الفهرس 
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Abstract
Conclusion • HCV infection results in the modulation of the hepatocyte specific miR-122 expression. The present study concluded that the serum level of miR-122 significantly increased in chronic HCV infected than in cirrhotic and HCC patients, hence it may represent novel, non-invasive biomarker for the diagnosis and evaluation of patients with chronic HCV, by permitting with a serum test.
• In addition, the data of our study indicate that the expression of miR‑122 is reduced in patients with hepatic decompensation. Therefore, serum miR-122 is a new potential parameter for liver function and a prognostic parameter in patients with liver cirrhosis.• The present study also provides evidence that miR-122 serum levels reflect necro-inflammatory activity in the liver of patients with CHC, thus it has a strong potential to serve as a useful biomarker for liver injury and for grading of CHC and potentially other liver diseases, as there was a need for new serum markers for staging and grading of chronic hepatitis.
• Our study also suggested that serum miR-122 could indicate the degree of liver cell damage, changes in serum miR-122 most accurately reflected the degree of fibrosis as it was significantly downregulated during disease progression from liver fibrosis (F1-F3) to cirrhosis (F4) to HCC, suggesting that serum miR-122 monitoring could be of clinical relevance as a new independent marker for prediction of survival of patients with liver cirrhosis and as a potential diagnostic biomarker for fibrosis progression to cirrhosis to HCC.• Furthermore, our findings indicate that serum levels of miR-122 significantly decreased in HCC patients, thus can differentiate HCC from CHC and LC, suggesting that serum miR-122 may serve as a promising biomarker for prediction of development of malignancy and for early detection of HCV-induced HCC.• Moreover, based on the ROC curves, miR-122 had high efficiency compared to other noninvasive indices in prediction of HCV, development of malignancy, and prognosis of HCC. Thus, serum miR-122 could be used alone or may be combined with 𝛼-FP for a better and early diagnosis of HCV-related HCC.
• Clinical proof-of-concept studies have demonstrated that miR-122 inhibitors efficiently reduced viral load in chronically infected HCV patients without detectable resistance. Furthermore, multiple mechanistic researches have revealed that artificially upregulated miR-122 obviously suppressed the proliferation, metastasis, and drug resistance of HCC tumor cells.Recommendations
• Circulating miRNAs are secreted in different body fluids through exosomes and macrovesicles. Serum/plasma miRNAs are very stable and last for long time and their determination methods are easy to perform. So, these miRNAs have an increased advantage over traditional biomarkers, making them potential and relatively non-invasive biomarkers for the detection of different stages of a disease. Moreover, their detection in serum/plasma can clearly define the progression of the disease. Thus, as there is an urgent need for new serum markers, future researches for better understanding of their functions will be of great value.
• In light of this, research focused on studying circulating miR-122 as a representative liver-specific miRNA candidate with potential clinical significance for liver disease conditions because its expression is dysregulated in liver diseases, its abnormal presence in the serum can indicate liver injuries.
• Assessing circulating miR-122 levels has shown promise in diagnosing liver pathogenesis in various diseases such as HIV, HBV and/or HCV-associated chronic viral hepatitis, chronic liver infections, and acts as a promising novel diagnostic and prognostic biomarker for chronic HCV infection and HCV- induced HCC, and as a potential therapeutic target as well.
• Evaluation of the diagnostic potential and prognostic significance of the serum miR-122 level, and its implication as a therapeutic tool or target in HCV-related chronic liver disease in clinical practice whether alone or as a supplement to other markers and different miRNAs panels will be beneficial to investigate establishing miRNAs as independent diagnostic and prognostic markers of HCV-related chronic liver disease, because there is a need for new serum markers such as miRNAs for staging and grading of chronic hepatitis as the routinely used surrogate parameters to evaluate patients with CHC fail to predict the progression of hepatic necroinflam¬matory activity and fibrosis in a considerable number of patients.• Identification of miRNAs associated with HCV is crucial to aid in the development of a new strategy to prevent the chronicity of HCV infection and the development of hepatocellular carcinoma, and for developing new diagnostic and therapeutic tools with high specificity and sensitivity in order to combat this vicious human cancer.
Summary
Hepatitis C virus represents an important health burden throughout the world. Egypt recorded the highest prevalence of HCV worldwide and a high morbidity and mortality from HCV-related chronic liver disease, cirrhosis, and hepatocellular carcinoma.
MicroRNAs are small non-coding RNAs that mediate post-transcriptional gene silencing by promoting mRNA degradation and repressing translation. Circulating microRNAs in human peripheral blood have been increasingly regarded as potential indicators of a variety of physiological and pathological conditions, including liver injury induced by hepatotoxic agents and viral hepatitis.MiR-122 is a liver-specific microRNA abundantly expressed in hepatocytes. A unique character of the HCV life-cycle is its reliance on miR-122, which promotes translation and stabilizes the viral genomic RNA.
