Assessment of circulating levels of microRNA-326, microRNA-424, and microRNA-511 as biomarkers for hepatocellular carcinoma in Egyptians

ARTICLE HIGHLIGHTS

Research background

Hepatocellular carcinoma (HCC) is lethal and is the fifth most common cancer. Differential expression of microRNAs (miRNAs)-326, miRNA-424, and miRNA-511 has been associated with the diagnosis and prognosis of HCC in different populations. However, limited information is available regarding their expression in Egyptian HCC patients.

Research motivation

This study aimed to assess the role of circulating miRNAs-326, miRNA-424, and miRNA-511 from plasma as a non-invasive route of detection and to explore the impact of these miRNAs in Egyptian HCC patients.

Research objectives

The study objectives were to assess the role of circulating miRNAs-326, miRNA-424, and miRNA-511 in Egyptian HCC patients by a non-invasive method using plasma samples.

Research methods

This prospective observational study included 70 adult Egyptian patients who developed HCC on top of HCV-related liver cirrhosis and 25 healthy age- and sex-matched participants who were seronegative for HCV and HBV, and served as controls. HCC was diagnosed according to the American Association for the Study of Liver Diseases updated practice guidelines, or was histo-pathologically confirmed. All patients were recruited from the multidisciplinary HCC clinic, the Endemic Medicine Department, Kasr al Ainy School of Medicine, Cairo University, and were HCC-treatment naïve at the time of enrollment. Exclusion criteria included; HBV co-infection, any cause of chronic liver disease other than HCV, any associated malignancies other than HCC, and prior treatment for HCV or HCC. Data collection: All patients were subjected to clinical assessment, laboratory investigations [complete blood count, prothrombin time and concentration, liver and kidney function tests, alpha-fetoprotein (AFP), hepatitis markers], ultrasound examination, and triphasic CT with documentation of HCC site, size, and number as well as the presence of PVT. At the time of enrolment into the study, blood samples were obtained from each participant, and Child-Pugh-score and BCLC stage were assessed based on the clinical, laboratory, and imaging data obtained. Whole blood samples were collected from each participant in 5-mL sterile RNAase-free vacutainer tubes containing EDTA. Blood samples were collected on ice and processed within 30 min of collection. To separate the plasma, each blood sample was centrifuged for 10 min at 1900g at 4 ℃. Plasma samples were carefully transferred into sterile RNase-free tubes. Plasma samples were then centrifuged for 10 min at 12000g at 4 ℃ to remove cellular nucleic acid contamination, and haemolysed plasma samples were excluded. Samples were separated into aliquots and immediately stored at 80 ℃ until processed. RNA extraction: Total RNA was extracted from 200 µL plasma using the miRNeasy Serum/plasma cell lysates kit, according to the manufacturer's instructions. RNA concentration and purity were monitored using a Nanodrop spectrophotometer device. Quantitative RT-PCR: The reverse transcription reaction was performed using TaqMan™ MicroRNA Reverse Transcription Kit, according to the manufacturer's instructions. miRNA-326, miRNA-511, and miRNA-424 quantifications were carried out using quantitative real-time PCR. The qRT-PCR for each sample was carried out in duplicate using TaqMan 2x universal master mix II and TaqMan microRNA Assay Mix containing PCR primers and TaqMan probes for each miRNA. The expression level of RNU6B was used as an endogenous control for normalization. To determine miRNA relative expression, it was reported as a fold change (ΔCt and ΔΔCt calculations). Patient management and follow-up: After blood sample withdrawal, all the patients were managed according to the BCLC guidelines after a case-by-case discussion. All HCC patients were followed up for a period of 24 mo. The response of HCC to treatment was evaluated according to mRECIST. The overall survival time of the patients was defined as the period from the initial presentation to the last follow-up or death.

Research results

The mean age of the studied cohort was 62.0 ± 7.6 years. Most of the patients were male (68.8%), non-diabetic (74.3%), non-smokers (80%), and Child A (82.9%). According to the BCLC staging system, most patients were in the early stage (48.6%). Most of the HCC lesions were single (70%), present in the right hepatic lobe (84.3%), and not associated with PVT (92.9%). That is why most of the patients were subjected to hepatectomy and microwave ablation (MWA), and most of them showed complete response according to mRECIST. All HCC patients were followed up for a period of 24 mo or until death. The mean survival time was 367.1 ± 173.9 days. The median values of miRNA-326, miRNA-511, and miRNA-424 were 35, 1.2, and 5.1, respectively. Correlation of miRNAs with different parameters in HCC patients: There was no significant association between the studied miRNAs levels and gender, smoking, or the patients' performance status. The serum level of miRNA-424 was significantly elevated in diabetic patients. There was a significant difference in miRNA-326 between Child grades A and B. Moreover, there was a significant difference in miRNA-424 between early and intermediate HCC. Diagnostic efficiency of miRNAs in our patients: On comparing the miRNAs levels between healthy participants and HCC patients, it was found that HCC patients showed significantly higher levels of miRNA-326 (P = 0.001) and significantly lower levels of miRNA-511 (P = 0.02). ROC analysis revealed the diagnostic performance of the studied miRNAs. miRNA-326 showed the best diagnostic performance, diagnosing HCC at a cut-off value of 1.165 with a sensitivity of 97.1%, specificity of 52%, PPV of 85%, and NPV of 86.7%. The AUC was 78.4% (67.7-89.1), and the overall accuracy was 85.3% (P < 0.001). Association of miRNAs with response to treatment and survival: Despite finding no statistically significant differences in the studied miRNAs between surviving and non-surviving HCC patients, we found that miRNA-326 > 1.165 was significantly associated with overall survival (P = 0.001). Moreover, there was a significant association between the BCLC stage as well as the response to treatment according to mRECIST and overall survival. It was also found that miRNA-511 was significantly associated with response to treatment according to mRECIST.

Research conclusions

We conclude that miRNA-326, miRNA-424, and miRNA-511 have diagnostic and prognostic roles in Egyptian patients with HCV-related HCC and should be considered for better disease management.

Research perspectives

miRNA-326, miRNA-424, and miRNA-511 can be detected from plasma and have diagnostic and prognostic roles in Egyptian patients with HCV-related HCC and should be considered for better disease management.