Serum microRNA 143 as a potential biomarker for the diagnosis of hepatitis C virus-related hepatocellular carcinoma

Background Hepatocellular carcinoma (HCC) is the fifth most frequently diagnosed cancer worldwide. Early recognition of the onset of HCC would help to select more effective therapies for patients leading to a better prognosis and life span. So, the development of effective markers for the diagnosis of HCC could have an impact on HCC-related cancer mortality. MicroRNAs (miRNAs) are reported as a group of small noncoding RNAs that can function as endogenous RNA interference to regulate the expression of targeted genes. Aim To study serum miR-143 expression level in patients with hepatitis C virus (HCV)-related HCC. Patients and methods The present study was conducted on 60 participants classified into group A: 30 patients with HCV-related cirrhosis with HCC; group B: 15 patients with HCV-related cirrhosis without HCC; and group C: healthy participants as control. Expression of miR-143 in the serum of all participants was obtained in all groups. Total serum RNA was extracted with small RNA enrichment followed by reverse transcription real-time PCR. Expression of miR-143 in the serum of all participants was obtained using the comparative cycle threshold method (2–ΔΔCT) after normalization for the expression of Syn-cel-miR-39 mi script miRNA mimic as control. MiR-143 expression levels were then compared in different groups. Results The mean serum miR-143 levels were significantly higher in cirrhotic patients without and with HCC than in healthy participants (1.69±0.64, 13.0±6.23 vs. 0.56±0.29, P˂0.001, respectively) and in patients with HCC than in patients without HCC (P<0.001). The mean serum miR-143 levels were significantly higher in cirrhotic patients with HCC Barcelona Clinic Liver Cancer stage D than stage B (19.91±2.40, 13.41±1.80, P˂0.001, respectively) and in stage B compared with stage A (13.41±1.80, 5.68±1.86, P<0.001, respectively). Conclusion The significantly higher serum level of miR-143 in cirrhotic patients with HCC compared with those without and to normal participants may suggest its role in hepatocarcinogenesis and may have a value as a potential biomarker for early diagnosis of HCV- related HCC.


Background
Hepatocellular carcinoma (HCC) is the fifth most frequently diagnosed cancer worldwide. Early recognition of the onset of HCC would help to select more effective therapies for patients leading to a better prognosis and life span. So, the development of effective markers for the diagnosis of HCC could have an impact on HCC-related cancer mortality. MicroRNAs (miRNAs) are reported as a group of small noncoding RNAs that can function as endogenous RNA interference to regulate the expression of targeted genes. Aim To study serum miR-143 expression level in patients with hepatitis C virus (HCV)related HCC.

Patients and methods
The present study was conducted on 60 participants classified into group A: 30 patients with HCV-related cirrhosis with HCC; group B: 15 patients with HCVrelated cirrhosis without HCC; and group C: healthy participants as control. Expression of miR-143 in the serum of all participants was obtained in all groups. Total serum RNA was extracted with small RNA enrichment followed by reverse transcription real-time PCR. Expression of miR-143 in the serum of all participants was obtained using the comparative cycle threshold method (2-ΔΔCT) after normalization for the expression of Syn-cel-miR-39 mi script miRNA mimic as control. MiR-143 expression levels were then compared in different groups.

Results
The mean serum miR-143 levels were significantly higher in cirrhotic patients without and with HCC than in healthy participants (1.69±0.64, 13.0±6.23 vs. 0.56 ±0.29, P< 0.001, respectively) and in patients with HCC than in patients without HCC (P<0.001). The mean serum miR-143 levels were significantly higher in cirrhotic patients with HCC Barcelona Clinic Liver Cancer stage D than stage B (19.91±2.40, 13.41±1.80, P< 0.001, respectively) and in stage B compared with stage A (13.41±1.80, 5.68±1.86, P<0.001, respectively).

