- Email: [email protected]
Relationship Between Serum microRNA155 and Telomerase Expression in Hepatocellular Carcinoma
Archives of Medical Research 47 (2016) 349e355
ORIGINAL ARTICLE
Relationship Between Serum microRNA155 and Telomerase Expression in Hepatocellular Carcinoma Wafaa M. Ezzat,a Khalda Said Amr,b Haiam Abdel Raouf,c Yasser A. Elhosary,a Abdelfattah E. Hegazy,d Hoda H. Fahim,e and Refaat R. Kamelf b
a Department of Internal Medicine, National Research Centre, Cairo, Egypt Department of Medical Molecular Genetics, National Research Centre, Cairo, Egypt c Department of Immunogenetics, National Research Centre, Cairo, Egypt d Department of Surgery, Elsahel Teaching Hospital, Cairo, Egypt e Department of Anesthesia, Elsahel Teaching Hospital, Cairo, Egypt f Department of Surgery, Faculty of Medicine, Ain Shams University, Cairo, Egypt
Received for publication January 24, 2016; accepted August 4, 2016 (ARCMED-D-16-00042).
Background and Aims. Activation of telomerase reverse transcriptase (hTERT) has a role in liver carcinogenesis where telomerase is normally suppressed in most human somatic tissues after birth. In the current study we aimed to detect the significance of hTERT mRNA in early detection of hepatocellular carcinoma (HCC) and to determine the relationship between serum microRNA155 and telomerase expression. Methods. Serum and liver tissue samples were collected from 40 patients (17 samples from patients with liver cirrhosis and 23 samples from patients with HCC) and 12 blood samples from healthy subjects were collected. Serum miRNA155 levels and blood and tissue hTERT mRNA were detected by real-time quantitative reverse-transcriptase PCR (RT-qPCR) among liver cirrhosis and HCC patients. Moreover, miR-155, blood and tissue hTERT levels were analyzed in relation to clinical and pathological features. Results. Calculated expression of miRNA155 revealed that relative quantity (RQ) miR 155 was overexpressed in sera of HCC patients when compared to patients with liver cirrhosis and controls ( p !0.0001). The median values of serum telomerase were significantly increased among HCC patients than in patients with liver cirrhosis and controls ( p 5 0.04). Moreover, tissue expression of telomerase was significantly higher in malignant tissue more than adjacent nonmalignant tissue among HCC patients ( p 5 0.02). It was also found that tissue expression of telomerase was significantly decreased in tissue of liver cirrhosis patients ( p 5 0.03). Interestingly, we found that blood telomerase was directly correlated with serum miRNA155 ( p 5 0.003). Conclusions. Both mir 155 and telomerase may have a role in development of HCC and mir 155 could regulate telomerase expression during liver carcinogenesis. Ó 2016 IMSS. Published by Elsevier Inc. Key Words: miR 155, Telomerase, hTERT-HCC.
Introduction Liver carcinogenesis is a major health problem in Egypt. Incidence of hepatocellular carcinoma (HCC) is increasing and is becoming clinically more and more dangerous (1). Address reprint requests to: Wafaa M. Ezzat, Professor of Gastroenterology and Hepatology, Internal Medicine Department National Research Center, Elbohoos St., Dokki, P.O. 12311, Cairo, Egypt; Phone: 01006063558; FAX: 202-33371433; E-mail: [email protected].
Telomerase is a ribonucleoprotein enzyme consisting of two components: a catalytic component (human telomerase reverse transcriptase, hTERT) and a ribonucleic acid (RNA) format. It contributes to settle the length of the telomere in stem cells, reproductive cells and malignant cells through gathering TTAGGG repeats onto the telomeres utilizing its fundamental RNA as a matrix for reverse transcription. Moreover, it is a component that can decrease telomerase activity (2,3).
0188-4409/$ - see front matter. Copyright Ó 2016 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2016.08.003
350
Ezzat et al./ Archives of Medical Research 47 (2016) 349e355
Telomeres of vertebrate are composed of TTAGGG similar repeats, which contain the specialized protein complex ‘‘shelterin’’ (4). Shelterin regulates several telomere functions and consists of six core components. Telomere Repeat Factor 1 (TRF1) and Telomere Repeat Factor 2 (TRF2) bind double-stranded DNA and Protection of Telomere 1 (POT1) binds to the single-stranded telomeric 3’ overhang (5). DNA binding shelterin proteins cooperate with Repressor/Activator Protein 1 (RAP1), TINT1, PTOP, PIP1 (TPP1) and TRF1- and TRF2-Interacting Nuclear Protein 2 (TIN2) to make a feasible composition. TRF1, TRF2 and POT1 have a key function in telomere saving by concealing the activation of the DNA deterioration coding and repair pathways at chromosome ends (6). Moreover, shelterin regulates telomere length and recurrence of homologous recombination between telomeric sister chromatids (7). In early tumorigenesis, cells tolerate comprehensive growth until telomere length becomes shortened (8). Telomere shortening occurs early during liver carcinogenesis, occurring in premalignant dysplastic nodules. Shortened telomeres have been reported to cause chromosomal instability in liver cells, especially useful in viral-related liver carcinogenesis (9). Furthermore, miRNAs are pivotal modulators of gene expression that work through several carcinogenesis pathways and render as dynamic promising biomarkers (10). Although telomere function is a cornerstone in cancer and aging, miRNAs that influence control of telomere function in human cancer have recently been determined (10). Looking at breast cancer model cell lines, Dinami et al. proved that miR-155 regulates telomere function and homeostasis by decreasing the expression of TRF1. Specifically, it was found that miR-155 upregulates chromosome fragility at telomeric repeats, encourages telomeric sister chromatid fusions and pushes telomere dysfunction as detected by a recruitment of DNA damage factors at chromosome ends (11). With this in mind, the function of TRF1 is a negative regulator of telomere length, reducing TRF1 expression by ectopic introduction of miR-155 mediating telomere elongation (12). This indicates that miR-155 can increase genomic instability by suppressing telomere function in human breast cancer. These results showed for the first time that miRNAs can modulate the role of shelterin in human cancer and assume the existence of additional ‘‘telo-miRNAs’’ that can gather key events in carcinogenesis with telomere function and homeostasis (11). In the current study we aim to clarify the relation of circulating microRNA155 to blood and tissue expression of telomerase in both HCC and liver cirrhosis compared to a control group. We found that both miR-155 and telomerase may have a role in development of HCC and miR-155 could regulate telomerase expression during liver carcinogenesis.
Patients and Methods HCC tissues and adjacent non-tumor tissues (NTs) used for qRT-PCR were collected from 23 HCC patients with liver cirrhosis (Figure 1) and tissues of chronic hepatitis from 17 patients who underwent liver resection or living donor liver transplantation (LDLT) between June 2011 and June 2013. Tissues were snap frozen in Qiazol immediately after resection and then stored at 80 C until use. At the same time, 10 mL blood samples were withdrawn from every patient and from 12 healthy subjects with no evidence of liver diseases and collected in tubes containing EDTA, kept on ice and transferred to the laboratory for processing of extraction of total RNA and miRNA. Adult patients 18e70 years old of both sexes who were suffering from chronic liver diseases with and without primary HCC and were eligible for LDLT or liver resection were enrolled in the current study. Patients with other chronic debilitating diseases, those O70 years old, patients with malignant diseases other than HCC, patients with metastatic liver tumors or patients who were ineligible for LDLTor liver resection were excluded from the study. Demographic and clinical data of patients are presented in Table 1. The study was approved by the local ethics committee at the National Research Center and all patients provided written informed consent. Histopathological Examination Tumor staging was performed according to the American Joint Committee on Cancer and International Union against Cancer (AJCC/UICC) staging system (6th edition, 2002). Histological tumor grading was performed according to the Edmondson-Steiner classification: grade 1e2 (well differentiated), grade 3 (moderately differentiated), and grade 4 (poorly differentiated) (13).
