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miR-744 is a potential prognostic marker in patients with hepatocellular carcinoma
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miR-744 is a potential prognostic marker in patients with hepatocellular carcinoma Yu-Le Tan a, Zhi-Gang Bai a,b, Wei-Long Zou c, Xue-Mei Ma a, Ting-Ting Wang a, Wei Guo a, Jun Liu a, Jiang-She Li a, Jie-Yin a, Yun-Jin Zang c,∗∗, Zhong-Tao Zhang a,∗ a
Department of General Surgery, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China b Beijing Key Laboratory of Translational Medicine in Liver Cirrhosis, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China c Organ Transplantation Institute, the General Hospital of Chinese People’s Armed Police Forces, Beijing 100039 China
Summary Identification of new biomarkers for aggressiveness of hepatocellular carcinoma (HCC) to supplement the current group of prognosis algorithms is a significant clinical need. To clarify expression levels of microRNA-744 (miR-744) in HCC tissues and to explore its clinicopathological significance in HCC patients following liver transplantation (LT), we quantified miR-744 using real-time quantitative reverse transcription polymerase chain reaction in 96 paired cancerous tissues and para-cancerous normal liver tissues. We investigated relationships among miR-744 expression, clinicopathological parameters, and overall survival (OS). Of 96 paired samples, 68 cancer tissues expressed low miR-744 compared with their matched normal liver tissues. Patients with microvascular invasion or multi-tumor nodules showed significantly lower miR-744 expression; miR-744 was further decreased in patients with post-LT HCC recurrence compared with non-recurring patients. Patients with lower miR-744 expression showed significantly poorer recurrence-free survival and OS than individuals with higher miR-744 levels. Multivariate analysis revealed that lower miR-744 was an independent predictor of poor prognosis. Our results associate decreased miR-744 expression with HCC recurrence and prognosis, and also suggest that miR-744 is an independent predictor of survival in HCC patients after LT and may therefore be a potential biomarker for their prognosis. © 2014 Published by Elsevier Masson SAS.
∗ Corresponding author. Department of General Surgery, Beijing Friendship Hospital, Capital Medical University (CCMU), 95, Yong-an Road, Xicheng District, Beijing 100050, China. ∗∗ Corresponding author. Organ Transplantation Institute, the General Hospital of Chinese People’s Armed Police Forces (GHCAPF), 69, Yongding Road, Beijing 100039, China. E-mail addresses: [email protected] (Y.-J. Zang), [email protected] (Z.-T. Zhang).
http://dx.doi.org/10.1016/j.clinre.2014.09.010 2210-7401/© 2014 Published by Elsevier Masson SAS.
Please cite this article in press as: Tan Y-L, et al. miR-744 is a potential prognostic marker in patients with hepatocellular carcinoma. Clin Res Hepatol Gastroenterol (2014), http://dx.doi.org/10.1016/j.clinre.2014.09.010
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Introduction Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is a major cause of cancer mortalities particularly in Africa and Asia [1,2]. The most common causes of HCC are chronic infection with hepatitis B or hepatitis C virus [3]. Mean survival of patients with HCC is estimated to be 6—20 months without intervention [4]. Curative treatment of liver cancer is currently limited to surgical resection and liver transplantation (LT) [5]. However, only a small percentage of HCCs can be surgically resected [6]. LT is considered a potential cure for unresectable HCC, because it removes both the tumor and diseased liver at risk of malignant transformation. HCC recurrence is the main factor countering the curative effect fold for patients with HCC. Considering the limitations of imaging studies and the limited sensitivity of the laboratory analyses available, the identification of new prognostic markers may be useful in guiding post-LT surveillance. MicroRNAs (miRNAs) are small noncoding RNAs of 18—24 nucleotides that modulate gene expression by recognition of complementary sequences within the 3-untranslated region of targeted genes. MiRNAs are key components of functional pathways that control important cellular process, such as tumor cell proliferation, differentiation, development, apoptosis, and invasiveness [7]. Abnormal miRNA expressions function as both oncogenes and tumor-suppressor genes [7,8]. Although several miRNAs have been shown to be aberrantly expressed in HCC [9—11], their involvement in hepatocarcinogenesis and HCC development is not well understood. Recently, a tumor-suppressor factor, miR-744, was found to be downregulated in several tissues [12—16]. MiR-744 can inhibit HCC cell growth by targeting c-Myc [16]. However, to our knowledge, no study has reported on the association between miR-744 and HCC prognosis after LT. In our current study, we evaluated miR-744 expression levels in 96 paired cancerous tissues and their adjacent non-cancerous hepatic tissues using quantitative reverse transcription polymerase chain reaction (RT-PCR) of FFPE samples, and found miR-744 to be significantly downregulated in HCC tissues. We also investigated the association of miR-744 expression with HCC patients after LT prognosis, and its clinical variables.
