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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 35  |  Issue : 1  |  Page : 104-109

The role of serum microRNA-192 for early diagnosis of hepatitis C virus-related hepatocellular carcinoma


1 Department of Internal Medicine, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Clinical Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt
4 Department of Internal Medicine, Al-Ahrar Teaching Hospital, Sharkia, Egypt

Date of Submission24-Aug-2020
Date of Decision30-Nov-2020
Date of Acceptance08-Dec-2020
Date of Web Publication18-Apr-2022

Correspondence Address:
Hany Abu Zeid Ismail
Menia El-Kamh, Sharkia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_287_20

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  Abstract 


Objectives
To elucidate the serum level of microRNA (miRNA)-192 in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC).
Background
Early diagnosis of HCC presents a challenge owing to lack of reliable biomarkers, stressing the need for new early diagnostic tools. The identification of new high-sensitivity and high-specificity markers for HCC is essential. We aimed to identify serum miRNA-192 as a biomarker to be used in diagnosing HCV-related HCC.
Patients and methods
We investigated serum miRNA-192 expression in 40 patients with HCC, 40 HCV-infected patients, and 20 healthy controls. An initial screening of miRNA-192 expressions by Illumina sequencing was performed using serum samples pooled from patients with HCC, HCV-infected patients, and controls. Quantitative reverse-transcriptase PCR was used to evaluate the expression of miRNA-192. Demographic, radiological, and laboratory analyses were recorded.
Results
MiRNA-192 was significantly increased in HCC group in comparison with HCV and control groups, with cutoff level of 62.06.
Conclusion
MiRNA-192 could be used as a diagnostic biomarker for early detection of HCC in HCV-related cirrhosis, as it showed significant upregulation in patients with HCC in comparison with non-HCC patients.

Keywords: hepatitis C virus, hepatocellular carcinoma, microRNA-192, microRNAs


How to cite this article:
Abd-Elatty E, Elshayeb E, El-Ela DA, Hamdy M, AbdEl-Salam L, Zeid Ismail HA. The role of serum microRNA-192 for early diagnosis of hepatitis C virus-related hepatocellular carcinoma. Menoufia Med J 2022;35:104-9

How to cite this URL:
Abd-Elatty E, Elshayeb E, El-Ela DA, Hamdy M, AbdEl-Salam L, Zeid Ismail HA. The role of serum microRNA-192 for early diagnosis of hepatitis C virus-related hepatocellular carcinoma. Menoufia Med J [serial online] 2022 [cited 2024 Mar 28];35:104-9. Available from: http://www.mmj.eg.net/text.asp?2022/35/1/104/343115




  Introduction Top


Hepatocellular carcinoma (HCC) is currently the third leading cause of cancer-related deaths in the world, with mortality rates reaching up to 500 000 deaths per annum. Patients with HCC show the shortest survival time among patients with different forms of cancer, with most patients dying within 12 months of developing the tumor [1]. HCC usually arises in the setting of cirrhosis or bridging fibrosis in hepatitis C virus (HCV)-associated chronic liver disease [2]. Low survival of patients with HCC is attributed to late diagnosis, tumor recurrence, and metastasis. Novel biomarkers for early diagnosis are urgently needed; therefore, prognosis and survival rates are improved significantly with early diagnosis [3].

Current methods for the diagnosis of HCC fall into two main categories: imaging and biomarker tests. Currently, only 30–40% of patients with HCC are found eligible for potentially curative intervention at diagnosis, owing to late clinical presentation and the lack of effective early detection measures. Therefore, the identification of new markers with high sensitivity and specificity for HCC is the need of the hour [4].

