | |
Tumor size, cm | ||||||
≤5 | 25 | 2 | 22.22 | 23 | 46.94 | 0.169 |
>5 | 33 | 7 | 77.78 | 26 | 53.06 | |
Tumor number | ||||||
Single | 52 | 8 | 88.89 | 44 | 89.80 | 0.935 |
Multiple | 6 | 1 | 11.11 | 5 | 10.20 | |
PVTT | ||||||
Absent | 54 | 8 | 88.89 | 46 | 93.88 | 0.587 |
Present | 4 | 1 | 11.11 | 3 | 6.12 | |
Preoperative AFP level, ng/ml | ||||||
≤400 | 34 | 7 | 77.78 | 27 | 55.10 | 0.204 |
>400 | 24 | 2 | 22.22 | 22 | 44.90 | |
Histopathological grade | ||||||
Well + moderately | 27 | 5 | 55.56 | 22 | 44.90 | 0.556 |
Poorly | 27 | 4 | 44.44 | 27 | 55.10 | |
TNM stage | ||||||
I + II | 52 | 7 | 77.78 | 45 | 91.84 | 0.203 |
III + IV | 6 | 2 | 22.22 | 4 | 8.16 | |
Milan criteria ( | ||||||
Within criteria | 24 | 2 | 22.22 | 22 | 44.90 | 0.204 |
Beyond criteria | 34 | 7 | 77.78 | 27 | 55.10 | |
UCSF criteria ( | ||||||
Within criteria | 31 | 3 | 33.33 | 28 | 57.14 | 0.188 |
Beyond criteria | 27 | 6 | 66.67 | 21 | 42.86 | |
Hangzhou criteria ( | ||||||
Within criteria | 46 | 6 | 66.67 | 40 | 81.63 | 0.308 |
Beyond criteria | 12 | 3 | 33.33 | 9 | 18.37 |
P<0.05 was considered to indicate statistical significance a statistically significant difference, according to the χ2 test.
HCC, hepatocellular carcinoma; HBV, hepatitis B virus; PVTT, portal vein tumor thrombosis; AFP, α-fetoprotein; TNM, tumor-node-metastasis; UCSF, University of California, San Francisco.
Clinicopathological correlation of PVT1 expression in human HCC (cohort two).
Low PVT1 expression | High PVT1 expression | |||||
---|---|---|---|---|---|---|
|
|
|||||
Factor | Cases, n | n | % | n | % | P-value |
Age, years | ||||||
≤60 | 187 | 49 | 85.96 | 138 | 87.90 | 0.707 |
>60 | 27 | 8 | 14.04 | 19 | 12.10 | |
Gender | ||||||
Female | 19 | 4 | 7.02 | 15 | 9.55 | 0.564 |
Male | 195 | 53 | 92.98 | 142 | 90.45 | |
HBV | ||||||
Negative | 5 | 2 | 3.51 | 3 | 1.91 | 0.494 |
Positive | 210 | 55 | 96.49 | 154 | 98.09 | |
Cirrhosis | ||||||
No | 2 | 1 | 1.75 | 1 | 0.64 | 0.453 |
Yes | 212 | 56 | 98.25 | 156 | 99.36 | |
Tumor size, cm | ||||||
≤5 | 122 | 33 | 57.89 | 89 | 56.69 | 0.875 |
>5 | 92 | 24 | 42.11 | 68 | 43.31 | |
Tumor number | ||||||
Single | 89 | 28 | 49.12 | 61 | 38.85 | 0.178 |
Multiple | 125 | 29 | 50.88 | 96 | 61.15 | |
PVTT | ||||||
Absent | 142 | 40 | 70.18 | 102 | 64.97 | 0.476 |
Present | 72 | 17 | 29.82 | 55 | 35.03 | |
Preoperative AFP level, ng/ml | ||||||
≤400 | 108 | 37 | 64.91 | 71 | 45.22 | 0.011 |
>400 | 106 | 20 | 35.09 | 86 | 54.78 | |
Histopathological grading | ||||||
Well + moderately | 110 | 35 | 61.40 | 75 | 47.77 | 0.078 |
Poorly | 104 | 22 | 38.60 | 82 | 52.23 | |
TNM stage | ||||||
I + II | 99 | 30 | 52.63 | 69 | 43.95 | 0.260 |
III + IV | 115 | 27 | 47.37 | 88 | 56.05 | |
Recurrence | ||||||
No | 100 | 36 | 63.16 | 64 | 40.76 | 0.004 |
Yes | 114 | 21 | 36.84 | 93 | 59.24 | |
Milan criteria ( | ||||||
Within criteria | 75 | 24 | 42.11 | 51 | 32.48 | 0.192 |
Beyond criteria | 139 | 33 | 57.89 | 106 | 67.52 | |
UCSF criteria ( | ||||||
Within criteria | 93 | 27 | 47.37 | 66 | 42.04 | 0.487 |
Beyond criteria | 121 | 30 | 52.63 | 91 | 57.96 | |
Hangzhou criteria ( | ||||||
Within criteria | 105 | 31 | 54.39 | 74 | 47.13 | 0.348 |
Beyond criteria | 109 | 26 | 45.61 | 83 | 52.87 |
P<0.05 was considered to indicate a statistically significant difference, according to the χ2 test.
HCC, hepatocellular carcinoma; HBV, hepatitis B virus; PVTT, portal vein tumor thrombosis; AFP, serum α-fetoprotein; TNM, tumor-node-metastasis; UCSF, University of California, San Francisco.
Cox univariate and multivariate analysis of predictors of recurrence in hepatocellular carcinoma patients following liver transplant.
Univariate analysis | Multivariate analysis | |||||
---|---|---|---|---|---|---|
|
| |||||
Variable for tumor recurrence | HR | 95% CI | P-value | HR | 95% CI | P-value |
Age, years (>60 vs. ≤60) | 0.669 | 0.349–1.280 | 0.224 | |||
Gender (male vs. female) | 1.577 | 0.768–3.239 | 0.214 | |||
HBV | 21.091 | 0.159–2793 | 0.221 | |||
Cirrhosis | 1.039 | 0.145–7.443 | 0.970 | |||
Tumor size, cm (>5 vs. ≤5) | 3.431 | 2.340–5.029 | 0.000 | 2.462 | 1.652–3.671 | 0.000 |
Tumor number (multiple vs. single) | 2.393 | 1.597–3.582 | 0.000 | 1.802 | 1.194–2.719 | 0.005 |
Histopathological grade (poorly vs. well + moderately) | 1.665 | 1.148–2.415 | 0.007 | |||
PVTT (present vs. absent) | 2.826 | 1.947–4.102 | 0.000 | 2.075 | 1.418–3.037 | 0.000 |
Preoperative AFP level, ng/ml (>400 vs. ≤400) | 2.380 | 1.622–3.492 | 0.000 | 1.539 | 1.027–2.305 | 0.037 |
TNM stage (III + IV vs. I + II) | 4.584 | 2.987–7.034 | 0.000 | |||
PVT1 expression (high vs. low) | 1.738 | 1.082–2.792 | 0.022 | 1.653 | 1.019–2.681 | 0.042 |
P<0.05
HR, hazard ratio; CI, confidence interval; HBV, hepatitis B virus; PVTT, portal vein tumor thrombosis; AFP, α-fetoprotein; TNM, tumor-node-metastasis.