Jacobs Journal of Cancer Science and Research

Evaluation of the Diagnostic Role of Human Telomerase Reverse Transcriptase (Htert) and Microrna 122a in Hepatocellular Carcinoma Patients

*Nihal MS El-Assaly
Department Of Oncology And Pathology, Theodor Bilharz Research Institute, Egypt

*Corresponding Author:
Nihal MS El-Assaly
Department Of Oncology And Pathology, Theodor Bilharz Research Institute, Egypt
Email:khaled_mabrouk@hotmail.com

Published on: 2019-02-18

Abstract

Background: Hepatocellular carcinoma (HCC) is currently the fifth most common solid tumor worldwide and the third leading cause of cancer related death. miRNAs are 19- to 25-nucleotide-long RNAs, able to bind complementary sequences in 3′-untranslated regions (3′-UTR) of several target mRNAs to induce their degradation or translational repression. miR- 122a, a hepato-specific miRNA, resulted down-regulated in the majority of HCCs and in all examined HCC-derived cell lines. The high frequency of aberrant regulation of these miRNAs in HCC versus non-tumor liver suggests that they might play an important role in hepatocarcinogenesis. Telomeres are specialized DNA–protein structures that cap the ends of linear chromosomes. Telomerase is a large ribonucleoprotein (RNP) complex that maintains telomeric DNA. Over 80% of human cancers show an activation of telomerase. Aim of the work: The present study was performed to evaluate the genetic role of miRNA 122a and Telomerase as reliable, non – invasive biochemical markers for early detection of hepato-cellular carcinoma Subjects and Methods: Analysis of the expression level of mature miRNA 122a (miR- 122a) and Telomerase in serum of 30 HCC patients ,30 HCV patients and 20 healthy subjects as control using quantitative reverse-transcription real time PCR (qRT-PCR). Results: Concerning miRNA-122a, the median fold change was statistically significant elevated in HCC patients when compared to both HCV and control groups. There was significant positive correlation between serum miRNA 122a and AST in HCV group. Regarding Telomerase, there was significant elevation in median fold change of serum Telomerase in HCC patients when compared to both HCV and control groups. A significant positive correlation was found between serum telomerase and total & direct bilirubin in HCC group. Conclusion: Current data suggest significant increase in miRNA 122a and Telomerase in serum of HCC patients.

Keywords

Hepatocellular carcinoma- microRNA 122a-Telomerase

Introduction

Hepatocellular carcinoma represents the 6th most common cancer worldwide and the 3rd most common cause of cancer death. Infection with hepatitis C virus (HCV) is a leading etiological factor for the developing HCC especially in Egypt. The early screening of HCC depends on imaging techniques including mainly ultrasonography and laboratory tests involving mainly serum alpha-fetoprotein (AFP). However, ultrasonography is an operator-dependent procedure with varied diagnostic accuracy; in addition, it fails to detect small tumors. As well, the accuracy of AFP as a diagnostic biomarker for the screening of HCC patients at the early stage is modest with the sensitivity of 60%–80% and with the specificity of 70%–90%. The lack of good diagnostic biomarkers for early-stage HCC accounts for low five-years survival rate from the time of diagnosis. Thus, discovering minimally invasive sensitive and specific biomarkers for the early detection of HCC improving the prognosis of HCC patients is recommended. MicroRNAs are small, non-coding RNA molecules; act as post-transcriptional regulators for expression of genes involved in diverse biological processes that underlie physiological and pathological conditions.

The differential expression of microRNAs deregulation has been reported in the development of many cancer types including HCC [8]. Many studies have reported a number of circulating miRNAs as potential biomarkers for HCC diagnosis and/or good prognosis [9-13]. For example, miR-122, a liver-specific miRNA, may function as a tumor suppressor gene and its expression is commonly down or lost in HCC cells [14] contributing to the tumorigenic phenotype of these cells. We began to study the involvement of miRNAs as noninvasive biomarkers in developing HCC of Egyptian patients [15, 16]. Telomeres cap and protect the ends of linear chromosomes from aberrant double-stranded DNA repair and detrimental end-to-end fusions, as well as ensuring proper genetic partitioning into daughter cells. The structure of telomeres is composed of hexanucleotide repeats bound specifically by ‘shelterin’ proteins [17]. Telomeres function as ‘mitotic clocks’ and shorten with subsequent mitotic cell divisions. Upon reaching a critically short length, a cellular signaling cascade involving p53 and p21 arrests cellular replication and induces senescence [18, 19]. The telomerase enzyme complex counterbalances telomere attrition by de novo synthesizing telomere repeats, maintaining telomere length in high proliferative cells.