Materials and methods Patients and tissue samples From June 2009 to December 2013, 96 patients underwent LT for HCC at the Liver Transplantation Surgery Unit, Chinese Armed Police General Hospital, Beijing, China. We obtained follow-up data about these patients. Inclusion criteria included the ‘‘up-to-seven’’ transplantation criteria for HCC [17]. Patients with large, liver vessel HCC invasions or extra-hepatic metastases, or who were aged younger than18 years were excluded. Posttransplant monitoring of recurrence was performed by
typical imaging and/or biopsy. Human primary HCC and adjacent non-tumor liver tissues (> 1 cm from the tumor) were collected from formalin-fixed paraffin-embedded (FFPE) tissue blocks. Tumor tissues and adjacent nontumor liver tissues were confirmed histologically. Clinical information was collected from patient records (Table 1). Median recurrence-free period was 10 months for patients with HCC recurrence and 23 months for patients without recurrence. Our study was approved by the Bioethics Committee of the Chinese Armed Police General Hospital, and each patient gave prior written informed consent and approval.
Isolation of total RNA FFPE tissue Total cellular RNA with efficient recovery of small RNA was isolated from 8 × 10 m sections of FFPE tissues using the RecoverAll Total Nucleic Acid Isolation Kit (P/N, AM1975; Invitrogen, Shanghai, China) according to the manufacturer’s instructions. RNA concentrations were determined spectrophotometrically. RNA quality was confirmed using a Fisher Scientific NanoDrop 2000 Spectrophotometer.
Quantitative real-time RT-PCR For reverse transcription, we used 200 ng of total RNA and specific stem-loop RT primer for miR-744; and for endogenous control, U6snRNA from a Taqman miR-744/U6snRNA assay (ID: 000395 and ID: 002324; Life Technologies, Foster City, CA, USA) in each reaction and mixed with the TaqMan MicroRNA Reverse Transcription Kit (P/N 4366597; Life Technologies) with the following thermal conditions: 16 ◦ C for 30 min, 42 ◦ C for 30 min, and 85 ◦ C for 5 min. Quantitative RT-PCR amplification reactions used TaqMan microRNA assays (P/N 4440887; Life Technologies) as described previously [18]. U6snRNA was used as an internal control to normalize and quantify miR-744 expression. Relative expression levels were calculated by the 2−Ct method. Quantitative PCR was quantified with an ABI PRISM 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA).
Statistical analysis Results represent mean ± standard deviation of three independent experiments. Two-sided Student’s t-test and the Mann—Whitney U test were applied to evaluate differences between groups. The chi2 test or the Fisher’s exact test was used for analysis of categorical data. Multivariate Cox proportional hazard regression analysis was used to evaluate the contribution of independent factors to patient’s survival; only factors that were found to be significant in the univariate analysis were taken as covariates. All calculations were performed by GraphPad Prism 6 software (GraphPad Software, Inc, CA, USA). P < 0.05 was considered statistically significant.
Please cite this article in press as: Tan Y-L, et al. miR-744 is a potential prognostic marker in patients with hepatocellular carcinoma. Clin Res Hepatol Gastroenterol (2014), http://dx.doi.org/10.1016/j.clinre.2014.09.010
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miR-744 is a potential prognostic marker in patients with hepatocellular carcinoma Table 1
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Correlation between miR-744 expression and clinicopathological characteristics of HCC patients following LT.
Parameter
Age Sex Male Female Etiology of liver disease HBV Others Cirrhosis Yes No Meld score Milan criteria In Out Histologic grade Well/moderately Poorly Tumor stage I + II III Tumor size (cm) ≤5 >5 Tumor nodes Multi Single Microvascular invasion Yes No Pre-LT serum AFP level > 400 g/L ≤ 400 g/L HCC recurrence Overall survival
n
MiR-744 relevant expression level
P value
Low
High
96
51.54 ± 8.882
54.04 ± 8.285
0.157a
86 10
46 4
40 6
0.513b
85 11
45 5
40 6
0.640b
90
45 3 11.23 ± 5.412
1.000c
96
46 2 10.13 ± 4.788
50 46
26 22
24 24
0.683b
80 18
44 10
36 8
0.966b
69 27
35 15
34 12
0.670b
60 36
29 19
31 17
0.673b
40 56
25 23
15 33
0.038b
23 73
16 32
7 41
0.031b
36 60 33/96 64/96
19 29 25/48 23/48
17 31 8/48 41/48
0.673b
0.292a
— —
AFP: alpha-fetoprotein, MELD: model for end-stage liver disease. a Unpaired student’s t-test. b Chi2 test. c Fisher’s exact test.
Results MiR-744 expression in HCC is lower than in matched adjacent non-cancerous hepatic tissues To understand the expression and significance of miR-744 in HCC, we first detected miR-744 expression levels in a training set of 96 paired cancerous tissues and adjacent non-cancerous hepatic tissues, using real-time RT-PCR. MiR744 expression was significantly downregulated in 70.8% (68/96, P = 0.009) of HCC tissues (Fig. 1A) compared with their adjacent tissues. We next examined correlation of miR-744 expression with clinicopathologic factors (Table 1). Patients with microvascular invasion or multi-tumor nodules expressed significantly lower MiR-744 levels than those without them (P < 0.05; Fig. 1F and G). Moreover, miR-744 expression was further decreased in patients with post-LT
HCC recurrence compared with those with non-recurrence (P = 0.002; Fig. 1B). A similar trend was seen in patients who died after LT compared with patients who still survive (P = 0.007; Fig. 1C).