Figure 1. Ultasonographic picture of hepatocellular carcinoma with hypoechoic area with liver cirrhosis as a background.
microRNA155 and Telomerase in Hepatocellular Carcinoma Table 1. Demographic, clinical, biochemical and pathological data of the two study groups of patients
Variables mean ± SD Age (years) Sex Male no. (%) Female no. (%) PH OF anti-Sch.ttt Negative no. (%) Positive no. (%) AST IU/L median (range) ALT IU/L median (range) Albumin g/dL median (range) TB mg/dL median (range) AFP ng/mL median (range) CA19-9 IU/mL median (range) CEA IU/mL median (range) CHOL mg/dL mean SD TRG mg/dL mean SD HDL mg/dL mean SD LDL mg/dL mean SD Cause of chronic liver disease HCV No (%) HBV No (%) PBC No (%) PSC no. (%) HCC lesions Number 1 no. (%) Multiple No (%) Site Rt. lobe no. (%) Lt. lobe no. (%) Both no. (%) Size cm
HCC group n [ 23
Chronic hepatitis group n [ 17
54.27 9.18
45.23 9.36
22 (95.7) 1 (4.3)
15 (88.2) 2 (11.8)
9 14 98 31.5 2
(39.1) (60.9) (33e300) (16e213) (1.5e3)
9 8 53 59 2.5
(52.9) (47.1) (33e83) (26e102) (1.8e3.5)
1.7 (1e2.3) 21 (8e7460) 32.4 (2e55)
1.5 (1e2.1) 6 (1.60e84) 45.8 (17.67e100.2)
2.1 (0.08e3.4) 108.00 16.35 91.20 44.69 41.00 18.29 42.30 18.46
3.04 (1.17e16.8) 121.66 33.29 74.00 13.11 28.00 15.11 114.00 16.35
20 (86.9) 3 (13.1)
10 5 1 1
(58.8) (29.4) (5.9) (5.9)
11 (47.8) 12 (42.2)
took place immediately within 2 h from blood sample collection. Extracted miRNA were subjected to reverse transcription. miRNA RT-PCR TaqMan miRNA assays (Applied Biosystems, Foster City, CA) was used to quantify the expression levels of mature miR-155 by miRNA reverse transcription Kit (Applied Biosystems) was reverse transcribed in a reaction mixture containing miR-specific stem-loop RT primers. qPCR was performed with 3 mL of each cDNA on a Step One TMPlus Real-Time PCR System (ABI) in duplicate reactions containing the prepared cDNA and TaqMan specific primers in Universal Master Mix without AmpErase UNG (Applied Biosystems) and threshold cycles (CT) were calculated using Sequence Detection Software (SDS v2.2.1, Applied Biosystems). miRNA quantification data were normalized to 18S RNA. All miRNA data are expressed relative to a RNU48 small nuclear (sn) RNA TaqMan PCR performed on the same samples. Fold expression was calculated from the mean CT values and relative quantity (RQ) of miRNA155 was calculated by the formula: RQ52DDCt where Ct is defined as the fractional cycle number at which the fluorescence generated by cleavage of the probe passes a fixed threshold above baseline. Total RNA Extraction and Quantification
10 (43.5) 1 (4.3) 12 (42.2) 3.65 1.75
Grade I no. (%) II no. (%) Type Trabecular no. (%) Mixed no. (%) Liver background 1-Mixed cirrhosis with mild activity, no. (%) 2-Mixed cirrhosis with moderate activity, no. (%)
351
4 (17.4) 19 (82.6) 9 (39.1) 14 (40.9) 2 (8.7)
5 (29.4)
21 (91.3)
12 (70.6)
PH OF anti-Sch.ttt, past history of antischistosomal treatment; AST, aspartate transaminase; ALT, alanine transaminase; AFP, a-fetoprotein; TB, total bilirubin; CHOL, cholesterol; TRG, triglycerides; HDL, high-density lipoprotein; LDL, low-density lipoprotein.
miRNA Extraction and Quantification Isolation of miRNAs from (tissue and blood) of patients and blood only of controls followed the protocol for miRNA easy RNA isolation kit (Qiagen). Separation of serum
Total RNA was extracted from blood samples and from 10e25 mg of tested tissue by disruption and homogenization using a RNA extraction kit (Qiagen) according to the manufacturer’s instructions and the extracted total RNA was eluted in 30 mL of RNase-free water. cDNA was synthesized using High-Capacity cDNA reverse transcription kit (Applied Biosystems). In brief, 10 mL of RNA (1 mg) was reverse transcribed in a 20-mL reaction containing 2 mL RT buffer, 0.8 mL dNTPs, 2 mL random hexmar primers, 1 mL RT enzyme MultiScribeTM reverse transcriptase and 4.2 mL nuclease-free H2O. The reverse transcription was performed on Perkin Elmer 9700 thermocycler condition (25 C for 10 min, 37 C for 120 min followed by 85 C for 5 min and hold at 4 C). hTERT mRNA qRT-PCR Assay To determine hTERT mRNA, real-time qRT-PCR assays were constructed using TaqManÒ Gene Expression Assay (Applied Biosystems). The target gene sequence was the catalytic subunit of hTERT and the calibrator sample is healthy control. The reference gene (housekeeping gene) was glyceraldehyde phosphate dehydrogenase (GAPDH). A singleplex reaction was used in this study. RT-PCR was carried out using TaqMan Universal PCR Master Mix.
352
Ezzat et al./ Archives of Medical Research 47 (2016) 349e355
PCR reaction mix was added to the cDNA samples in a sterile 48-well PCR plate. The prepared reaction components were done in One Step PCR (Applied Biosystems) using real-time cycler conditions of 50 C for 2 min (reverse transcription), 95 C, 10 min (initial denaturation) followed by 40 cycles of 95 C, 15 sec, 60 C, 1 min and 72 C, 1 min for denaturation, annealing, extension steps, respectively, using assay primers (Applied Biosystems). Total RNA hTERT gene quantification data were normalized to GAPDH gene and data analyzed according to the RQ manager program ABI SDS software (ABI 7900) using the previous RQ formula (Livak and Schmittgen, 2001). Statistical Analysis Data are expressed as median or mean SD unless otherwise indicated. Categorical data are described as frequency of the subjects with a specific characteristic. c2 test or Fisher’s exact test was used for comparing categorical data and Student t-test, Mann-Whitney U test, one way ANOVA or Kruskal-Wallis test, when appropriate, was used for comparing continuous variables. ROC curve was conducted to determine the specificity and sensitivity of serum mir 155 and serum telomerase as diagnostic markers for HCC. Twotailed p values !0.05 were considered statistically significant. Statistical analysis was performed using SPSS software v.12.0 (SPSS Inc., Chicago, IL). Results Two groups of patients were enrolled in the current study; HCC group consists of 23 patients (22 males and one female) with a mean age of 54.27 9.18 years. Chronic hepatitis group consisted of 17 patients (15 males and 2 females) with a mean age of 45.23 9.36 years. Twelve healthy subjects were enrolled as a control group. Frequency of past history of schistosomiasis was 60.9% among HCC group, whereas it was 47.1% among patients of the chronic hepatitis group. Distribution of causes of chronic liver diseases among the studied groups of patients revealed that the most common cause was HCV infection, especially among the HCC group followed by HBV infection. One case of primary biliary cirrhosis (PBC) and another case
of primary sclerosing cholangitis (PSC) were found among the chronic hepatitis group as shown in Table 1. Statistical analysis revealed that there were no significant differences between the two studied groups of patients as regards liver function tests. As a traditional marker for HCC, a-fetoprotein (AFP) was significantly increased among the HCC group of patients. Macroscopic examination of HCC lesions revealed that distribution of these lesions were more prevalent in both right and left lobes, with a mean size of 3.65 1.75 cm. Microscopic examination of liver tissues of the HCC group of patients revealed that grade 2 differentiation and mixed type were more prevalent than grade 1 and trabecular type. With regard to the liver background, it was found that the frequency of mixed cirrhosis with moderate activity was higher in the two studied groups of patients than mixed cirrhosis with mild activity (Table 1). Calculated expression of miRNA155 revealed that RQ miR 155 was overexpressed in sera of patients with HCC compared to patients with chronic hepatitis and controls ( p !0.0001). miRNA 155 was directly correlated to AFP ( p 5 0.001, significant) and serum total bilirubin ( p 5 0.037, significant). The median values of blood telomerase were significantly higher among HCC patients than cirrhotic patients and controls ( p 5 0.04). Moreover, tissue expression of telomerase was significantly higher in malignant tissue more than adjacent nonmalignant tissue among HCC patients ( p 5 0.02). We also found that tissue expression of telomerase was significantly lower in tissue of chronic hepatitis group ( p 5 0.03) (Table 2). There was a significant direct correlation between tissue and serum telomerase ( p 5 0.007). Interestingly, we found a significant direct correlation between serum telomerase and miRNA155 ( p 5 0.003) (Table 3). Our results also revealed that there was no significant impact of tumor characteristics regarding number; site; size and microscopic features of blood; tissue expression of telomerase or serum values of miRNA155. It was found that miR 155 could predict HCC at a level of 6.3 with 95.5% sensitivity and 76.5% specificity. Blood telomerase could predict HCC at a level of 1.08 with a sensitivity of 81.2% and specificity of 62.5% (Table 4).