Conclusion
The significantly higher serum level of miR-143 in cirrhotic patients with HCC compared with those without and to normal participants may suggest its role in Introduction Hepatocellular carcinoma (HCC) is the most common primary liver cancer with an estimated incidence of one million cases per year. It is the fifth most common cancer worldwide and the third leading cause of cancer death after lung and stomach cancer [1].
Because of detection at an advanced, nonoperable stage, the prognosis of HCC is usually poor. This limits the potential for curative surgical treatment only for cases with small HCC malignancies. This problem calls for more surveillance strategies targeted to the population at risk, to be used for early detection of HCC [2]. Currently, the diagnosis of HCC depends on clinical information, hepatic ultrasonography, and measurement of serum alphafetoprotein (AFP) at 6-12 month intervals. However, neither of the aforementioned is adequate for detecting very small (<2 cm) HCC tumors. The sensitivity of AFP is low particularly in the detection of early-stage HCC. As such, no marker has been proven to be reliable to detect small HCC tumors (2 cm or less). Therefore, it is urgently needed to identify novel biomarkers with high efficacy for early detection and therapeutic monitoring of HCC [3].
MicroRNAs (miRNAs) are small noncoding RNAs that can function as post-transcriptional regulators of expression of their targeted genes [4]. In addition to their presence in tissues, miRNAs were recently shown to be secreted from the cells and can thereby gain access to different body fluids such as plasma, serum, urine, saliva, sputum, etc. [5]. Nevertheless, they were shown to remain highly stable upon exposure to conditions such as boiling, extremes of pH, and long-duration storage that induce immediate degradation of free RNA [6].
The advent of highly sensitive real-time (RT) PCR assay detection methods aided the detection of miRNAs in blood. They were also proven as minimally invasive and their results showed high reproducibility. Circulating miRNAs can therefore be used as noninvasive, sensitive disease biomarkers. Today, prediction and diagnosis of HCC via determining disease-specific circulating miRNAs has become the focus of many studies. Researchers have previously reported the potential clinical application of circulating miRNAs (such as miR-122) as diagnostic and prognostic HCC biomarkers. Nonetheless, further investigation of aberrant miRNA expression could lead to the discovery of novel miRNA biomarkers for HCC [7].
Among the large number of miRNAs, miR-143, owing to its association with malignancies, has been widely studied. Mir-143 is a short RNA molecule highly conserved in vertebrates and has been implicated in several cancers [8]. The expression of miR-143 was shown to be downregulated in breast, gastric, and colorectal cancers [9][10][11]. However, it was found that miR-143 was overexpressed in other cancers such as pancreatic ductal adenocarcinoma [12] and during the differentiation of prostate cancer stem cells and could promote prostate cancer metastasis by repressing fibronectin-type III domain containing 3B expression [13].
Few previous studies were focused on the relationship between miR-143 expression and the diagnosis of HCC. So, the aim of this study was to study serum miR-143-expression level in patients with hepatitis C virus (HCV)-related HCC.