Decreased miR-744 expression is associated with tumor malignancy We analyzed associations between miR-744 expression (based on real-time PCR readings) and clinicopathological features, and found decreased miR-744 expression in HCC to be associated with significantly malignant behavior, such as microvascular invasion (P = 0.031) and multiple tumor nodes (P = 0.038) (Table 1); and conversely, MiR-744 expression in patients with microvascular invasion or multi-tumor nodules was significantly lower than in those without them (P < 0.05; Fig. 1A and B).
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Figure 1 Downregulation of miR-744 in HCC is associated with tumor recurrence and poor prognosis following LT. A. The expression of miR-744 was examined in cancerous tissues (T) and their adjacent non-cancerous hepatic tissues (N). B. The expression levels of miR-744 were further downregulated in HCC samples of patients with tumor recurrence after LT. C. The expression levels of miR-744 were much lower in the patients who had died after LT than the patients who still survived. D and E. Kaplan—Meier analyses of survival curve for patients with high and low miR-744 expression levels. F. The expression of miR-744 was analyzed statistically in patients with microvascular invasion or without. v: microvascular invasion; G. The expression of miR-744 was analyzed statistically in patients with multi-tumor nodules and single tumor nodule. M: multi-tumor nodules; S: single tumor nodule.
Decreased miR-744 expression correlates with HCC recurrence and poor prognosis Kaplan—Meier analysis showed decreased miR-744 expression (i.e.< median level) to correlate with shorter recurrence-free survival (RFS; P = 0.006; Fig. 1D) and
overall survival (OS; P = 0.015; Fig. 1E) for post-LT HCC patients. Univariate Cox regression analyses showed miR-744 expression to be significant associated with OS (P < 0.001; hazard ratio [HR]: 5.947; 95% confidence interval [CI]: 2.542—13.913; Table 3), and RFS (P < 0.001; HR: 5.609; 95% CI: 2.385—13.191; Table 2).
Please cite this article in press as: Tan Y-L, et al. miR-744 is a potential prognostic marker in patients with hepatocellular carcinoma. Clin Res Hepatol Gastroenterol (2014), http://dx.doi.org/10.1016/j.clinre.2014.09.010
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miR-744 is a potential prognostic marker in patients with hepatocellular carcinoma Table 2
Univariate and multivariate Cox regression analyses of recurrence-free survival in 96 HCC patients following LT.
Parameter
Univariate analysis
Age Gender HBV infection Cirrhosis MELD score Histologic grade Tumor diameter (> 5 cm) Milan criteria (out) AFP > 400 (g/L) Microvascular invasion MiR-744 (low) *
Multivariable analysis
HR
95% CI
P value
HR
95% CI
P value
1.044 2.007 1.355 1.573 1.022 1.042 3.060 4.078 3.528 6.932 5.609
0.853—1.806 0.478—8.422 0.412—4.450 0.228—12.286 0.958—1.091 0.401—2.709 1.503—6.232 1.828—9.136 1.718—7.247 3.121—15.393 2.385—13.191
0.156 0.341 0.617 0.613 0.511 0.932 0.002 0.001 0.001 < 0.001 < 0.001
— — — — — — 2.839 1.283 2.438 3.784 4.196
— — — — — — 1.070—7.538 0.402—4.096 1.090—5.457 1.635—8.761 1.736—10.138
— — — — — — 0.036* 0.674 0.030* 0.002* 0.001*
Statistically significant difference.
MiR-744 independently predicts survival of post-LT HCC patients Some LT studies have recommended various candidate selection criteria and compared them with the standard Milan rules [19,20]. Maximum tumor size and biological factors, such as alfa-fetoprotein (AFP) have been extensively used to predict OS in post-LT HCC patients [21—24]. Univariate Cox regression for OS and RFS in the present patient cohort revealed that Milan criteria, tumor diameter, AFP, and microvascular invasion were strongly predictive of OS and RFS (Tables 2 and 3). Multivariate Cox regression analysis showed tumor diameter (HR = 2.716, P = 0.034), microvascular invasion (HR = 5.921, P < 0.001), and decreased miR-744 expression (HR = 4.177, P = 0.002) were independently associated with poor survival (Table 3).