Table 2. Comparison between HCC group and chronic hepatitis group as regards mean values of serum; tissue telomerase and serum miR 155
mirRNAs Serum miR 155 mean SD Serum telomerase Tissue telomerase
HCC group n [ 23
Chronic hepatitis group n [ 17
Control group n [ 12
10.25 3.31 1.59 (0.06e25.0) Malignant tissues Nonmalignant tissues 13.08 (0.15e942.1) 9.78 (0.19e783.8) 0.02*
5.18 2.45 1.02 (0.001e5.45) 2.87 (0.23e15.45)
1.1 0.7 0.17 (0.015e0.72)
*p value is significant; **p value is highly significant.
p 0.0001** 0.04* 0.03*
microRNA155 and Telomerase in Hepatocellular Carcinoma Table 3. Correlations between serum miRNA 155 and serum telomerase Variables Serum miR 155
Serum telomerase
Tissue telomerase
Correlation coefficient: 0.535 p 5 0.003a
Correlation coefficient: 0.373 p 5 0.073 Correlation coefficient: 0.668 p 5 0.007a
Serum telomerase a
p value was significant.
Discussion HCC is a complex malignancy with many risk factors and underlying mechanisms. Promising diagnostic markers are needed for liver carcinogenesis (14). Liver cirrhosis with its regenerative nodules usually represents the background for HCC development. The existence of large regenerative nodules frequently interferes with the accurate diagnosis of small HCC. Therefore, useful markers for the diagnosis of HCC have been sought (15). Important biomarkers such as AFP-b1-mRNA, insulin growth factor 2 (IGF-2) mRNA, telomerase, etc. were secreted by malignant hepatocytes. These molecular markers could be used for early HCC detection (16). In agreement with Waguri et al. who proved that the existence of circulating malignant cells detached from original HCC tissues in blood and can detect hTERT mRNA in blood (17), the present study suggests that quantification of hTERT mRNAs in blood has diagnostic implications for HCC as the study results indicated that the mean blood values of hTERT mRNAs were significantly increased among patients with HCC when compared to patients with liver cirrhosis. Moreover, tissue expression of telomerase was significantly increased in cancerous tissue when compared to adjacent nonmalignant tissue among patients with HCC. In a previous Egyptian study, Abdel Hady et al. concluded that the telomerase activity assay could be used for liver cancer diagnosis and is more sensitive than other diagnostic markers. Therefore, activity of telomerase is a promising, available diagnostic and prognostic marker for liver carcinogenesis diagnosis (18). Kong et al. analyzed hTERT in peripheral blood in 343 Korean patients with liver cancer. hTERT expression was not related to clinical Table 4. Diagnostic performance of serum miR 155 and telomerase in comparison with a-fetoprotein
Group HCC
Parameter
AUC
Cutoff value
Sensitivity %
Specificity %
Serum miR 155 Serum telomerase Serum a-fetoprotein
0.924 0.724 0.832
6.3 1.08 8.050
95.5 81.2 100.0
76.5 62.5 69.2
HCC, hepatocellular carcinoma; AUC, area under the curve.
353
manifestations and AFP. They suggested that AFP and hTERT mRNA expression in peripheral blood could not be used as diagnostic markers (19). Telomerase is considered a useful specific candidate tumor marker; it could not be applied practically because telomerase expression has not been detected stably in body fluid (20). In serum, the hTERT mRNA derived from cancer cells seemed to be undetectable because it becomes instable by RNase in blood. Because RNAs in serum are interestingly stable within 24 h after drawing blood due to particle-associated complex in structure (21,22), it has been suggested that they can be generally detected even in RNase-rich blood. Actually, hTERT mRNA can be detected in serum from breast cancer patients and its sensitivity and specificity are, at most, 40 and 100%, respectively (23). The sensitivity in patients with HCC rose to 89.7% in the semiquantitative assay. A previous study reported that hTERT expression was very low in serum from normal persons because lymphocytes and circulating normal cells express very low levels of hTERT mRNA (9). As hTERT mRNA in lymphocytes is very low, increased hTERT mRNA values in serum may signify that hTERT mRNA is derived from malignant cells. As we can detect very small amounts of lymphocyte markers after three steps of centrifugation of blood samples, the RNA extraction procedure appeared to clear lymphocytes properly. Moreover, healthy or injured hepatocytes express very small amounts of hTERT (24,25). The significant correlation of hTERT mRNA expression between malignant tissue and serum was then proven (26). These data suggest that hTERT mRNA determined in serum is delivered from cancer cells. In the current study we found a direct correlation of telomerase in blood with telomerase in tissue samples, which certifies the usefulness of telomerase as a noninvasive marker for early HCC detection. All studied patients underwent liver transplantation. Our results also revealed that there was no significant impact of tumor characteristics as regards number, site, size and microscopic features in blood; tissue expression of telomerase or serum values of miRNA155.This finding may be interpreted as most HCC patients show the same pathological characteristics as size, grade 2 of differentiation and mixed type. We think that more aggressive types of HCC make patients unable to withstand liver transplantation surgery. Therefore, we cannot judge the impact of HCC characteristics on the studied markers among patients who underwent liver transplantation as we did not have reasonable numbers with different HCC characteristics. To understand the underlying mechanism by which telomerase shares in HCC development, some authors suggested that telomere shortening results in chromosomal instability (CIS), which leads to cancer development. Previous studies suggested that telomeres were shorter in malignant liver cells when compared to regenerative nodules and
354
Ezzat et al./ Archives of Medical Research 47 (2016) 349e355
healthy liver tissue and that, within the group of HCC, hepatocyte telomere length of aneuploid was shorter than that of diploid tumors. On the other hand, advancement of carcinogenesis needs an effective telomere signaling. Other studies (27) found a slight increase of telomere length in low differentiated liver cancer when compared to highly differentiated liver cancer, suggesting a reactivation of telomerase and recovered chromosomal stability to a level convenient to malignant cell survival. Accordingly, several studies have proved that telomerase activity was determined in nearly 90% of malignant liver tissues as compared to only 21% of non-malignant tissue and was accompanied with the increase of TERT mRNA values, implicating the feasibility that TERT mRNA expression could detect early HCC (28e31). With the advancing research in miRNA biology, the prospective role of miRNAs in carcinogenesis will probably expand in the near future (32). In the current study, it was found that miR155 was upregulated in sera of HCC patients. In regard to the contribution of miR-155 in the pathogenesis of malignancy, it targets numerous molecules in key coding pathways like glutathione metabolism, stressactivated protein kinase/c-Jun N-terminal kinases (SAPK/ JNK), toll-like receptor (TLR), extracellular-signalregulated kinases/mitogen-activated protein kinase (ERK/ MAPK), and B-cell receptor signaling. MiR-155 was also applied in increasing programmed cell death through targeting anti-apoptotic operators (33). TRF1 is a telomere repeat binding protein that works to shorten telomere length, contributes to mitotic stability, and protects from replication-associated DNA damage (6,34). In regard to the relation between miR 155 and telomerase in carcinogenesis, Dinami et al. demonstrated that miR155 directly controls TRF1 expression levels by specifically targeting a partially conserved sequence motif in the 30 untranslational region (30 UTR) of TRF1, leading to translational repression. They attempted to link miR-155 to molecular pathways that modulate TRF1-related aspects of telomere function. To test whether alteration of miR155 expression levels not only impacts on global TRF1 expression levels but also alters TRF1 abundance at telomeres, they transiently transfected SK-BR-3 luminal breast cancer cells with mimic-miR-155, antago-miR-155 siRNAs, or TRF1-specific siRNAs and performed quantitative immunofluorescence analysis using anti-TRF1 antibodies. They found that ectopically introduced miR-155 significantly reduced fluorescence signal intensity, indicative for a reduced abundance of TRF1 at telomeres. Together they concluded that miR-155-dependent regulation of TRF1 is an efficient mechanism to control telomere fragility and genomic stability in human breast cancer cells. The consistent upregulation of miR-155 in breast cancer suggests that impaired telomere function is a cornerstone of the carcinogenic mission of miR-155 that increases genomic instability in human breast cancer and contributes to decreased
distant metastasis-free survival and recurrence-free survival in ERþ breast cancer (35). In a trial to change basic research into applied research, we performed this study to clarify more than one factor cooperating in HCC development. Liver carcinogenesis is multistep with underlying multiple factors, which may act in parallel to each other or they may act step by step, each in its role. Altogether, these players represent a big challenge in the face of conventional therapeutic modalities for HCC. The time has come to try to discover the underlying mechanisms for hepatocarcinogenesis to pave the way for development of tailored therapy for those patients. According to our findings, serum mir 155 and blood telomerase have diagnostic abilities and could be used as early detectors for HCC. From a diagnostic point of view, miR has reasonable sensitivity and specificity for HCC detection. Depending on senescence blocking, telomerase reactivation to block the attrition of the hepatocyte capacity of regeneration may be useful, relying on the effect of this method on liver carcinogenesis. More efforts must be made in this area of research for understanding telomere biology in human disease and malignancies and to detect the most appropriate and effective therapy (36). Although miR155 has a suggestive role in regulation of telomerase expression during liver carcinogenesis, therapeutic intervention targeting miR155 may, at the same time, control telomerase activity. In conclusion, both miR 155 and telomerase expression may play a role in development of HCC and miR 155 could regulate telomerase expression during liver carcinogenesis.