HCC risk predictors that identify the subset of advanced fibrosis and cirrhosis patients with the highest risk of HCC are needed. Accordingly, monitoring of fibrosis is mandatory as it reflects disease progression, ultimately to HCC. As liver biopsy for fibrosis staging presents invasiveness, sample variability, and other limitations, circulating microRNAs (miRNAs) were proposed as novel noninvasive methods to assess histological disease severity in chronic HCV [5].

miRNAs are an emerging class of highly conserved, noncoding small RNAs that regulate gene expression at the posttranscriptional level. It is now clear that miRNA scan potentially regulate every aspect of cellular activity, including differentiation and development, metabolism, and proliferation; they also play a role in regulating apoptotic cell death, cellular responses to viral infection, and tumorigenesis [6].

Recent studies provide clear evidence that miRNAs are abundant in the liver and modulate a diverse spectrum of liver functions [7].

MiRNAs are extremely stable and protected from RNAase-mediated degradation in body fluids; they, therefore, have emerged as candidate biomarkers for many diseases [8]. The use of miRNAs as noninvasive biomarkers is of particular interest in the diagnosis of liver diseases [5]. Many studies have demonstrated that miRNA expression profiles in HCC and nontumor tissue are significantly different [9].

MiRNA-192 is hepatocyte-specific miRNA; therefore, its hepatic and circulating levels are clinically a prognostic marker in patients with HCC. Its hepatocyte-specific expression and its release from damaged hepatocytes early in liver injury in animal models have made miRNA-192 an attractive biomarker for liver damage. The huge amount of miRNA-192 in the liver is considered an advantage for all HCV serotypes, as it activates replication and translation of their genome [10].

The aim of this study is to identify serum miRNA-192 as biomarker to be used in diagnosing HCV-related HCC.


  Patients and methods Top


A cross-sectional observational study was performed on patients selected from the outpatient hepatology clinics and HCC early detection clinic in Al-Ahrar Teaching Hospital, Sharkia, Egypt, from October 2017 to October 2018. All patients were enrolled in the study after signing an informed written consent. Approval from the medical ethics research committee, Faculty of Medicine, Menoufia University, was obtained.

This study included 80 HCV-positive patients, comprising 40 patients without HCC and 40 patients with HCC. Moreover, 20 healthy participants were included as a control group. The following patients were excluded from the study: patients with other identifiable cause for liver cirrhosis other than HCV, patients with associated malignancies other than HCC, patients who received previous treatment for HCC, patients who received antiviral therapy for HCV, and pregnant or lactating woman. All included patients were subjected to the following: full history, with stress on personal history (including special habits; smoking, alcoholics, or drug addiction), the complaint and reason of attendance, history of present illness, presence of comorbidities like diabetes mellitus or hypertension, past history of admission to any hospital for any cirrhosis-related complication like hepatic encephalopathy, gastrointestinal bleeding, etc., history of blood transfusion, history of previous operation, previous treatment for HCV, and history of previous treatment for HCC. Detailed clinical examination of all body systems was done, with stress on signs of liver cell failure and portal hypertension (such as jaundice, palmar erythema, gynecomastia, and lower limb edema) and palpation of the liver and the spleen. Evaluation of performance status was done by the scale of the Eastern Co-operative Group (in HCC cases only). Moreover, they underwent laboratory investigations, including hemoglobin (Hb) (g/dl), red blood cell count (10/mm), white blood cell count (10/mm), platelet counts (10/mm), alanine aminotransferase (U/l), aspartate aminotransferase (U/l), serum albumin (g/dl), total and direct bilirubin (mg/dl), prothrombin time, international normalized ratio, serum creatinine, fasting blood sugar, HCV PCR, HbsAg, alpha-fetoprotein (AFP), miRNA-192, and pregnancy test for female in childbearing period. Abdominal ultrasonography was done to evaluate the liver, the spleen, the diameter, and patency of the portal vein and presence of ascites. Moreover, triphasic computed tomography abdomen was done to assess the tumor size and number and portal vein thrombosis.

Total RNA, including miRNAs, was extracted by miRNeasy extraction kit (Qiagen, Valencia, California, USA) using QIAzollysis reagent according to the manufacturer's instructions.