Discussion MicroRNAs are critical to both physiological and pathological processes, including tumor development and progression
Table 3
[7,9,25]. Some miRNAs have even been identified as cancer markers or prognostic markers for cancer patients [9,10,26]. Some downregulated miRNAs contribute to tumor progression by decreasing apoptosis and promoting invasiveness and proliferation, which rely on their target genes. Certain miRNAs are associated with clinical outcomes in colorectal cancer [26], pancreatic cancer [27], breast cancer [28], and HCC [29]. miR-744 is known to suppress tumor factors and was found to be downregulated in breast cancer tissues and renal cancer tissues [12,13]. Here, we are the first to describe miR-744 expression levels and their correlation with different clinicopathological parameters in post-LT HCC patients. Post-transplant HCC recurrence can appear in two situations: when extra-hepatic metastasis was not detectable during the pre-transplant work-up; and as a consequence of circulating HCC cells engrafting and growing in a target organ during the peri-transplant period [30]. These two mechanisms may be associated with tumor cell aggressiveness. Expression profiling of microRNAs may be a more accurate method of classifying cancer subtypes
Univariate and multivariate Cox regression analyses of overall survival in 96 HCC patients following LT.
Parameter
Age Gender HBV infection Cirrhosis MELD score Tumor grade Tumor diameter (> 5 cm) Milan criteria (out) AFP > 400 (g/L) Microvascular invasion MiR-744 (low) *
5
Univariate analysis
Multivariable analysis
HR
95%CI
P value
HR
95%CI
P value
1.098 2.182 1.260 1.022 1.025 1.179 3.825 4.202 3.388 12.494 5.947
0.734—2.985 0.521—9.146 0.384—4.137 0.138—7.592 0.958—1.097 0.454—3.063 1.844—7.935 1.873—9.423 1.643—6.986 5.307—15.416 2.542—13.913
0.574 0.286 0.704 0.983 0.473 0.736 < 0.001 < 0.001 0.001 < 0.001 < 0.001
— — — — — — 2.716 1.776 1.623 5.921 4.177
— — — — — — 1.076—6.858 0.594—5.308 0.703—3.746 2.256—15.543 1.670—10.452
— — — — — — 0.034* 0.304 0.257 < 0.001* 0.002*
Statistically significant difference.
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than using the expression profiles of protein-coding genes [31]. Therefore, identification of novel miRNAs to predict aggressiveness in HCC is an important clinical need. Although the existence of miR-744 was confirmed by Landgraf et al. [32], investigations of its function have only appeared in the last few years. Studies that have associated downregulated miR-744 with tumor cell proliferation and invasion/migration [12,14,16] are consistent with our findings that miR-744 expression is decreased in HCC tissues. Moreover, decreased miR-744 expression was associated with tumor malignancy. Decreased miR744 expression was also correlated with HCC recurrence and poor prognosis. To the best of our knowledge, this is the first study showing a clear relationship between miR-744 and invasiveness, metastasis, and prognosis in HCC. Biomarkers for tumor behavior may more accurately predict patient outcomes than conventional pathology-based assessments. Circulating and serum miRNA levels have been reported as prognostic markers in HCC [10,33], and could potentially augment HCC diagnostic techniques, which in some cases are based on imaging techniques without tumor biopsy. A study of patients with gastric cancer (GC) showed serum miR-744 levels could distinguish early GCs from controls, and revealed a trend of increased miR-744 levels during GC development [34]. Serum miR-744 levels also reportedly correlate with OS in patients with myeloma [35]. In agreement with those studies, we found miR-744 expression in HCC tissues to correlate with prognosis in post-LT HCC patients, and to independently predict their survival. Unfortunately, serum miR-744 levels have not been studied in HCC patients, although investigations to detect serum miR-744 levels in patients with liver diseases and to validate their prognostic significance are clearly warranted. Mechanisms of miR-744 downregulation in various tumor tissues could be related to different target genes for miR744, which have been reported in some cancers. Eukaryotic translation elongation factor 1A2 (EEF1A2) is a reported target of miR-744 in MCF7 breast cancer cells; miR-744 overexpression reduced EEF1A2 mRNA by 50%, thus retarding MCF7 cell proliferation [14]. In the HER2-amplified breast cell lines KPL-4 and JIMT-1, HER2 was a direct target of miR-744 [12]. Cyclin B1 was identified as a positive regulation target of miR-744 in mouse cells, in which short-term miR-744 overexpression enhanced proliferation, whereas prolonged expression caused chromosomal instability and in vivo tumor suppression [15]. Our studies correlated decreased miR-744 expression in HCC with microvascular invasion and multiple tumor nodes. Deregulated miRNAs can contribute to tumor proliferation, survival, or invasiveness, depending on their target genes. However, the list of direct targets of miR-744 is far from complete. Moreover, miR744 targets have not been fully understood in HCC; their identification and function warrant study going forward, as they may open up new therapeutic targets or biomarkers for cancers. In conclusion, we have shown that miR-744 expression levels were decreased in FFPE HCC tissues and are a significant prognostic factor for HCC patients. Our results indicate that miR-744 is a potential biomarker for survival of post-LT HCC patients.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgments The study was supported by Beijing Key Laboratory of Translational Medicine in Liver Cirrhosis, Beijing Friendship Hospital, Capital Medical University.