References 1. Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74e108. 2. Nakayama J, Tahara H, Tahara E, et al. Telomerase activation by hTERT in human normal fibroblasts and hepatocellular carcinomas. Nat Genet 1998;18:65e68. 3. Nakamura TM, Morin GB, Chapman KB, et al. Telomerase catalytic subunit homologs from fission yeast and human. Science 1997;277: 955e959. 4. Palm W, de Lange T. How shelterin protects mammalian telomeres. Annu Rev Genet 2008;42:301e334. 5. Denchi EL, de Lange T. Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1. Nature 2007;448: 1068e1071. 6. Sfeir A, Kosiyatrakul ST, Hockemeyer D, et al. Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication. Cell 2009;138:90e103. 7. Cesare AJ, Reddel RR. Alternative lengthening of telomeres: models, mechanisms and implications. Nat Rev Genet 2010;11:319e330. 8. Murnane JP, Sabatier L. Chromosome rearrangements resulting from telomere dysfunction and their role in cancer. Bioassays 2004;26: 1164e1174. 9. Oh BK, Kim H, Park YN, et al. High telomerase activity and long telomeres in advanced hepatocellular carcinoma with poor prognosis. Lab Invest 2008;88:144e152. 10. Iorio MV, Croce CM. MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review. EMBO Mol Med 2012;4:143e159.
microRNA155 and Telomerase in Hepatocellular Carcinoma 11. Dinami R, Petti E, Sestito R, et al. microRNAs control the function of telomeres in cancer. RNA Dis 2014;1:e282. 12. Mu~ noz P, Blanco R, de Carcer G, et al. TRF1 controls telomere length and mitotic fidelity in epithelial homeostasis. Mol Cell Biol 2009;29: 1608e1625. 13. Greene FL, Page DL, Fleming ID. AJCC Cancer Staging Manual. 6th ed New York: Springer; 2002. 14. Aravalli RN, Steer CJ, Cressman ENK. Molecular mechanisms of hepatocellular carcinoma. Hepatology 2008;48:2047e2063. 15. Takahashi S, Kitamoto M, Takaishi H, et al. Expression of telomerase component genes in hepatocellular carcinomas. Eur J Cancer 2000;36: 496e502. 16. Miura N, Shiota G, Nakagawa T, et al. Sensitive detection of hTERT mRNA in the serum of patients with hepatocellular carcinoma. Oncology 2003;64:430e434. 17. Waguri N, Suda T, Nomoto M, et al. Sensitive and specific detection of circulating cancer cells in patients with hepatocellular carcinoma; detection of human telomerase reverse transcriptase messenger RNA after immunomagnetic separation. Clin Cancer Res 2003;9:3004e3011. 18. Hady AA, El Shanawany F, Hassan MM, et al. Evaluation of human telomerase activity as a novel tumor marker for hepatocellular carcinoma. Life Sci J 2010;7:153e161. 19. Kong SY, Park JW, Kim JO, et al. Alpha-fetoprotein and human telomerase reverse transcriptase mRNA levels in peripheral blood of patients with hepatocellular carcinoma. J Cancer Res Clin Oncol 2009; 135:1091e1098. 20. Tatsuma T, Goto S, Kitano S, et al. Telomerase activity in peripheral blood for diagnosis of hepatoma. J Gastroenterol Hepatol 2000;15: 1064e1070. 21. Ng EK, Tsui NB, Lam NY, et al. Presence of filterable and nonfilterable mRNA in the plasma of cancer patients and healthy individuals. Clin Chem 2002;48:1212e1217. 22. Tsui NB, Ng EK, Lo YM. Stability of endogeneous and added RNA in blood specimens, serum, and plasma. Clin Chem 2002;48:1647e1653. 23. Chen XQ, Bonnefoi H, Pelte MF, et al. Telomerase RNA as a detection marker in the serum of breast cancer patients. Clin Cancer Res 2000;6: 3823e3826.
355
24. Onishi T, Nouso K, Higashi T, et al. Cellular distribution of telomerase reverse transcriptase in human hepatocellular carcinoma. J Gastroenterol Hepatol 2003;18:1168e1174. 25. Wege H, Chui MS, Le HT, et al. In vitro expansion of human hepatocytes is restricted by telomere-dependent replicative aging. Cell Transplant 2003;12:897e906. 26. Miura N, Maeda Y, Kanbe T, et al. Serum human telomerase reverse transcriptase messenger RNA as a novel tumor marker for hepatocellular carcinoma. Clin Cancer Res 2005;1:3205e3209. 27. Urabe Y, Nouso K, Higashi T, et al. Telomere length in human liver diseases. Liver 1996;16:293e297. 28. Nagao K, Tomimatsu M, Endo H, et al. Telomerase reverse transcriptase mRNA expression and telomerase activity in hepatocellular carcinoma. J Gastroenterol 1999;34:83e87. 29. Shimojima M, Komine F, Hisatomi H, et al. Detection of telomerase activity, telomerase RNA component, and telomerase reverse transcriptase in human hepatocellular carcinoma. Hepatol Res 2004;29: 31e38. 30. Lee CM, Hsu CY, Eng HL, et al. Telomerase activity and telomerase catalytic subunit in hepatocellular carcinoma. Hepatogastroenterology 2004;51:796e800. 31. Kojima H, Yokosuka O, Imazeki F, et al. Telomerase activity and telomere length in hepatocellular carcinoma and chronic liver disease. Gastroenterology 1997;112:493e500. 32. Baek S, Cho KJ, Ro SW. Functional analysis of micro RNAs overexpressed in hepatocellular carcinoma. RNA Dis 2015;2:e728. 33. Higgs G, Slack F. The multiple roles of microRNA-155 in oncogenesis. J Clin Bioinforma 2013;3:17. 34. Martınez P, Thanasoula M, Mu~noz P, et al. Increased telomere fragility and fusions resulting from TRF1 deficiency lead to degenerative pathologies and increased cancer in mice. Genes Dev 2009;23: 2060e2075. 35. Dinami R, Ercolani C, Petti E, et al. miR-155 drives telomere fragility in human breast cancer by targeting TRF1. Cancer Res 2014;74: 4145e4156. 36. Carulli L, Anzivino C. Telomere and telomerase in chronic liver disease and hepatocarcinoma. World J Gastroenterol 2014;20:6287e6292.