The quality of RNA was determined using NanoDrop2000 (Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA USA 02451). Reverse transcription (RT) was carried out on 100 ng of total RN in a final volume of 2 μl RT reactions (incubated for 60 min at 37°C and 5 min at 95°C) using miScript II RT Kit (Qiagen) according to the manufacturer's instructions. Quantitative real-time PCR was done.

Serum expression levels of mature miRNAs were evaluated using miScriptmiRNA PCR custom array (Qiagen) according to the manufacturer's protocol. The housekeeping miScript PCR miRNA SNORD68, was used as the internal control.

For real-time PCR of miRNA-192, 2.5 μl of diluted RT products was mixed with 5.5 μl RNase-free water, 10 μl QuantiTect SYBR Green PCR Master Mix, and 2 μl miScript universal primer (reverse primer), and then added to a custom Rotor-Disc 100 miRNA PCR array, which contains miRNA-specific miScript primer assays (Qiagen).

The Rotor-Disc was sealed with optical thin-walled strips. Real-time PCR was performed using Rotor gene Q Real-Time PCR System (Qiagen) with the following conditions: 95°C for 30 min, followed by 40 cycles at 94°C for 15 s, 55°C for 30 s, and 70°C for 30 s. The cycle threshold (Ct) is the number of cycles required for the fluorescent signal to cross the threshold in real-time PCR.

Then calculations of Child–Turcotte–Pugh score, APRI score, FIB.4 formula, and MELD score were done to evaluate the outcome of liver cirrhosis.

Statistical analysis

Clinical data were recorded on a report form. These data were tabulated and analyzed using the computer program Microsoft Office 2003 (excel) and Statistical Package for the Social Sciences, version 16, IBM (SPSS Inc., Chicago, Illinois, USA). Descriptive data were presented in the form of mean and SD for quantitative data and frequency and distribution for qualitative data. Analytical statistics were done for the statistical comparison between the different groups. The significance of difference was tested using one of the following tests: Student's t test and Mann–Whitney test were used to compare mean of two groups of quantitative data, whether parametric or nonparametric, respectively. Intergroup comparison of categorical data was performed by using χ2 test.

Correlation coefficient was used to find relationships between variables. Receiver operating characteristic curve was used to find validity of miRNA-192 value. P value less than 0.05 was considered statistically significant, whereas more than 0.05 was considered statistically insignificant, and P value less than 0.01 was considered highly significant.


  Results Top


Serum levels of aspartate aminotransferase, alanine aminotransferase, and total bilirubin (P < 0.001, for each) were significantly higher, whereas hepatic synthetic functions (albumin and prothrombin concentration) tended to decrease significantly (P < 0.001, for each) in patients with HCC versus the other two groups [Table 1].
Table 1: Liver and kidney functions among the studied groups

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MiRNA-192 was significantly increased in HCC group in comparison with HCV and control groups (mean ± SD 193.62 ± 257.56, 44.19 ± 35.12, and 23.01 ± 18.93, respectively) [Table 2] and [Figure 1] and [Figure 2].
Figure 1: ROC curve of miRNA-192 to differentiate HCC cases from healthy controls. HCC, hepatocellular carcinoma; miRNA, microRNA.

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Figure 2: ROC curve analysis of miRNA-192 to differentiate HCC cases from HCV cases. HCC, hepatocellular carcinoma; HCV, hepatitis C virus; miRNA, microRNA.

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Table 2: MicroRNA-192 among the studied groups

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The best cutoff level of serum miRNA-192 for distinguishing HCC group from HCV group was 62.06 (P < 0.001), with specificity and sensitivity of 70%, with area under the curve of 0.799 [Table 3].
Table 3: Validity of microRNA-192 to differentiate hepatocellular carcinoma cases from hepatitis C virus cases

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The best cutoff level of serum miRNA-192 for distinguishing HCC group from control group was 34.575 (P < 0.001), with specificity and sensitivity of 70 and 87.5%, respectively, with area under the curve of 0.884 [Table 4].
Table 4: Validity of microRNA-192 to differentiate hepatocellular carcinoma cases from healthy controls

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There was a significant difference in the level of serum AFP between different groups, with higher level in HCC group in comparison with non-HCC (P < 0.001) [Table 1].