In addition, miR-122 expression is typically observed to be lost in hepatocellular carcinoma, and miR-122 knockout mice developed persistent hepatosteatosis, fibrosis, and HCC, suggesting that miR-122 also has an important role as a tumor suppressor.This study aimed to: 1) measure & compare the levels of miR-122 in peripheral blood between healthy controls, chronic hepatitis C (CHC), cirrhotic & hepatocellular carcinoma (HCC) patients. 2) Correlate serum miR-122 with serum inflammatory markers of the liver disease; alanine transaminase (ALT) and aspartate transaminase (AST). 3) Investigate whether the serum levels of miR‑122 may be useful as a diagnostic marker of liver injury in patients with chronic HCV infection and whether it can be used as a marker for the early detection of HCC. 4) Study miR-122 as biomarker of HCV disease progression to open the field for prognostic or therapeutic intervention in HCV.
The current cross-sectional study was conducted in Medical Microbiology and Immunology, Internal Medicine departments, and Central Research Laboratory, Faculty of Medicine, Sohag University Hospital (SUH) during the period from January 2018 to January 2021. This study involved one hundred and eighteen patients attended the outpatient clinic or admitted to inpatient department of Internal Medicine Department of SUH and twenty age and sex matched apparently healthy persons as a control group.
Patients were categorized into twenty six patients with chronic hepatitis C, twenty eight patients with compensated cirrhosis, thirty four patients with decompensated cirrhosis, and thirty hepatocellular carcinoma patients. Informed written consent was obtained from all participants. The study protocol was approved by the Ethics Committee of Sohag Faculty of Medicine.
The studied patients who fulfilled the defined inclusion criteria were subjected to complete medical history, thorough clinical examination, and laboratory investigations including complete blood count, liver and kidney function tests, hepatitis markers and AFP. In addition to abdominal ultrasonography and triphasic CT scan of the abdomen (was done if a hepatic focal lesion was detected by ultrasonography to establish the diagnosis of HCC). Then, blood samples were collected and estimation of serum levels of miR-122 was performed by real time reverse transcription polymerase chain reaction (RT-PCR) for all participants.
The results obtained in our study revealed that serum miR‑122 expression showed significantly increased expression level in CHC, compensated cirrhosis and decompensated cirrhosis patients’ sera in comparison with the control group. On the other hand, mean serum miR-122 level was significantly lower in HCC patients compared to both CHC patients and controls (P=0.0001). Comparison between compensated and decompensated cirrhosis patients revealed that mean serum miR-122 level was significantly reduced in patients with hepatic decompensation (P=0.0001).
Serum miR-122 expression level showed statistically significant positive correlation with serum necro-inflammatory markers of the liver AST and ALT in non-HCC patients, and significant negative correlation with AFP in HCC patients.
Regarding its relation to liver fibrosis grades, serum miR-122 revealed gradually decreased levels with progression of fibrosis stage, with more significant decrease in late stages of fibrosis; F3 and F4 (P=0.01). Comparing groups revealed statistically significant increase of serum miR-122 levels in F0 compared to F4 (P=0.02).
In addition, there was a trend toward lower mean levels of miR-122 with increased Child score and with advanced HCC stage, but didn’t reach statistical significance. While serum miR‑122 expression was higher in patients with high viremia than in those with moderate and low viremia, although insignificant (P=0.87).
Our results also disclosed that miR-122 could predict HCV with sensitivity 76.9%, specificity 100%, accuracy 88.5%, and cut off value >5.7 (RQ) (P<0.0001). Furthermore, miR-122 could predict HCC with sensitivity 93.3%, specificity 100%, accuracy 96.65%, and cut off value ≤2.1 (RQ) (P<0.0001).
The main goal of our study was to investigate the expression of serum miR-122 as potential and reliable non-invasive biomarker that can differentiate between different stages of HCV infection in Egyptian population and can be used as a sensitive and specific biomarker for HCC. Our data revealed that miR-122 results are significantly des-regulated among the studied categories.
Our study is unique as we demonstrated serum miR-122 signatures during stepwise progression form chronic HCV to cirrhosis to HCC. We hypothesized that the miR-122 would be a biomarker of disease progression. Further studies to validate our findings are recommended.
The process of examination of serum miR¬NAs as diagnostic and prognostic biomarkers is still at its infancy, but consider¬ing the rapid progress in this field, development of miRNA-based blood diagnostics is likely to be successful in order to introduce this new marker into clinical practice in the near future.
Interestingly, based on the importance of miR-122 in HCV infection and HCC progression, several clinical trials of miR-122 are ongoing to introduce miR-122 as a new therapeutic target as well.
In conclusion, manipulation of miR-122 represents a major breakthrough in diagnosis, prognosis, and treatment of HCV-related chronic liver disease.