ORIGINAL ARTICLE
Relationship Between Serum microRNA155 and Telomerase Expression in Hepatocellular Carcinoma Wafaa M. Ezzat,a Khalda Said Amr,b Haiam Abdel Raouf,c Yasser A. Elhosary,a Abdelfattah E. Hegazy,d Hoda H. Fahim,e and Refaat R. Kamelf b
a Department of Internal Medicine, National Research Centre, Cairo, Egypt Department of Medical Molecular Genetics, National Research Centre, Cairo, Egypt c Department of Immunogenetics, National Research Centre, Cairo, Egypt d Department of Surgery, Elsahel Teaching Hospital, Cairo, Egypt e Department of Anesthesia, Elsahel Teaching Hospital, Cairo, Egypt f Department of Surgery, Faculty of Medicine, Ain Shams University, Cairo, Egypt
Received for publication January 24, 2016; accepted August 4, 2016 (ARCMED-D-16-00042).
Background and Aims. Activation of telomerase reverse transcriptase (hTERT) has a role in liver carcinogenesis where telomerase is normally suppressed in most human somatic tissues after birth. In the current study we aimed to detect the significance of hTERT mRNA in early detection of hepatocellular carcinoma (HCC) and to determine the relationship between serum microRNA155 and telomerase expression. Methods. Serum and liver tissue samples were collected from 40 patients (17 samples from patients with liver cirrhosis and 23 samples from patients with HCC) and 12 blood samples from healthy subjects were collected. Serum miRNA155 levels and blood and tissue hTERT mRNA were detected by real-time quantitative reverse-transcriptase PCR (RT-qPCR) among liver cirrhosis and HCC patients. Moreover, miR-155, blood and tissue hTERT levels were analyzed in relation to clinical and pathological features. Results. Calculated expression of miRNA155 revealed that relative quantity (RQ) miR 155 was overexpressed in sera of HCC patients when compared to patients with liver cirrhosis and controls ( p !0.0001). The median values of serum telomerase were significantly increased among HCC patients than in patients with liver cirrhosis and controls ( p 5 0.04). Moreover, tissue expression of telomerase was significantly higher in malignant tissue more than adjacent nonmalignant tissue among HCC patients ( p 5 0.02). It was also found that tissue expression of telomerase was significantly decreased in tissue of liver cirrhosis patients ( p 5 0.03). Interestingly, we found that blood telomerase was directly correlated with serum miRNA155 ( p 5 0.003). Conclusions. Both mir 155 and telomerase may have a role in development of HCC and mir 155 could regulate telomerase expression during liver carcinogenesis. Ó 2016 IMSS. Published by Elsevier Inc. Key Words: miR 155, Telomerase, hTERT-HCC.
Introduction Liver carcinogenesis is a major health problem in Egypt. Incidence of hepatocellular carcinoma (HCC) is increasing and is becoming clinically more and more dangerous (1). Address reprint requests to: Wafaa M. Ezzat, Professor of Gastroenterology and Hepatology, Internal Medicine Department National Research Center, Elbohoos St., Dokki, P.O. 12311, Cairo, Egypt; Phone: 01006063558; FAX: 202-33371433; E-mail: [email protected].
Telomerase is a ribonucleoprotein enzyme consisting of two components: a catalytic component (human telomerase reverse transcriptase, hTERT) and a ribonucleic acid (RNA) format. It contributes to settle the length of the telomere in stem cells, reproductive cells and malignant cells through gathering TTAGGG repeats onto the telomeres utilizing its fundamental RNA as a matrix for reverse transcription. Moreover, it is a component that can decrease telomerase activity (2,3).
0188-4409/$ - see front matter. Copyright Ó 2016 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2016.08.003
350
Ezzat et al./ Archives of Medical Research 47 (2016) 349e355
Telomeres of vertebrate are composed of TTAGGG similar repeats, which contain the specialized protein complex ‘‘shelterin’’ (4). Shelterin regulates several telomere functions and consists of six core components. Telomere Repeat Factor 1 (TRF1) and Telomere Repeat Factor 2 (TRF2) bind double-stranded DNA and Protection of Telomere 1 (POT1) binds to the single-stranded telomeric 3’ overhang (5). DNA binding shelterin proteins cooperate with Repressor/Activator Protein 1 (RAP1), TINT1, PTOP, PIP1 (TPP1) and TRF1- and TRF2-Interacting Nuclear Protein 2 (TIN2) to make a feasible composition. TRF1, TRF2 and POT1 have a key function in telomere saving by concealing the activation of the DNA deterioration coding and repair pathways at chromosome ends (6). Moreover, shelterin regulates telomere length and recurrence of homologous recombination between telomeric sister chromatids (7). In early tumorigenesis, cells tolerate comprehensive growth until telomere length becomes shortened (8). Telomere shortening occurs early during liver carcinogenesis, occurring in premalignant dysplastic nodules. Shortened telomeres have been reported to cause chromosomal instability in liver cells, especially useful in viral-related liver carcinogenesis (9). Furthermore, miRNAs are pivotal modulators of gene expression that work through several carcinogenesis pathways and render as dynamic promising biomarkers (10). Although telomere function is a cornerstone in cancer and aging, miRNAs that influence control of telomere function in human cancer have recently been determined (10). Looking at breast cancer model cell lines, Dinami et al. proved that miR-155 regulates telomere function and homeostasis by decreasing the expression of TRF1. Specifically, it was found that miR-155 upregulates chromosome fragility at telomeric repeats, encourages telomeric sister chromatid fusions and pushes telomere dysfunction as detected by a recruitment of DNA damage factors at chromosome ends (11). With this in mind, the function of TRF1 is a negative regulator of telomere length, reducing TRF1 expression by ectopic introduction of miR-155 mediating telomere elongation (12). This indicates that miR-155 can increase genomic instability by suppressing telomere function in human breast cancer. These results showed for the first time that miRNAs can modulate the role of shelterin in human cancer and assume the existence of additional ‘‘telo-miRNAs’’ that can gather key events in carcinogenesis with telomere function and homeostasis (11). In the current study we aim to clarify the relation of circulating microRNA155 to blood and tissue expression of telomerase in both HCC and liver cirrhosis compared to a control group. We found that both miR-155 and telomerase may have a role in development of HCC and miR-155 could regulate telomerase expression during liver carcinogenesis.
Patients and Methods HCC tissues and adjacent non-tumor tissues (NTs) used for qRT-PCR were collected from 23 HCC patients with liver cirrhosis (Figure 1) and tissues of chronic hepatitis from 17 patients who underwent liver resection or living donor liver transplantation (LDLT) between June 2011 and June 2013. Tissues were snap frozen in Qiazol immediately after resection and then stored at 80 C until use. At the same time, 10 mL blood samples were withdrawn from every patient and from 12 healthy subjects with no evidence of liver diseases and collected in tubes containing EDTA, kept on ice and transferred to the laboratory for processing of extraction of total RNA and miRNA. Adult patients 18e70 years old of both sexes who were suffering from chronic liver diseases with and without primary HCC and were eligible for LDLT or liver resection were enrolled in the current study. Patients with other chronic debilitating diseases, those O70 years old, patients with malignant diseases other than HCC, patients with metastatic liver tumors or patients who were ineligible for LDLTor liver resection were excluded from the study. Demographic and clinical data of patients are presented in Table 1. The study was approved by the local ethics committee at the National Research Center and all patients provided written informed consent. Histopathological Examination Tumor staging was performed according to the American Joint Committee on Cancer and International Union against Cancer (AJCC/UICC) staging system (6th edition, 2002). Histological tumor grading was performed according to the Edmondson-Steiner classification: grade 1e2 (well differentiated), grade 3 (moderately differentiated), and grade 4 (poorly differentiated) (13).