On the contrary, there were significant lower levels of albumin, international normalized ratio, Hb, and platelets. Regarding white blood cells, there was no significant statistical difference between HCV and HCC groups [Table 5].
Table 5: Complete blood count results among the studied groups

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  Discussion Top


Sensitive and specific cancer biomarkers are essential for early detection and diagnosis of HCC, as well as for developing preventive screening. However, current methods are insufficient to detect HCC in the early stages. Advances in magnetic resonance imaging and computed tomography have greatly improved imaging of focal hypervascular masses consistent with HCC, but these procedures are costly and not readily available in developing countries. Laboratory data including serum AFP level have been used as HCC biomarkers for a long time. However, the accuracy of AFP is modest (sensitivity: 39–65%; specificity: 76–94%). One-third of cases of early-stage HCC (tumors <3 cm) are missed using AFP analysis, and serum AFP levels are also elevated in patients with benign liver diseases, such as hepatitis and cirrhosis [11].

Many miRNAs are dysregulated in HCC; thus, it is to be expected that circulating miRNA levels are also affected by HCC progression. The high stability of miRNAs in circulation makes them perfect biomarkers, especially for detection of early-stage, presymptomatic disease. MiRNA-192 is considered an important tumor suppressor, and therefore, its hepatic and circulating levels are clinically a prognostic marker in patients with HCC. Its hepatocyte-specific expression and its release from damaged hepatocytes early in liver injury in animal models have made miRNA-192 an attractive biomarker for liver damage.

Most of our patients with HCC have statistically significant male predominance (67.5%). This is concomitant with the results published by Abd-Elsalam et al. [12], Ferlay et al. [13], Abou El Azm et al. [14], Ma et al. [15], and Kanda et al. [16]. Approximately 60% of patients with HCC and 75% of HCV-infected patients in our study were from rural areas. This was in agreement with Ibrahim et al. [17] and Abou El Azm et al. [14]. The platelets count and Hb level were lower significantly in HCC group, and these results cope with the results reached by Tang et al. [17].

The majority of our patients with HCC were classified as Child B and C (62.5 and 32%, respectively), and most cases in HCV-infected patients were Child A (95%), a result that was in agreement with Allison et al. [18], Salama et al. [19], and Mohamed et al. [11]. Performance status of most cases of HCV-infected patients was stage 0 (82.5%) and stage 2 in most cases of HCC (37.5%), a result that was in agreement with Abou El Azm et al. [14] and Ma et al. [15]. Most cases were staged terminal, intermediate, and advanced, representing 37.5, 32.5, and 20%, respectively, according to BCLC scoring system, a result that was in agreement with El-Houseini et al. [20], Neamatallah et al. [21], and Mohamed et al. [11]. In HCC group, the incidence of single lesion was more than multiple lesions (65 and 32.5%, respectively), and 77.8% of HCC cases have focal lesion size more than 3 cm. This result was hand in hand with Shiani et al. [22] and El-Houseini et al. [20]. There is a significant difference in serum AFP level between different groups. It was significantly higher in HCC group (P < 0.001). These results are in agreement with the results of Mohamed et al. [11] and Hickey et al. [23].

Our study proved that miRNA-192 could be used as a biomarker for early detection of HCC, as it was found that miRNA-192 level showed significant increase in HCC group and mild increase in HCV group. Comparison of serum level of miRNA-192 among the three groups revealed that there was a significant change in level of miRNA-192 among HCV, HCC, and control. Moreover, miRNA-192 was significantly upregulated in patients with HCC in comparison with HCV and control groups (P < 0.001), and specificity and sensitivity were 70 and 87.5%, respectively.


  Conclusion Top


MiRNA-192 could be used as a diagnostic biomarker for early detection of HCC in HCV-related cirrhosis, as it showed significant upregulation in patients with HCC in comparison with non-HCC patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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