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ORIGINAL ARTICLE
miR-744 is a potential prognostic marker in patients with hepatocellular carcinoma Yu-Le Tan a, Zhi-Gang Bai a,b, Wei-Long Zou c, Xue-Mei Ma a, Ting-Ting Wang a, Wei Guo a, Jun Liu a, Jiang-She Li a, Jie-Yin a, Yun-Jin Zang c,∗∗, Zhong-Tao Zhang a,∗ a
Department of General Surgery, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China b Beijing Key Laboratory of Translational Medicine in Liver Cirrhosis, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China c Organ Transplantation Institute, the General Hospital of Chinese People’s Armed Police Forces, Beijing 100039 China
Summary Identification of new biomarkers for aggressiveness of hepatocellular carcinoma (HCC) to supplement the current group of prognosis algorithms is a significant clinical need. To clarify expression levels of microRNA-744 (miR-744) in HCC tissues and to explore its clinicopathological significance in HCC patients following liver transplantation (LT), we quantified miR-744 using real-time quantitative reverse transcription polymerase chain reaction in 96 paired cancerous tissues and para-cancerous normal liver tissues. We investigated relationships among miR-744 expression, clinicopathological parameters, and overall survival (OS). Of 96 paired samples, 68 cancer tissues expressed low miR-744 compared with their matched normal liver tissues. Patients with microvascular invasion or multi-tumor nodules showed significantly lower miR-744 expression; miR-744 was further decreased in patients with post-LT HCC recurrence compared with non-recurring patients. Patients with lower miR-744 expression showed significantly poorer recurrence-free survival and OS than individuals with higher miR-744 levels. Multivariate analysis revealed that lower miR-744 was an independent predictor of poor prognosis. Our results associate decreased miR-744 expression with HCC recurrence and prognosis, and also suggest that miR-744 is an independent predictor of survival in HCC patients after LT and may therefore be a potential biomarker for their prognosis. © 2014 Published by Elsevier Masson SAS.
∗ Corresponding author. Department of General Surgery, Beijing Friendship Hospital, Capital Medical University (CCMU), 95, Yong-an Road, Xicheng District, Beijing 100050, China. ∗∗ Corresponding author. Organ Transplantation Institute, the General Hospital of Chinese People’s Armed Police Forces (GHCAPF), 69, Yongding Road, Beijing 100039, China. E-mail addresses: [email protected] (Y.-J. Zang), [email protected] (Z.-T. Zhang).
http://dx.doi.org/10.1016/j.clinre.2014.09.010 2210-7401/© 2014 Published by Elsevier Masson SAS.
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Introduction Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is a major cause of cancer mortalities particularly in Africa and Asia [1,2]. The most common causes of HCC are chronic infection with hepatitis B or hepatitis C virus [3]. Mean survival of patients with HCC is estimated to be 6—20 months without intervention [4]. Curative treatment of liver cancer is currently limited to surgical resection and liver transplantation (LT) [5]. However, only a small percentage of HCCs can be surgically resected [6]. LT is considered a potential cure for unresectable HCC, because it removes both the tumor and diseased liver at risk of malignant transformation. HCC recurrence is the main factor countering the curative effect fold for patients with HCC. Considering the limitations of imaging studies and the limited sensitivity of the laboratory analyses available, the identification of new prognostic markers may be useful in guiding post-LT surveillance. MicroRNAs (miRNAs) are small noncoding RNAs of 18—24 nucleotides that modulate gene expression by recognition of complementary sequences within the 3-untranslated region of targeted genes. MiRNAs are key components of functional pathways that control important cellular process, such as tumor cell proliferation, differentiation, development, apoptosis, and invasiveness [7]. Abnormal miRNA expressions function as both oncogenes and tumor-suppressor genes [7,8]. Although several miRNAs have been shown to be aberrantly expressed in HCC [9—11], their involvement in hepatocarcinogenesis and HCC development is not well understood. Recently, a tumor-suppressor factor, miR-744, was found to be downregulated in several tissues [12—16]. MiR-744 can inhibit HCC cell growth by targeting c-Myc [16]. However, to our knowledge, no study has reported on the association between miR-744 and HCC prognosis after LT. In our current study, we evaluated miR-744 expression levels in 96 paired cancerous tissues and their adjacent non-cancerous hepatic tissues using quantitative reverse transcription polymerase chain reaction (RT-PCR) of FFPE samples, and found miR-744 to be significantly downregulated in HCC tissues. We also investigated the association of miR-744 expression with HCC patients after LT prognosis, and its clinical variables.