Figure 1. Ultasonographic picture of hepatocellular carcinoma with hypoechoic area with liver cirrhosis as a background.
microRNA155 and Telomerase in Hepatocellular Carcinoma Table 1. Demographic, clinical, biochemical and pathological data of the two study groups of patients
Variables mean ± SD Age (years) Sex Male no. (%) Female no. (%) PH OF anti-Sch.ttt Negative no. (%) Positive no. (%) AST IU/L median (range) ALT IU/L median (range) Albumin g/dL median (range) TB mg/dL median (range) AFP ng/mL median (range) CA19-9 IU/mL median (range) CEA IU/mL median (range) CHOL mg/dL mean SD TRG mg/dL mean SD HDL mg/dL mean SD LDL mg/dL mean SD Cause of chronic liver disease HCV No (%) HBV No (%) PBC No (%) PSC no. (%) HCC lesions Number 1 no. (%) Multiple No (%) Site Rt. lobe no. (%) Lt. lobe no. (%) Both no. (%) Size cm
HCC group n [ 23
Chronic hepatitis group n [ 17
54.27 9.18
45.23 9.36
22 (95.7) 1 (4.3)
15 (88.2) 2 (11.8)
9 14 98 31.5 2
(39.1) (60.9) (33e300) (16e213) (1.5e3)
9 8 53 59 2.5
(52.9) (47.1) (33e83) (26e102) (1.8e3.5)
1.7 (1e2.3) 21 (8e7460) 32.4 (2e55)
1.5 (1e2.1) 6 (1.60e84) 45.8 (17.67e100.2)
2.1 (0.08e3.4) 108.00 16.35 91.20 44.69 41.00 18.29 42.30 18.46
3.04 (1.17e16.8) 121.66 33.29 74.00 13.11 28.00 15.11 114.00 16.35
20 (86.9) 3 (13.1)
10 5 1 1
(58.8) (29.4) (5.9) (5.9)
11 (47.8) 12 (42.2)
took place immediately within 2 h from blood sample collection. Extracted miRNA were subjected to reverse transcription. miRNA RT-PCR TaqMan miRNA assays (Applied Biosystems, Foster City, CA) was used to quantify the expression levels of mature miR-155 by miRNA reverse transcription Kit (Applied Biosystems) was reverse transcribed in a reaction mixture containing miR-specific stem-loop RT primers. qPCR was performed with 3 mL of each cDNA on a Step One TMPlus Real-Time PCR System (ABI) in duplicate reactions containing the prepared cDNA and TaqMan specific primers in Universal Master Mix without AmpErase UNG (Applied Biosystems) and threshold cycles (CT) were calculated using Sequence Detection Software (SDS v2.2.1, Applied Biosystems). miRNA quantification data were normalized to 18S RNA. All miRNA data are expressed relative to a RNU48 small nuclear (sn) RNA TaqMan PCR performed on the same samples. Fold expression was calculated from the mean CT values and relative quantity (RQ) of miRNA155 was calculated by the formula: RQ52DDCt where Ct is defined as the fractional cycle number at which the fluorescence generated by cleavage of the probe passes a fixed threshold above baseline. Total RNA Extraction and Quantification
10 (43.5) 1 (4.3) 12 (42.2) 3.65 1.75
Grade I no. (%) II no. (%) Type Trabecular no. (%) Mixed no. (%) Liver background 1-Mixed cirrhosis with mild activity, no. (%) 2-Mixed cirrhosis with moderate activity, no. (%)
351
4 (17.4) 19 (82.6) 9 (39.1) 14 (40.9) 2 (8.7)
5 (29.4)
21 (91.3)
12 (70.6)
PH OF anti-Sch.ttt, past history of antischistosomal treatment; AST, aspartate transaminase; ALT, alanine transaminase; AFP, a-fetoprotein; TB, total bilirubin; CHOL, cholesterol; TRG, triglycerides; HDL, high-density lipoprotein; LDL, low-density lipoprotein.
miRNA Extraction and Quantification Isolation of miRNAs from (tissue and blood) of patients and blood only of controls followed the protocol for miRNA easy RNA isolation kit (Qiagen). Separation of serum
Total RNA was extracted from blood samples and from 10e25 mg of tested tissue by disruption and homogenization using a RNA extraction kit (Qiagen) according to the manufacturer’s instructions and the extracted total RNA was eluted in 30 mL of RNase-free water. cDNA was synthesized using High-Capacity cDNA reverse transcription kit (Applied Biosystems). In brief, 10 mL of RNA (1 mg) was reverse transcribed in a 20-mL reaction containing 2 mL RT buffer, 0.8 mL dNTPs, 2 mL random hexmar primers, 1 mL RT enzyme MultiScribeTM reverse transcriptase and 4.2 mL nuclease-free H2O. The reverse transcription was performed on Perkin Elmer 9700 thermocycler condition (25 C for 10 min, 37 C for 120 min followed by 85 C for 5 min and hold at 4 C). hTERT mRNA qRT-PCR Assay To determine hTERT mRNA, real-time qRT-PCR assays were constructed using TaqManÒ Gene Expression Assay (Applied Biosystems). The target gene sequence was the catalytic subunit of hTERT and the calibrator sample is healthy control. The reference gene (housekeeping gene) was glyceraldehyde phosphate dehydrogenase (GAPDH). A singleplex reaction was used in this study. RT-PCR was carried out using TaqMan Universal PCR Master Mix.
352
Ezzat et al./ Archives of Medical Research 47 (2016) 349e355
PCR reaction mix was added to the cDNA samples in a sterile 48-well PCR plate. The prepared reaction components were done in One Step PCR (Applied Biosystems) using real-time cycler conditions of 50 C for 2 min (reverse transcription), 95 C, 10 min (initial denaturation) followed by 40 cycles of 95 C, 15 sec, 60 C, 1 min and 72 C, 1 min for denaturation, annealing, extension steps, respectively, using assay primers (Applied Biosystems). Total RNA hTERT gene quantification data were normalized to GAPDH gene and data analyzed according to the RQ manager program ABI SDS software (ABI 7900) using the previous RQ formula (Livak and Schmittgen, 2001). Statistical Analysis Data are expressed as median or mean SD unless otherwise indicated. Categorical data are described as frequency of the subjects with a specific characteristic. c2 test or Fisher’s exact test was used for comparing categorical data and Student t-test, Mann-Whitney U test, one way ANOVA or Kruskal-Wallis test, when appropriate, was used for comparing continuous variables. ROC curve was conducted to determine the specificity and sensitivity of serum mir 155 and serum telomerase as diagnostic markers for HCC. Twotailed p values !0.05 were considered statistically significant. Statistical analysis was performed using SPSS software v.12.0 (SPSS Inc., Chicago, IL). Results Two groups of patients were enrolled in the current study; HCC group consists of 23 patients (22 males and one female) with a mean age of 54.27 9.18 years. Chronic hepatitis group consisted of 17 patients (15 males and 2 females) with a mean age of 45.23 9.36 years. Twelve healthy subjects were enrolled as a control group. Frequency of past history of schistosomiasis was 60.9% among HCC group, whereas it was 47.1% among patients of the chronic hepatitis group. Distribution of causes of chronic liver diseases among the studied groups of patients revealed that the most common cause was HCV infection, especially among the HCC group followed by HBV infection. One case of primary biliary cirrhosis (PBC) and another case
of primary sclerosing cholangitis (PSC) were found among the chronic hepatitis group as shown in Table 1. Statistical analysis revealed that there were no significant differences between the two studied groups of patients as regards liver function tests. As a traditional marker for HCC, a-fetoprotein (AFP) was significantly increased among the HCC group of patients. Macroscopic examination of HCC lesions revealed that distribution of these lesions were more prevalent in both right and left lobes, with a mean size of 3.65 1.75 cm. Microscopic examination of liver tissues of the HCC group of patients revealed that grade 2 differentiation and mixed type were more prevalent than grade 1 and trabecular type. With regard to the liver background, it was found that the frequency of mixed cirrhosis with moderate activity was higher in the two studied groups of patients than mixed cirrhosis with mild activity (Table 1). Calculated expression of miRNA155 revealed that RQ miR 155 was overexpressed in sera of patients with HCC compared to patients with chronic hepatitis and controls ( p !0.0001). miRNA 155 was directly correlated to AFP ( p 5 0.001, significant) and serum total bilirubin ( p 5 0.037, significant). The median values of blood telomerase were significantly higher among HCC patients than cirrhotic patients and controls ( p 5 0.04). Moreover, tissue expression of telomerase was significantly higher in malignant tissue more than adjacent nonmalignant tissue among HCC patients ( p 5 0.02). We also found that tissue expression of telomerase was significantly lower in tissue of chronic hepatitis group ( p 5 0.03) (Table 2). There was a significant direct correlation between tissue and serum telomerase ( p 5 0.007). Interestingly, we found a significant direct correlation between serum telomerase and miRNA155 ( p 5 0.003) (Table 3). Our results also revealed that there was no significant impact of tumor characteristics regarding number; site; size and microscopic features of blood; tissue expression of telomerase or serum values of miRNA155. It was found that miR 155 could predict HCC at a level of 6.3 with 95.5% sensitivity and 76.5% specificity. Blood telomerase could predict HCC at a level of 1.08 with a sensitivity of 81.2% and specificity of 62.5% (Table 4).