Patients and methods
An informed consent was obtained from all participants included in the study. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki and Good Clinical Practice guidelines and was approved by the Alexandria Faculty of Medicine Human Research Committee. This study was conducted on 60 participants allocated into three groups as follows: Group A: 30 patients with HCV-related cirrhosis with HCC classified according to the Barcelona Clinic Liver Cancer (BCLC) staging system into 10 patients with early-stage BCLC A; 10 patients with intermediatestage BCLC B; and 10 patients with end-stage BCLC D. Group B: 15 patients with HCV-related cirrhosis without HCC. Group C: 15 age-matched and sexmatched healthy participants with no evidence of liver disease were included as the control group.
The diagnosis of chronic HCV infection with cirrhosis was based on the following criteria: (a) positive test for HCV antibody and (b) detectable serum HCV RNA and the diagnosis of cirrhosis was determined by clinical, biochemical, and ultrasonographic evidence.
The diagnosis of HCC was based on serum levels of AFP, ultrasonography, and triphasic computed tomography. All the patients of groups A and B were selected from the Hepatobiliary Unit of Alexandria University Hospital.
Patients who have the following criteria: seropositivity for HBV infection, other causes of chronic liver diseases, chronic illness, for example, collagen diseases, diabetes mellitus, autoimmune diseases, cardiac, respiratory, renal diseases, chronic alcoholism, and other malignancies were excluded from the study.
All patients included in the study were evaluated clinically as regards history taking and clinical examination focusing on symptoms and signs of chronic liver disease (previous upper gastrointestinal bleeding, jaundice, ascites, hepatic encephalopathy, and bleeding diathesis), presence of palpable focal hepatic lesions, and liver and spleen size. Blood samples were collected from all patients and healthy participants and the following tests were performed: complete blood picture, serum creatinine, liver test profile (serum aspartate aminotransferase) and alanine aminotransferase, serum albumin, serum bilirubin, serum gamma glutamyl transpeptidase and prothrombin time (PT), serum HCV RNA levels using RT-PCR and serum AFP levels using standardized enzyme-linked immunosorbent assay kit. The severity of the liver disease in patients with HCV-related cirrhosis was graded according to Child-Pugh classification. Radiological examination using ultrasonographic/triphasic computed tomography examination was performed for the assessment of liver echo pattern, liver and spleen size, presence of ascites, and tumor characteristics in patients with HCC (maximum diameter, echo pattern, number of nodules, extension, portal vein invasion, and intrahepatic metastasis). The stage of HCC was determined according to the BCLC staging system. RNA extraction to perform expression of serum microRNA 143 The blood samples were collected and centrifuged at 3000 rpm for 5 min to separate serum and then stored at −80°C until RNA extraction. Total RNA was isolated from 100 μl of serum previously thawed on ice using an miRNAeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The RNA was eluted with 40 μl of RNAse-free water and was stored at −80°C until RT-PCR reaction. The concentration of all RNA samples was quantified using NanoDrop 1000 (Thermo Scientific, Wilmington, Delaware, USA). Thereafter, TaqMan miRNA assay was used to perform the expression levels of serum miRNA-143. All reagents, primers, and probes were obtained from Applied Biosystems (Foster City, California, USA). A measure of 5 μl of total RNA was used from each sample for individual assays in 15 μl reactions containing reverse transcription mixture and primers. The mix was incubated at 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min; miRNA expression levels were quantified using Applied Biosystems Step One RT-PCR System, thermal cycling (Applied Biosystems). For this purpose, 1 μl of reverse transcription reaction was mixed with 10 μl of 2× TaqMan PCR mixtures, 1 μl of TaqMan primer mix and probe and 8 μl water to obtain a final volume of 20 μl. An RT-PCR was done in triplicate, including nontemplate controls. The relative expression of mature miRNA was calculated using the comparative cycle threshold (2−ΔΔCT) method. Syncel-miR-39 mi script miRNA mimic was used as a control to normalize the data.

Statistical analysis
Statistical analysis of the data was performed using SPSS program (version 20.0) (IBM-SPSS Inc., New York, New York, USA) for Windows. Statistical significance was assessed at P value less than 0.05. Sensitivity and specificity of serum miR-143 levels in discriminating cirrhotic patients with and without HCC were assessed by plotting a receiver-operating characteristic (ROC) curve and determining its cutoff value. Table 1 shows the demographic data of the studied groups. There was no statistically significant difference between the three groups regarding sex and age. Serum level of total and direct bilirubin, alanine aminotransferase, aspartate aminotransferase, and gamma glutamyl transpeptidase were significantly higher in group A and group B patients compared with group C, but no significant difference was found between group A and group B patients.

Results
Mean serum albumin level was significantly lower in cirrhotic patients with and without HCC than in Table 1 Comparison between the studied groups according to the demographic data    (Fig. 1).  ROC curve analysis (Fig. 2) shows that the sensitivity and specificity of serum miR-143 expression level in discriminating cirrhotic patients with HCC from those without were 96.67 and 100%, respectively, at a cutoff level of more than 3.26 and area under the curve (AUC) was 0.996 indicating good discriminative power with P value less than or equal to 0.001. These results show that miR-143 could serve as a valuable biomarker for the diagnosis of HCC in HCV-related cirrhosis.
ROC curve analysis (Fig. 3) shows that the sensitivity and specificity of serum miR-143 in discriminating cirrhotic patients from healthy control group was 100 and 93.33%, respectively, at a cutoff level of more than <0.001* AFP, alpha-fetoprotein; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma glutamyl transpeptidase; Hb, hemoglobin; HCC, hepatocellular carcinoma; PT, prothrombin time; WBC, white blood cell count. P 1 , P value for groups A and B. P 2 , P value between groups A and C. P 3 , P value between groups B and C. Statistically significant at P value of less than or equal to 0.05.
1 and AUC equal to 0.996 indicating good discriminative power and statistically significant at P value less than or equal to 0.001. These results indicated that miR-143 could serve as a valuable biomarker for differentiating HCV-related cirrhosis from healthy participants.
ROC curve (Fig. 4) compares serum miR-143 and AFP in cirrhotic patients with HCC and patients without HCC. It shows a significant difference between AUC of miR-143 and AFP (P<0.001) with a sensitivity and specificity of 96.67 and 100% for miR-143, respectively, and 80 and 100% for AFP, respectively, AUC=0.996 for miR-143 and 0.929 for AFP at a cutoff level of more than 3.26 for miR-143 and more than 72 for AFP. These results indicate that miR-143 has better diagnostic performance than AFP in the detection of HCC.