Materials and methods Patients and tissue samples From June 2009 to December 2013, 96 patients underwent LT for HCC at the Liver Transplantation Surgery Unit, Chinese Armed Police General Hospital, Beijing, China. We obtained follow-up data about these patients. Inclusion criteria included the ‘‘up-to-seven’’ transplantation criteria for HCC [17]. Patients with large, liver vessel HCC invasions or extra-hepatic metastases, or who were aged younger than18 years were excluded. Posttransplant monitoring of recurrence was performed by
typical imaging and/or biopsy. Human primary HCC and adjacent non-tumor liver tissues (> 1 cm from the tumor) were collected from formalin-fixed paraffin-embedded (FFPE) tissue blocks. Tumor tissues and adjacent nontumor liver tissues were confirmed histologically. Clinical information was collected from patient records (Table 1). Median recurrence-free period was 10 months for patients with HCC recurrence and 23 months for patients without recurrence. Our study was approved by the Bioethics Committee of the Chinese Armed Police General Hospital, and each patient gave prior written informed consent and approval.
Isolation of total RNA FFPE tissue Total cellular RNA with efficient recovery of small RNA was isolated from 8 × 10 m sections of FFPE tissues using the RecoverAll Total Nucleic Acid Isolation Kit (P/N, AM1975; Invitrogen, Shanghai, China) according to the manufacturer’s instructions. RNA concentrations were determined spectrophotometrically. RNA quality was confirmed using a Fisher Scientific NanoDrop 2000 Spectrophotometer.
Quantitative real-time RT-PCR For reverse transcription, we used 200 ng of total RNA and specific stem-loop RT primer for miR-744; and for endogenous control, U6snRNA from a Taqman miR-744/U6snRNA assay (ID: 000395 and ID: 002324; Life Technologies, Foster City, CA, USA) in each reaction and mixed with the TaqMan MicroRNA Reverse Transcription Kit (P/N 4366597; Life Technologies) with the following thermal conditions: 16 ◦ C for 30 min, 42 ◦ C for 30 min, and 85 ◦ C for 5 min. Quantitative RT-PCR amplification reactions used TaqMan microRNA assays (P/N 4440887; Life Technologies) as described previously [18]. U6snRNA was used as an internal control to normalize and quantify miR-744 expression. Relative expression levels were calculated by the 2−Ct method. Quantitative PCR was quantified with an ABI PRISM 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA).
Statistical analysis Results represent mean ± standard deviation of three independent experiments. Two-sided Student’s t-test and the Mann—Whitney U test were applied to evaluate differences between groups. The chi2 test or the Fisher’s exact test was used for analysis of categorical data. Multivariate Cox proportional hazard regression analysis was used to evaluate the contribution of independent factors to patient’s survival; only factors that were found to be significant in the univariate analysis were taken as covariates. All calculations were performed by GraphPad Prism 6 software (GraphPad Software, Inc, CA, USA). P < 0.05 was considered statistically significant.
Please cite this article in press as: Tan Y-L, et al. miR-744 is a potential prognostic marker in patients with hepatocellular carcinoma. Clin Res Hepatol Gastroenterol (2014), http://dx.doi.org/10.1016/j.clinre.2014.09.010
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miR-744 is a potential prognostic marker in patients with hepatocellular carcinoma Table 1
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Correlation between miR-744 expression and clinicopathological characteristics of HCC patients following LT.
Parameter
Age Sex Male Female Etiology of liver disease HBV Others Cirrhosis Yes No Meld score Milan criteria In Out Histologic grade Well/moderately Poorly Tumor stage I + II III Tumor size (cm) ≤5 >5 Tumor nodes Multi Single Microvascular invasion Yes No Pre-LT serum AFP level > 400 g/L ≤ 400 g/L HCC recurrence Overall survival
n
MiR-744 relevant expression level
P value
Low
High
96
51.54 ± 8.882
54.04 ± 8.285
0.157a
86 10
46 4
40 6
0.513b
85 11
45 5
40 6
0.640b
90
45 3 11.23 ± 5.412
1.000c
96
46 2 10.13 ± 4.788
50 46
26 22
24 24
0.683b
80 18
44 10
36 8
0.966b
69 27
35 15
34 12
0.670b
60 36
29 19
31 17
0.673b
40 56
25 23
15 33
0.038b
23 73
16 32
7 41
0.031b
36 60 33/96 64/96
19 29 25/48 23/48
17 31 8/48 41/48
0.673b
0.292a
— —
AFP: alpha-fetoprotein, MELD: model for end-stage liver disease. a Unpaired student’s t-test. b Chi2 test. c Fisher’s exact test.
Results MiR-744 expression in HCC is lower than in matched adjacent non-cancerous hepatic tissues To understand the expression and significance of miR-744 in HCC, we first detected miR-744 expression levels in a training set of 96 paired cancerous tissues and adjacent non-cancerous hepatic tissues, using real-time RT-PCR. MiR744 expression was significantly downregulated in 70.8% (68/96, P = 0.009) of HCC tissues (Fig. 1A) compared with their adjacent tissues. We next examined correlation of miR-744 expression with clinicopathologic factors (Table 1). Patients with microvascular invasion or multi-tumor nodules expressed significantly lower MiR-744 levels than those without them (P < 0.05; Fig. 1F and G). Moreover, miR-744 expression was further decreased in patients with post-LT
HCC recurrence compared with those with non-recurrence (P = 0.002; Fig. 1B). A similar trend was seen in patients who died after LT compared with patients who still survive (P = 0.007; Fig. 1C).