Table 2. Comparison between HCC group and chronic hepatitis group as regards mean values of serum; tissue telomerase and serum miR 155
mirRNAs Serum miR 155 mean SD Serum telomerase Tissue telomerase
HCC group n [ 23
Chronic hepatitis group n [ 17
Control group n [ 12
10.25 3.31 1.59 (0.06e25.0) Malignant tissues Nonmalignant tissues 13.08 (0.15e942.1) 9.78 (0.19e783.8) 0.02*
5.18 2.45 1.02 (0.001e5.45) 2.87 (0.23e15.45)
1.1 0.7 0.17 (0.015e0.72)
*p value is significant; **p value is highly significant.
p 0.0001** 0.04* 0.03*
microRNA155 and Telomerase in Hepatocellular Carcinoma Table 3. Correlations between serum miRNA 155 and serum telomerase Variables Serum miR 155
Serum telomerase
Tissue telomerase
Correlation coefficient: 0.535 p 5 0.003a
Correlation coefficient: 0.373 p 5 0.073 Correlation coefficient: 0.668 p 5 0.007a
Serum telomerase a
p value was significant.
Discussion HCC is a complex malignancy with many risk factors and underlying mechanisms. Promising diagnostic markers are needed for liver carcinogenesis (14). Liver cirrhosis with its regenerative nodules usually represents the background for HCC development. The existence of large regenerative nodules frequently interferes with the accurate diagnosis of small HCC. Therefore, useful markers for the diagnosis of HCC have been sought (15). Important biomarkers such as AFP-b1-mRNA, insulin growth factor 2 (IGF-2) mRNA, telomerase, etc. were secreted by malignant hepatocytes. These molecular markers could be used for early HCC detection (16). In agreement with Waguri et al. who proved that the existence of circulating malignant cells detached from original HCC tissues in blood and can detect hTERT mRNA in blood (17), the present study suggests that quantification of hTERT mRNAs in blood has diagnostic implications for HCC as the study results indicated that the mean blood values of hTERT mRNAs were significantly increased among patients with HCC when compared to patients with liver cirrhosis. Moreover, tissue expression of telomerase was significantly increased in cancerous tissue when compared to adjacent nonmalignant tissue among patients with HCC. In a previous Egyptian study, Abdel Hady et al. concluded that the telomerase activity assay could be used for liver cancer diagnosis and is more sensitive than other diagnostic markers. Therefore, activity of telomerase is a promising, available diagnostic and prognostic marker for liver carcinogenesis diagnosis (18). Kong et al. analyzed hTERT in peripheral blood in 343 Korean patients with liver cancer. hTERT expression was not related to clinical Table 4. Diagnostic performance of serum miR 155 and telomerase in comparison with a-fetoprotein
Group HCC
Parameter
AUC
Cutoff value
Sensitivity %
Specificity %
Serum miR 155 Serum telomerase Serum a-fetoprotein
0.924 0.724 0.832
6.3 1.08 8.050
95.5 81.2 100.0
76.5 62.5 69.2
HCC, hepatocellular carcinoma; AUC, area under the curve.
353
manifestations and AFP. They suggested that AFP and hTERT mRNA expression in peripheral blood could not be used as diagnostic markers (19). Telomerase is considered a useful specific candidate tumor marker; it could not be applied practically because telomerase expression has not been detected stably in body fluid (20). In serum, the hTERT mRNA derived from cancer cells seemed to be undetectable because it becomes instable by RNase in blood. Because RNAs in serum are interestingly stable within 24 h after drawing blood due to particle-associated complex in structure (21,22), it has been suggested that they can be generally detected even in RNase-rich blood. Actually, hTERT mRNA can be detected in serum from breast cancer patients and its sensitivity and specificity are, at most, 40 and 100%, respectively (23). The sensitivity in patients with HCC rose to 89.7% in the semiquantitative assay. A previous study reported that hTERT expression was very low in serum from normal persons because lymphocytes and circulating normal cells express very low levels of hTERT mRNA (9). As hTERT mRNA in lymphocytes is very low, increased hTERT mRNA values in serum may signify that hTERT mRNA is derived from malignant cells. As we can detect very small amounts of lymphocyte markers after three steps of centrifugation of blood samples, the RNA extraction procedure appeared to clear lymphocytes properly. Moreover, healthy or injured hepatocytes express very small amounts of hTERT (24,25). The significant correlation of hTERT mRNA expression between malignant tissue and serum was then proven (26). These data suggest that hTERT mRNA determined in serum is delivered from cancer cells. In the current study we found a direct correlation of telomerase in blood with telomerase in tissue samples, which certifies the usefulness of telomerase as a noninvasive marker for early HCC detection. All studied patients underwent liver transplantation. Our results also revealed that there was no significant impact of tumor characteristics as regards number, site, size and microscopic features in blood; tissue expression of telomerase or serum values of miRNA155.This finding may be interpreted as most HCC patients show the same pathological characteristics as size, grade 2 of differentiation and mixed type. We think that more aggressive types of HCC make patients unable to withstand liver transplantation surgery. Therefore, we cannot judge the impact of HCC characteristics on the studied markers among patients who underwent liver transplantation as we did not have reasonable numbers with different HCC characteristics. To understand the underlying mechanism by which telomerase shares in HCC development, some authors suggested that telomere shortening results in chromosomal instability (CIS), which leads to cancer development. Previous studies suggested that telomeres were shorter in malignant liver cells when compared to regenerative nodules and
354
Ezzat et al./ Archives of Medical Research 47 (2016) 349e355
healthy liver tissue and that, within the group of HCC, hepatocyte telomere length of aneuploid was shorter than that of diploid tumors. On the other hand, advancement of carcinogenesis needs an effective telomere signaling. Other studies (27) found a slight increase of telomere length in low differentiated liver cancer when compared to highly differentiated liver cancer, suggesting a reactivation of telomerase and recovered chromosomal stability to a level convenient to malignant cell survival. Accordingly, several studies have proved that telomerase activity was determined in nearly 90% of malignant liver tissues as compared to only 21% of non-malignant tissue and was accompanied with the increase of TERT mRNA values, implicating the feasibility that TERT mRNA expression could detect early HCC (28e31). With the advancing research in miRNA biology, the prospective role of miRNAs in carcinogenesis will probably expand in the near future (32). In the current study, it was found that miR155 was upregulated in sera of HCC patients. In regard to the contribution of miR-155 in the pathogenesis of malignancy, it targets numerous molecules in key coding pathways like glutathione metabolism, stressactivated protein kinase/c-Jun N-terminal kinases (SAPK/ JNK), toll-like receptor (TLR), extracellular-signalregulated kinases/mitogen-activated protein kinase (ERK/ MAPK), and B-cell receptor signaling. MiR-155 was also applied in increasing programmed cell death through targeting anti-apoptotic operators (33). TRF1 is a telomere repeat binding protein that works to shorten telomere length, contributes to mitotic stability, and protects from replication-associated DNA damage (6,34). In regard to the relation between miR 155 and telomerase in carcinogenesis, Dinami et al. demonstrated that miR155 directly controls TRF1 expression levels by specifically targeting a partially conserved sequence motif in the 30 untranslational region (30 UTR) of TRF1, leading to translational repression. They attempted to link miR-155 to molecular pathways that modulate TRF1-related aspects of telomere function. To test whether alteration of miR155 expression levels not only impacts on global TRF1 expression levels but also alters TRF1 abundance at telomeres, they transiently transfected SK-BR-3 luminal breast cancer cells with mimic-miR-155, antago-miR-155 siRNAs, or TRF1-specific siRNAs and performed quantitative immunofluorescence analysis using anti-TRF1 antibodies. They found that ectopically introduced miR-155 significantly reduced fluorescence signal intensity, indicative for a reduced abundance of TRF1 at telomeres. Together they concluded that miR-155-dependent regulation of TRF1 is an efficient mechanism to control telomere fragility and genomic stability in human breast cancer cells. The consistent upregulation of miR-155 in breast cancer suggests that impaired telomere function is a cornerstone of the carcinogenic mission of miR-155 that increases genomic instability in human breast cancer and contributes to decreased
distant metastasis-free survival and recurrence-free survival in ERþ breast cancer (35). In a trial to change basic research into applied research, we performed this study to clarify more than one factor cooperating in HCC development. Liver carcinogenesis is multistep with underlying multiple factors, which may act in parallel to each other or they may act step by step, each in its role. Altogether, these players represent a big challenge in the face of conventional therapeutic modalities for HCC. The time has come to try to discover the underlying mechanisms for hepatocarcinogenesis to pave the way for development of tailored therapy for those patients. According to our findings, serum mir 155 and blood telomerase have diagnostic abilities and could be used as early detectors for HCC. From a diagnostic point of view, miR has reasonable sensitivity and specificity for HCC detection. Depending on senescence blocking, telomerase reactivation to block the attrition of the hepatocyte capacity of regeneration may be useful, relying on the effect of this method on liver carcinogenesis. More efforts must be made in this area of research for understanding telomere biology in human disease and malignancies and to detect the most appropriate and effective therapy (36). Although miR155 has a suggestive role in regulation of telomerase expression during liver carcinogenesis, therapeutic intervention targeting miR155 may, at the same time, control telomerase activity. In conclusion, both miR 155 and telomerase expression may play a role in development of HCC and miR 155 could regulate telomerase expression during liver carcinogenesis.