Discussion
This study showed significantly higher levels of serum miR-143 in cirrhotic patients without HCC than in healthy participants. Similar to our study Murakami et al. [14] have found miR-143 to be downregulated in chronic hepatitis and upregulated in liver cirrhosis. This result may be explained by release from damaged hepatocytes which might be the major source of hepatocyte-derived miRNAs; so, the release of miRNAs upon damage might be higher than in patients with healthy liver tissue [15]. In comparison with liver-specific miR-122, Trebica et al. [16] reported significant decrease of miR-122 expression in cirrhosis compared with normal controls. The finding was explained by the abundance of miR-122 in hepatocytes and its presence in much lower levels in the circulation than in healthy participants. With hepatocyte injury, miR-122 is released in the circulation and serum levels rise, with eventual loss of hepatocyte and development of fibrosis, hence, the circulating miR-122 levels drop again.
In this study, serum miR-143 was shown to be significantly higher in patients with early HCC (BCLC stage A) than cirrhotic patients without HCC (P<0.001). As miRNAs play a key role in diverse biological processes, including development, cell proliferation, differentiation, and apoptosis, Significance between groups P 1 <0.001*, P 2 <0.001*, P 3 <0.001* r s (P) 0.941(<0.001*) BCLC, Barcelona Clinic Liver Cancer; KW χ 2 , χ 2 for Kruskal-Wallis test; miR-143, microRNA 143. Significance between groups was done using Mann-Whitney test. P 1 , P value for comparing between A and B. P 2 , P value for comparing between A and D. P 3 , P value for comparing between B and D. r s , Spearman's coefficient. *Statistically significant at P value less than or equal to 0.05, significance between groups altered miRNA expression is likely to contribute to human disease, including cancer. The underlying mechanisms of miRNAs deregulation are largely uncertain [17]. In parallel with our results, Zhu et al. [18] reported that elevated serum levels of miR17HG protein (a protein encoded by miR-17-92 cluster host gene) in HCC tissues may suggest that the miR-17-92 cluster is activated early in HCC, even at the precancerous cirrhosis stage.
Taken together, these data suggest that the miR-143 is activated in early HCV-related HCC and may play a role in hepatocarcinogenesis.
In the current work, we found that serum miR-143 levels positively correlate with BCLC staging as HCC stage D patients have a higher level of serum miR-143 than in stages A and B patients and HCC stage B patients have a higher serum level of miR-143 than stage A patients. In this regard, Zhang et al. [19] reported similar results showing that miR-143 is upregulated in HBV-HCC and promotes cancer cell invasion and migration.
In the present study, serum miR-143 levels were significantly higher in HCC patients than in the healthy group and cirrhotic patients without HCC. ROC curve analysis using miR-143 serum expression level for discriminating cirrhotic patients with HCC from cirrhotic patients without HCC showed that sensitivity and specificity were 96.67 and 100%, respectively at a cutoff level of more than 3.26 and the AUC was 0.996 indicating good discriminative power and was statistically significant (P≤0.001). This may suggest that serum miR-143 can be used as a biomarker for the diagnosis of HCV-related HCC. Zhang et al. [19] demonstrated that miR-143 might have a role in the regulation of target genes in HCC ROC curve for miR-143 to differentiate liver cirrhosis from healthy participants. miR-143, microRNA 143; ROC, receiver-operating characteristic.
where microvesicles packaged with miRNAs are released mainly from the tumor cells. They also reported that the levels of miR-143 were dramatically overexpressed in metastatic HBVrelated HCC of both transgenic mice and HCC patients and this overexpression is transcribed by nuclear factor kappa B that favors liver tumor cell invasive and metastatic behavior and they found that local liver metastasis and distant lung metastasis are significantly inhibited by blocking miR-143 and fibronectin-type III domain containing 3B.
To date, only few studies have reported the association between miR-143 and HCC. In agreement with our data, Zhang et al. [20] reported that the expression of miR-143 in serum was significantly upregulated in patients with HCC and serum miR-143 can be used as potential biomarkers for the diagnosis of HCC. On the other hand, Shen et al. [21] demonstrated that a number of miRNAs have not only diagnostic but also prognostic and therapeutic implications in HCC.
Contrary to our results Liu et al. [22] reported that miR-143 was underexpressed in hepatoma and so upregulating its expression level could inhibit its potential target gene toll-like receptor 2 and nuclear factor-κB in the downstream pathway, leading to the inhibition of hepatoma proliferation. Wang et al. [23] reported that the expression of miR-143 was significantly lower in HCC tissues than in paired adjacent normal tissues.
Since HCC is a complex disease and has an unpredictable behavior in the regulation of noncoding RNAs, the discrepancy between the results of our study and other studies may be due to the multiple risk factors that have been implicated in the development of HCC [24][25][26]. Also, the large ethnic and geographic variability in the incidence of HCC among the different populations may lead to etiology-related differences in miRNA expression. This may have contributed to the difference between our study and others. Moreover, there are different HCV genotypes other than genotype 4, which represents more than 90% of the cases in Egypt. In our study, we measured miR-143 in serum sample while other contradicting studies detected miR-143 in the tissue sample. So, the different sampling may provide another possible reason for the variation in results. Compared with miR-122 which is downregulated in primary HCC tissues, serum miR-122 is upregulated in HBV patients with HCC, possibly due to miR-122 release from tissues into circulation [27]. However, Mamdouh et al. [28] reported that serum miR-143 is upregulated in HCV-related HCC and can be used as a diagnostic and prognostic marker and may promote malignant progression of HCC in agreement with our results.
ROC curve analysis for serum miR-143 and AFP in discriminating cirrhotic HCC group A patients from non-HCC group B revealed a significant difference between AUC of miR-143 and AFP (P<0.001), with a sensitivity and specificity of 96.67 and 100% for miR-143 and 80 and 100% for AFP, respectively, AUC=0.996 for miR-143 and 0.929 for AFP at a cutoff level of more than 3.26 for miR-143 and more than 72 for AFP. This result may indicate that miRNA-143 has a better diagnostic performance than AFP in the detection of HCC. In agreement with this, Lin et al. [29] identified a set of seven miRNAs including miR-143 that were upregulated in the serum of patients with HBV-related HCC compared with patients with chronic HBV. Using a training cohort and two independent validation cohorts, they developed and validated a seven-miRNA panel including miR-29a, miR-29c, miR-133a, miR-143, miR-145, miR-192, and miR-505. This panel was more sensitive than AFP and could detect small AFP-negative HCC samples, providing hope for earlier detection of HCC.