Decreased miR-744 expression is associated with tumor malignancy We analyzed associations between miR-744 expression (based on real-time PCR readings) and clinicopathological features, and found decreased miR-744 expression in HCC to be associated with significantly malignant behavior, such as microvascular invasion (P = 0.031) and multiple tumor nodes (P = 0.038) (Table 1); and conversely, MiR-744 expression in patients with microvascular invasion or multi-tumor nodules was significantly lower than in those without them (P < 0.05; Fig. 1A and B).
Please cite this article in press as: Tan Y-L, et al. miR-744 is a potential prognostic marker in patients with hepatocellular carcinoma. Clin Res Hepatol Gastroenterol (2014), http://dx.doi.org/10.1016/j.clinre.2014.09.010
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Figure 1 Downregulation of miR-744 in HCC is associated with tumor recurrence and poor prognosis following LT. A. The expression of miR-744 was examined in cancerous tissues (T) and their adjacent non-cancerous hepatic tissues (N). B. The expression levels of miR-744 were further downregulated in HCC samples of patients with tumor recurrence after LT. C. The expression levels of miR-744 were much lower in the patients who had died after LT than the patients who still survived. D and E. Kaplan—Meier analyses of survival curve for patients with high and low miR-744 expression levels. F. The expression of miR-744 was analyzed statistically in patients with microvascular invasion or without. v: microvascular invasion; G. The expression of miR-744 was analyzed statistically in patients with multi-tumor nodules and single tumor nodule. M: multi-tumor nodules; S: single tumor nodule.
Decreased miR-744 expression correlates with HCC recurrence and poor prognosis Kaplan—Meier analysis showed decreased miR-744 expression (i.e.< median level) to correlate with shorter recurrence-free survival (RFS; P = 0.006; Fig. 1D) and
overall survival (OS; P = 0.015; Fig. 1E) for post-LT HCC patients. Univariate Cox regression analyses showed miR-744 expression to be significant associated with OS (P < 0.001; hazard ratio [HR]: 5.947; 95% confidence interval [CI]: 2.542—13.913; Table 3), and RFS (P < 0.001; HR: 5.609; 95% CI: 2.385—13.191; Table 2).
Please cite this article in press as: Tan Y-L, et al. miR-744 is a potential prognostic marker in patients with hepatocellular carcinoma. Clin Res Hepatol Gastroenterol (2014), http://dx.doi.org/10.1016/j.clinre.2014.09.010
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miR-744 is a potential prognostic marker in patients with hepatocellular carcinoma Table 2
Univariate and multivariate Cox regression analyses of recurrence-free survival in 96 HCC patients following LT.
Parameter
Univariate analysis
Age Gender HBV infection Cirrhosis MELD score Histologic grade Tumor diameter (> 5 cm) Milan criteria (out) AFP > 400 (g/L) Microvascular invasion MiR-744 (low) *
Multivariable analysis
HR
95% CI
P value
HR
95% CI
P value
1.044 2.007 1.355 1.573 1.022 1.042 3.060 4.078 3.528 6.932 5.609
0.853—1.806 0.478—8.422 0.412—4.450 0.228—12.286 0.958—1.091 0.401—2.709 1.503—6.232 1.828—9.136 1.718—7.247 3.121—15.393 2.385—13.191
0.156 0.341 0.617 0.613 0.511 0.932 0.002 0.001 0.001 < 0.001 < 0.001
— — — — — — 2.839 1.283 2.438 3.784 4.196
— — — — — — 1.070—7.538 0.402—4.096 1.090—5.457 1.635—8.761 1.736—10.138
— — — — — — 0.036* 0.674 0.030* 0.002* 0.001*
Statistically significant difference.
MiR-744 independently predicts survival of post-LT HCC patients Some LT studies have recommended various candidate selection criteria and compared them with the standard Milan rules [19,20]. Maximum tumor size and biological factors, such as alfa-fetoprotein (AFP) have been extensively used to predict OS in post-LT HCC patients [21—24]. Univariate Cox regression for OS and RFS in the present patient cohort revealed that Milan criteria, tumor diameter, AFP, and microvascular invasion were strongly predictive of OS and RFS (Tables 2 and 3). Multivariate Cox regression analysis showed tumor diameter (HR = 2.716, P = 0.034), microvascular invasion (HR = 5.921, P < 0.001), and decreased miR-744 expression (HR = 4.177, P = 0.002) were independently associated with poor survival (Table 3).