Telomerase is a ribonucleoprotein and is minimally composed of the catalytic telomerase reverse transcriptase (TERT) protein and intrinsic RNA component (TERC), which provides the template for telomere repeat synthesis. The human telomerase holoenzyme is composed of several accessory proteins, including the dyskerin protein complex [20-22]. Telomerase can maintain the telomeric length by acting as a reverse transcriptase. In humans, tumor cells escape programmed cell senescence through reactivation of telomerase [23]. These immortalized cells can compensate for telomeric shortening at each cell division, leading to progressive neoplastic evolution. Telomerase re-expression was found in 85 % of malignant tumors [24] A variety of studies demonstrated telomerase reactivation in different human cancers, including>80% of human hepatocellular carcinomas [25-27], indicating that telomerase reactivation is a rate-limiting step in carcinogenesis. Telomerase reactivation in HCC correlates with the upregulation of TERT [28] and TERC, [29] the two essential components of telomerase. In this context, our study aimed to identify the performance of two serum HCC-related markers (miR-122 and Telomerase), compared with the conventional marker serum AFP, in early prediction of primary HCC.

Study Design and Patient Samples

Subjects: Our study was conducted on a total of 80 subjects selected from tropical medicine department at Theodor Bilharz Research Institute (TBRI) in the period from April to June 2016. They were divided into the following groups: 30 Hepatocellular carcinoma (HCC) patients diagnosed by clinical examination, serum AFP level and typical findings on abdominal ultrasound, and abdominal triphasic CT scan; [30] patients with chronic hepatitis C virus (HCV) diagnosed by clinical examination, anti HCV Abs and ultrasonographic criteria; and finally, 20 apparently healthy age- and sex-matched volunteers representing the control group. All patients were free from any inflammatory conditions as spontaneous bacterial peritonitis, inflammatory bowel diseases and systemic sepsis and presence of any type of other malignancy. The study protocol was approved by the Ethics Committee of Kasr Alainy Hospitals and adhered to the tenets of the Declaration of Helsinki. Additionally, informed consent was obtained from participants for the use of their withdrawn samples in this study. Routine liver function tests including serum total bilirubin, direct bilirubin, alanine transaminase (ALT), aspartate transaminase (AST), total protein, albumin, alkaline phosphatase (ALP) and gamma glutamyl transferase (GGT) and Creatinine as for assessment of kidney function, Serum alpha fetoprotein (AFP) and HCV Ab and HBsAg were analyzed using automated analyzers according to manufacturer’s instructions. 

Serum Preparation and RNA Extraction

Small RNA was isolated from serum using Qiagen miRNeasy Mini Kit; Cat. no. 217004; according to kit instructions. Reverse Transcription (RT) and Quantitative PCR (qPCR) Using miScript II RT Kit (QIAGEN), Cat. no.21816. In a reverse transcription reaction with miScriptHiSpec Buffer, mature miRNAs are polyadenylated by poly(A) polymerase and converted into cDNA by reverse transcriptase with oligo-dT priming. The cDNA is then used for real-time PCR quantification of mature miRNA expression (McKnight et al., 2006).

Reverse Transcription (RT) and Quantitative PCR (qPCR)

Ready RT and qPCR kits for accurate miRNA examination (Applied Biosystems) were utilized to assess the expression of the chosen miRNA from serum samples. A Total volume of 15 μl RT master mix: 4 μl of 5x miScript HiSpec Buffer; 2 μl of 10x miScript Nucleics Mix, 7 μl of RNase-free water, 2 μl of miScript Reverse Transcriptase Mix, then 5 μl Template RNA was added to each tube containing reverse transcription master mix, gently mixed, briefly centrifuged, and then stored on ice according to manufacturer instructions. The cDNA was diluted in RNase-free water by adding 200 μl RNase- free water to the 20 μl reverse transcription reaction to proceed with real-time PCR. Real - time PCR quantification was done using miScript SYBR Green PCR Kit Qiagen; Catalog no. 218073 on Automated Step One real - time PCR (Applied Biosystem). Target miR-122a Sequences to be Studied and Amplified is as follows: UGGAGUGUGACAAUGGUGUUUG. 25 μl of reaction Mix for qPCR of miRNA 122a, consisted of 12.5 μl 2x, 2.5 μl -10x miScript Universal Primer, 2.5 μl 10x miScript Primer Assay, 5 μl RNase-free water, 2.5 μl Template cDNA QuantiTect SYBR Green PCR Master Mix.