Conclusion
The serum level of miR-143 is significantly higher in cirrhotic patients with early HCC than in cirrhotic patients without HCC, suggesting a possible role in hepatocarcinogenesis. Serum levels of miR-143 showed significant increase in HCV-related cirrhosis and HCC in comparison with non-HCC cirrhotic patients and healthy participants with high sensitivity and specificity in detecting HCC. This suggests its value as a potential biomarker for early diagnosis of HCV-related HCC.
Ahmed M. El-Gohary provided the study concept and design, study supervision, and approval of the final revision of the manuscript. Ahmed E. Zeid: analysis and interpretation of data, statistical analysis, manuscript drafting, and revision and approval of the final revision of the manuscript. Mohamed E. Ibrahim: performed imaging work of participants of the study, analysis and interpretation of data, manuscript revision and approval of the final revision of the manuscript. Fatma I. Dewedar: performed the laboratory work of participants of the study, analysis and interpretation of data, statistical analysis, manuscript writing, and revision and approval of the final revision of the manuscript. Essam A. Elzoheiry: acquisition and assembly of data, analysis and interpretation of data, statistical analysis, drafting and revision of the manuscript, and approval of the final revision of the manuscript.

Financial support and sponsorship
Nil.

Conflicts of interest
There are no conflicts of interest.