Discussion MicroRNAs are critical to both physiological and pathological processes, including tumor development and progression
Table 3
[7,9,25]. Some miRNAs have even been identified as cancer markers or prognostic markers for cancer patients [9,10,26]. Some downregulated miRNAs contribute to tumor progression by decreasing apoptosis and promoting invasiveness and proliferation, which rely on their target genes. Certain miRNAs are associated with clinical outcomes in colorectal cancer [26], pancreatic cancer [27], breast cancer [28], and HCC [29]. miR-744 is known to suppress tumor factors and was found to be downregulated in breast cancer tissues and renal cancer tissues [12,13]. Here, we are the first to describe miR-744 expression levels and their correlation with different clinicopathological parameters in post-LT HCC patients. Post-transplant HCC recurrence can appear in two situations: when extra-hepatic metastasis was not detectable during the pre-transplant work-up; and as a consequence of circulating HCC cells engrafting and growing in a target organ during the peri-transplant period [30]. These two mechanisms may be associated with tumor cell aggressiveness. Expression profiling of microRNAs may be a more accurate method of classifying cancer subtypes
Univariate and multivariate Cox regression analyses of overall survival in 96 HCC patients following LT.
Parameter
Age Gender HBV infection Cirrhosis MELD score Tumor grade Tumor diameter (> 5 cm) Milan criteria (out) AFP > 400 (g/L) Microvascular invasion MiR-744 (low) *
5
Univariate analysis
Multivariable analysis
HR
95%CI
P value
HR
95%CI
P value
1.098 2.182 1.260 1.022 1.025 1.179 3.825 4.202 3.388 12.494 5.947
0.734—2.985 0.521—9.146 0.384—4.137 0.138—7.592 0.958—1.097 0.454—3.063 1.844—7.935 1.873—9.423 1.643—6.986 5.307—15.416 2.542—13.913
0.574 0.286 0.704 0.983 0.473 0.736 < 0.001 < 0.001 0.001 < 0.001 < 0.001
— — — — — — 2.716 1.776 1.623 5.921 4.177
— — — — — — 1.076—6.858 0.594—5.308 0.703—3.746 2.256—15.543 1.670—10.452
— — — — — — 0.034* 0.304 0.257 < 0.001* 0.002*
Statistically significant difference.
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than using the expression profiles of protein-coding genes [31]. Therefore, identification of novel miRNAs to predict aggressiveness in HCC is an important clinical need. Although the existence of miR-744 was confirmed by Landgraf et al. [32], investigations of its function have only appeared in the last few years. Studies that have associated downregulated miR-744 with tumor cell proliferation and invasion/migration [12,14,16] are consistent with our findings that miR-744 expression is decreased in HCC tissues. Moreover, decreased miR-744 expression was associated with tumor malignancy. Decreased miR744 expression was also correlated with HCC recurrence and poor prognosis. To the best of our knowledge, this is the first study showing a clear relationship between miR-744 and invasiveness, metastasis, and prognosis in HCC. Biomarkers for tumor behavior may more accurately predict patient outcomes than conventional pathology-based assessments. Circulating and serum miRNA levels have been reported as prognostic markers in HCC [10,33], and could potentially augment HCC diagnostic techniques, which in some cases are based on imaging techniques without tumor biopsy. A study of patients with gastric cancer (GC) showed serum miR-744 levels could distinguish early GCs from controls, and revealed a trend of increased miR-744 levels during GC development [34]. Serum miR-744 levels also reportedly correlate with OS in patients with myeloma [35]. In agreement with those studies, we found miR-744 expression in HCC tissues to correlate with prognosis in post-LT HCC patients, and to independently predict their survival. Unfortunately, serum miR-744 levels have not been studied in HCC patients, although investigations to detect serum miR-744 levels in patients with liver diseases and to validate their prognostic significance are clearly warranted. Mechanisms of miR-744 downregulation in various tumor tissues could be related to different target genes for miR744, which have been reported in some cancers. Eukaryotic translation elongation factor 1A2 (EEF1A2) is a reported target of miR-744 in MCF7 breast cancer cells; miR-744 overexpression reduced EEF1A2 mRNA by 50%, thus retarding MCF7 cell proliferation [14]. In the HER2-amplified breast cell lines KPL-4 and JIMT-1, HER2 was a direct target of miR-744 [12]. Cyclin B1 was identified as a positive regulation target of miR-744 in mouse cells, in which short-term miR-744 overexpression enhanced proliferation, whereas prolonged expression caused chromosomal instability and in vivo tumor suppression [15]. Our studies correlated decreased miR-744 expression in HCC with microvascular invasion and multiple tumor nodes. Deregulated miRNAs can contribute to tumor proliferation, survival, or invasiveness, depending on their target genes. However, the list of direct targets of miR-744 is far from complete. Moreover, miR744 targets have not been fully understood in HCC; their identification and function warrant study going forward, as they may open up new therapeutic targets or biomarkers for cancers. In conclusion, we have shown that miR-744 expression levels were decreased in FFPE HCC tissues and are a significant prognostic factor for HCC patients. Our results indicate that miR-744 is a potential biomarker for survival of post-LT HCC patients.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgments The study was supported by Beijing Key Laboratory of Translational Medicine in Liver Cirrhosis, Beijing Friendship Hospital, Capital Medical University.
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