Quantitative PCR (qPCR) of Telomerase Gene Expression

In a final volume of 25 μl of PCR reaction mix, 1.25 μl of Gene Expression Assay (20X working stock) mix, 10.0 μl of cDNA template and RNA free water, 12.5 μl of TaqMan Universal PCR Master Mix (2X), with AmpErase UNG, 1.25 μl of TaqMan Endogenous Control Assays Gene ID: 18S.All reactions were run using the following Cycling conditions: 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15s and 60 °C for 1 min.

Table 1: Sequence of the primers and probes for Telomerase

Calculation of results

Relative expression of miRNA was calculated using the comparative cycle threshold (CT) (2 -??C T) method; with Snord as the endogenous control to normalize the data. Relative telomerase expression in a sample was determined by subtracting the cycle threshold of the reference gene (GAPDH) from that of the target gene (telomerase) to get the normalized amount of the mRNA, then comparing this value to that of the calibrators. The CT is defined as the number of cycles required for the FAM signal to cross the threshold in real-time PCR. ?CT was calculated by subtracting the CT values of Snord from the CT values of miRNA122a. ??CT was then calculated by subtracting mean ?CT of the control samples from ?CT of tested samples. Fold change of miRNA122a was calculated by the equation 2 -??C T

Statistical analysis

All results were tabulated, analyzed using SPSS 10 statistical package for windows. Normality of numerical data distribution was examined. Parametric numerical variables were presented as mean ± SD and intergroup differences were compared using the using one-way analysis of variance (ANOVA). Non-parametric numerical variables were presented as median and interquartile range. Categorical variables were presented as number and percentage and intergroup differences were compared using the chi squared test for. Multivariable regression analysis was used to examine the relation between the miRNA 122a, Telomerase and HCC as adjusted for relevant confounding factors. P-value<0.05 was considered statistically significant.

Results

Subjects Characteristics A total of 80 participants including 30 HCC patients, 30 chronic HCV infection patients without HCC, and 20 normal subjects were recruited into this study (Table 1). There were no significant differences of age (t-test) and sex (Pearson x2 test) between cases and controls. In addition, the HCC group and the other two controls groups had statistically different laboratory results for ALB, T-Bil, ALT, AFP (p<0.05). With regard to clinicopathologic characteristics of HCC patients, single tumor was found in 22 patients (73.3%); tumor diameter was< 5 cm in 16 patients (53.3%) and 28(93.3%)patients were also with hepatic cirrhosis.

Table 2: Summary of Clinical, Laboratory and Radiological details of subjects used for miRNA and Telomerase analysis.

Identification of HCC-associated MiRNAs and Telomerase

The goal of the present study was to explore the potential use of serum miRNA 122a and Telomerase as biomarkers for HCC. To validate the 2 markers, RT-qPCR assays were developed to quantify miR-122a and Telomerase in serum among 30 HCC patients compared to 30 HCV patients and 20 healthy subjects. Our data indicated that expression levels of miR-122a in serum were significantly higher in HCC patients than in HCV and healthy subjects (p=0.001) (Figure 1). Levels of miR-122a were significantly elevated in HCC patients than in HCV patients without HCC and healthy controls (p=0.001), no significant difference was observed between HCV subjects and controls (p>0.05). In addition, levels of Telomerase activity were found to be significantly higher in HCC group when compared to HCV subjects and healthy control group (p=0.001) (Figure 2). To evaluate whether serum miR-122a can be used as a potential diagnostic marker for HCC, ROC curve analyses were performed. It was revealed that serum miR-122a was a potential marker for discriminating HCC patients from healthy controls with an area under the curve (AUC)=0.90 (P=0.001, 95% CI: 0.337-0.468) (Figure 3). At the cut-off value of 3.6, the sensitivity and specificity of this marker were 86% and 82% respectively. However, for serum Telomerase as a potential marker for discriminating HCC patients from healthy controls with an AUC=0.86 (P=0.001, 95% CI 0.277-0.433) (Figure 4). At the cut-off value of 1.4, the sensitivity and specificity of this marker were 90% and 66%, respectively. Relationship of Circulating miRNAs to Clinicopathological Parameters, it was reported that some unique miRNA signatures were associated with prognostic factors and disease progression in several cancers. Therefore, we examined the correlation between the expression of circulating miR122a and Telomerase with clinical parameters. A significant positive correlation between miRNA122a and Telomerase (r=0.263, p=0.04) in HCC and HCV groups was observed (Fig. 5). In HCC group there was significant positive correlation between Telomerase and both total bilirubin (r=0.466,p=0.01) and direct bilirubin (r=0.362, p=0.05). Regression analysis study was done for miRNA 122 and Telomerase and revealed an increase in 1 degree of miRNA122 increased the odds of being HCC by a factor of 1.35 ((OR=1.35; CI: 1.068-1.706 P=0.012), and also an increase in 1 degree of Telomerase increased the odds of being HCC by a factor of ((OR=1.43; CI: 1.158 -1.766 P=0.01).