References 1. Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74e108. 2. Nakayama J, Tahara H, Tahara E, et al. Telomerase activation by hTERT in human normal fibroblasts and hepatocellular carcinomas. Nat Genet 1998;18:65e68. 3. Nakamura TM, Morin GB, Chapman KB, et al. Telomerase catalytic subunit homologs from fission yeast and human. Science 1997;277: 955e959. 4. Palm W, de Lange T. How shelterin protects mammalian telomeres. Annu Rev Genet 2008;42:301e334. 5. Denchi EL, de Lange T. Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1. Nature 2007;448: 1068e1071. 6. Sfeir A, Kosiyatrakul ST, Hockemeyer D, et al. Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication. Cell 2009;138:90e103. 7. Cesare AJ, Reddel RR. Alternative lengthening of telomeres: models, mechanisms and implications. Nat Rev Genet 2010;11:319e330. 8. Murnane JP, Sabatier L. Chromosome rearrangements resulting from telomere dysfunction and their role in cancer. Bioassays 2004;26: 1164e1174. 9. Oh BK, Kim H, Park YN, et al. High telomerase activity and long telomeres in advanced hepatocellular carcinoma with poor prognosis. Lab Invest 2008;88:144e152. 10. Iorio MV, Croce CM. MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review. EMBO Mol Med 2012;4:143e159.
microRNA155 and Telomerase in Hepatocellular Carcinoma 11. Dinami R, Petti E, Sestito R, et al. microRNAs control the function of telomeres in cancer. RNA Dis 2014;1:e282. 12. Mu~ noz P, Blanco R, de Carcer G, et al. TRF1 controls telomere length and mitotic fidelity in epithelial homeostasis. Mol Cell Biol 2009;29: 1608e1625. 13. Greene FL, Page DL, Fleming ID. AJCC Cancer Staging Manual. 6th ed New York: Springer; 2002. 14. Aravalli RN, Steer CJ, Cressman ENK. Molecular mechanisms of hepatocellular carcinoma. Hepatology 2008;48:2047e2063. 15. Takahashi S, Kitamoto M, Takaishi H, et al. Expression of telomerase component genes in hepatocellular carcinomas. Eur J Cancer 2000;36: 496e502. 16. Miura N, Shiota G, Nakagawa T, et al. Sensitive detection of hTERT mRNA in the serum of patients with hepatocellular carcinoma. Oncology 2003;64:430e434. 17. Waguri N, Suda T, Nomoto M, et al. Sensitive and specific detection of circulating cancer cells in patients with hepatocellular carcinoma; detection of human telomerase reverse transcriptase messenger RNA after immunomagnetic separation. Clin Cancer Res 2003;9:3004e3011. 18. Hady AA, El Shanawany F, Hassan MM, et al. Evaluation of human telomerase activity as a novel tumor marker for hepatocellular carcinoma. Life Sci J 2010;7:153e161. 19. Kong SY, Park JW, Kim JO, et al. Alpha-fetoprotein and human telomerase reverse transcriptase mRNA levels in peripheral blood of patients with hepatocellular carcinoma. J Cancer Res Clin Oncol 2009; 135:1091e1098. 20. Tatsuma T, Goto S, Kitano S, et al. Telomerase activity in peripheral blood for diagnosis of hepatoma. J Gastroenterol Hepatol 2000;15: 1064e1070. 21. Ng EK, Tsui NB, Lam NY, et al. Presence of filterable and nonfilterable mRNA in the plasma of cancer patients and healthy individuals. Clin Chem 2002;48:1212e1217. 22. Tsui NB, Ng EK, Lo YM. Stability of endogeneous and added RNA in blood specimens, serum, and plasma. Clin Chem 2002;48:1647e1653. 23. Chen XQ, Bonnefoi H, Pelte MF, et al. Telomerase RNA as a detection marker in the serum of breast cancer patients. Clin Cancer Res 2000;6: 3823e3826.
355
24. Onishi T, Nouso K, Higashi T, et al. Cellular distribution of telomerase reverse transcriptase in human hepatocellular carcinoma. J Gastroenterol Hepatol 2003;18:1168e1174. 25. Wege H, Chui MS, Le HT, et al. In vitro expansion of human hepatocytes is restricted by telomere-dependent replicative aging. Cell Transplant 2003;12:897e906. 26. Miura N, Maeda Y, Kanbe T, et al. Serum human telomerase reverse transcriptase messenger RNA as a novel tumor marker for hepatocellular carcinoma. Clin Cancer Res 2005;1:3205e3209. 27. Urabe Y, Nouso K, Higashi T, et al. Telomere length in human liver diseases. Liver 1996;16:293e297. 28. Nagao K, Tomimatsu M, Endo H, et al. Telomerase reverse transcriptase mRNA expression and telomerase activity in hepatocellular carcinoma. J Gastroenterol 1999;34:83e87. 29. Shimojima M, Komine F, Hisatomi H, et al. Detection of telomerase activity, telomerase RNA component, and telomerase reverse transcriptase in human hepatocellular carcinoma. Hepatol Res 2004;29: 31e38. 30. Lee CM, Hsu CY, Eng HL, et al. Telomerase activity and telomerase catalytic subunit in hepatocellular carcinoma. Hepatogastroenterology 2004;51:796e800. 31. Kojima H, Yokosuka O, Imazeki F, et al. Telomerase activity and telomere length in hepatocellular carcinoma and chronic liver disease. Gastroenterology 1997;112:493e500. 32. Baek S, Cho KJ, Ro SW. Functional analysis of micro RNAs overexpressed in hepatocellular carcinoma. RNA Dis 2015;2:e728. 33. Higgs G, Slack F. The multiple roles of microRNA-155 in oncogenesis. J Clin Bioinforma 2013;3:17. 34. Martınez P, Thanasoula M, Mu~noz P, et al. Increased telomere fragility and fusions resulting from TRF1 deficiency lead to degenerative pathologies and increased cancer in mice. Genes Dev 2009;23: 2060e2075. 35. Dinami R, Ercolani C, Petti E, et al. miR-155 drives telomere fragility in human breast cancer by targeting TRF1. Cancer Res 2014;74: 4145e4156. 36. Carulli L, Anzivino C. Telomere and telomerase in chronic liver disease and hepatocarcinoma. World J Gastroenterol 2014;20:6287e6292.