Figure 1: Median fold change of miRNA 122a in the studied groups.

Figure 2: Median fold change of Telomerase in the studied groups.

Figure 3: ROC curve for miRNA122a, at cutoff 3.6, the sensitivity is 86% and specificity is 82%, and The AUC was 0.90 (P= 0.001, 95% CI 0.337 - 0.468).

Figure 4: ROC curve for Telomerase, at cutoff 1.4, the sensitivity is 90% and specificity is 66%, and The AUC was 0.86 (P= 0.001, 95% CI 0.277 - 0.433).

Figure 5: Correlation between miRNA122a and Telomerase in the HCC and HCV groups. (r= 0.263, p= 0.04).

Discussion

HCC is a complex disease with multiple underlying pathogenic mechanisms caused by a variety of risk factors and it is difficult to characterize it with a single biomarker. AFP has mainly been used for diagnosis of primary HCC; however, its sensitivity is not satisfactory [30]. Thus, signatures of combined biomarkers may be more valuable for the diagnosis, staging, and prognosis of HCC. MiR-122 is liver specific miRNA and plays a central role in hepatocyte development and differentiation [31]. Here, Serum miR-122 was reported to be significantly down-expressed in HCC patients compared to healthy controls and CHC patients supporting its function as a tumor suppressor gene [32, 33] MiR-122 has a central role in the suppression of HCC, [34] the role of miR-122 was suppression of oncogenic genes involved in diverse HCC hallmarks. Among these genes, Bcl-2 which can inhibit tumor cells apoptosis, Wnt1 which is responsible for the proliferation of cells, ADAM17 which is responsible for the metastasis and Ccgn1 which is responsible for the progression of the cell cycle [35]. Moreover, miR-122 can inhibit angiogenesis and intrahepatic metastasis by suppressing the expression of tumor necrosis factor-α-converting enzyme [36]. In contrary to these finding, circulated miR-122 was found to be up regulated in HCC in two studies by Varnholt et al and El-Garem et al [37, 38] They suggested that miR-122 may down-regulate target mRNA of obscure tumor suppressor genes and in this way prompt further tumor development.

In our study, miR-122 expression was reported to be nonsignificantly higher in CHC patients when compared to healthy controls. That could be explained by the leakage of this miRNA from apoptotic or necrotic cells into the blood. MiR-122 may contribute to the pathogenesis of chronic HCV due to its function in HCV replication, translation, and inflammation [39, 40] Interestingly, the statistical ROC curve analysis showed that miR-122 as potential biomarkers could predict HCC at the early stage. these findings were in similar with two studies which confirmed that they were to be better than the sensitivity and accuracy of AFP in the diagnosis of HCC, especially at the early stage [41, 42]

In the current work, serum miR-122 expressions showed higher diagnostic performance in distinguishing HCC from CHC patients (Figure 1). However, the conventional marker AFP for HCC failed to do this discrimination. The combination of AFP with miR-122 could increase the sensitivity (for miR-122 with 97.5%). These results supported by Muawia and his colleagues [43] which revealed that miR-122 presented a significant (AUC) of 0.705, sensitivity (63.64%) and specificity (75%) in distinguishing HCC patients from CHC patients. Telomerase is a ribonucleoprotein complex that catalyses the addition of TTAGGG repeats onto telomeres, repetitive DNA structures found at the ends of linear chromosomes. Telomerase expression thereby prevents telomeric shortening during cell division and allows cells to bypass replicative senescence [44]. The majority of human somatic tissues does not display TA and undergo telomeric short ening with consecutive divisions. It is regulated throughout human development, undergoing silencing in almost all organ systems from embryogenesis onwards [45]. There are indications that the level of activity might parallel tumor progression and be of prognostic relevance [46]. In HCC, telomerase activation is thought to be essential for cellular immortality and oncogenesis. It is a general finding with large differences in activity levels [47].ening with consecutive divisions. It is regulated throughout human development, undergoing silencing in almost all organ systems from embryogenesis onwards [45]. There are indications that the level of activity might parallel tumor progression and be of prognostic relevance [46]. In HCC, telomerase activation is thought to be essential for cellular immortality and oncogenesis. It is a general finding with large differences in activity levels [47].ening with consecutive divisions. It is regulated throughout human development, undergoing silencing in almost all organ systems from embryogenesis onwards [45]. There are indications that the level of activity might parallel tumor progression and be of prognostic relevance [46]. In HCC, telomerase activation is thought to be essential for cellular immortality and oncogenesis. It is a general finding with large differences in activity levels [47].ening with consecutive divisions. It is regulated throughout human development, undergoing silencing in almost all organ systems from embryogenesis onwards [45]. There are indications that the level of activity might parallel tumor progression and be of prognostic relevance [46]. In HCC, telomerase activation is thought to be essential for cellular immortality and oncogenesis. It is a general finding with large differences in activity levels [47].ening with consecutive divisions. It is regulated throughout human development, undergoing silencing in almost all organ systems from embryogenesis onwards [45]. There are indications that the level of activity might parallel tumor progression and be of prognostic relevance [46]. In HCC, telomerase activation is thought to be essential for cellular immortality and oncogenesis. It is a general finding with large differences in activity levels [47].

In this study, the serum hTERT mRNA in HCC patients was significantly higher than in HCV patient and also significantly higher than in healthy controls. In HCV patients, serum hTERT mRNA showed no significant elevation than healthy controls. In accordance with these results, Satra et al. [48], Miura et al. [49], and Zhou et al. [50] all reached the same final results parallel to results of present study that is; telomerase is significantly increased in HCC compared to non HCC liver disease and control subjects. This is to be explained by the striking positive correlation between high telomerase activity and tumors of different histological origins and types. That’s why although telomerase per se is not carcinogenic, it plays a direct role in oncogenesis by allowing the precancerous cells to proliferate continuously and become immortal. No significant differences in hTERT mRNA expression were detected between HCV patients and controls, which coincides with results reported by Tatsuma et al. [50] and Miura et al. [51] and explained by normal hepatocytes that may express negligible amount of hTERT mRNA and inflamed hepatocytes still express weaker than hepatocellular carcinoma cells [52]. On the other hand, Kong et al. [53] reported that cell-free circulating hTERT mRNA in plasma can be detected only in 14% of patients with HCC. They detected hTERT mRNA in the plasma of 48 out of 343 (14%) HCC patients.

Also, the present study showed that hTERT mRNA at a cut-off level of 1.4, prediction of HCC showed a sensitivity of 90% and specificity of 66%. Similar results were reported by Miura et al. which showed sensitivity of 88.2%& specificity 72.4%. Also, Deng-Fu et al. [54] reported 68.4% for sensitivity& 92% for specificity. In an Egyptian study Atia et al. [55] reported a sensitivity of 77.3% &specificity 96.8%, PPV 98%, NPV 78%. Additionally, higher sensitivity rates of hTERT mRNA in prediction of HCC was reported by Masaki et al. [56] who reported a sensitivity of 100%, but their study measured hTERT mRNA in HCC tissue & not in peripheral blood, which might indicate that the locally expressed hTERT mRNA may be a more sensitive predictor of HCC in tissue than in peripheral blood, but still blood sample is much more easily obtained for monitoring vulnerable subjects for HCC than the tissue biopsy which need an expert hand and may be contraindicated in suspected HCC patients. Attia et al. reported that combined hTERT mRNA and/or AFP could increase accuracy for HCC diagnosis to 90.5%. Deng Fu et al., also reported that AFP in combination with telomerase expression in peripheral blood could increase accuracy for HCC diagnosis to 92.6%.

Conclusions

Serum miR-122 and Telomerase can be used as feasible noninvasive early detectors biomarkers for HCC at the early stage among patients with chronic HCV infection because of their sensible sensitivity and specificity for HCC, however, larger patient cohort analysis is recommended for clinical value. Telomerase constitutes an additional step in oncogenesis that most tumors require for their ongoing proliferation. In our study, the hTRET was significantly higher in the HCC group than HCV and the control group

We suggest paying more endeavors in the exploration zone of microRNAs which are identified with HCC planning to recognize the genetic mark of this cancerous disease; to investigate the riddle of HCC pathogenesis and to make ready for improvement of custom fitted treatment for HCC relying upon the contributing microRNAs Combination of more than one epigenetic marker may prove valuable as a tumor marker of HCC.

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