A molecular study on the role of microRNAs as a prognostic marker for Hepatitis B virus infection

Hepatitis B virus (HBV) is a virus that attacks the liver, which can cause acute and chronic liver disorders that can progress to liver cirrhosis and hepatocellular carcinoma (HCC). HBV remains a leading cause of disease and death globally. MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression at the post-transcriptional level by affecting both the stability and translation of complementary mRNAs. MicroRNAs (miRNAs) play an important role in the regulation of gene function. Numerous findings have shown that miR-101 and some miRNAs such as miR-122 and miR-221 are involved in HBV replication and HBV-associated disease, including the development of HBV-associated HCC. The aims of this study were to determine HBV genotyping in Iraqi HBV-positive patients and to evaluate the expression level of some microRNAs (101, 122 and 221) in plasma as a diagnostic marker of liver damage in chronic hepatitis B. This study included four subject groups: 25 healthy individuals, 25 chronic HBV carrier patients, 25 HBV positive patients under treatment and 25 patients with occult HBV infection. The study was started on 15/2/2023 by taking blood samples from Bakuba Teaching Hospital / Gastroenterology and Hepatology Department and Diyala Health Directorate Central Blood Bank Laboratory Virology Unit. HBV genotype, viral load and miRNAs (101, 122 and 221) in plasma were measured using real-time PCR. Statistical analysis was performed using SPSS software version 25 and a P value ≤ 0.05 was considered significant. These results showed that the mean ± SE expression of microRNA-101 in the treatment group was 33.8 ± 1.2, which was significantly higher compared to the control group (P= 0.042). In chronic HBV patients, the mean ± SE expression of microRNA-101 was 31 ± 1.4, which was also significantly higher than the control group (P= 0.012). Similarly, the mean ± SE expression of microRNA-101 in the occult HB group was significantly higher than in the control group (29.4 ± 1.5 vs 28.8 ± 2.2, P = 0.01). The results also showed that the mean ± SE expression of microRNA-122 was significantly higher in the treatment group compared to the control group (43 ± 3.5 Vs 32.3 ± 2.4, P = 0.001). Similarly, the mean ± SE expression of microRNA-122 in the chronic HB group was significantly higher than in the control group (35.8 ± 3.2 Vs 32.3 ± 2.4, P= 0.012). Finally, the mean ± SE of microRNA-122 expression was also significantly higher in the occult HB group compared to the control group (38.2 ± 2.7 Vs 32.3 ± 2.4, P= 0.01). Furthermore, the mean ± SE value of microRNA-221 expression in the treatment group was 36.6 ± 0.10, which was significantly lower than the control group (39.8 ± 0.11) (P= 0.004). The mean ± SE value of microRNA 221 expression in the chronic HB group was 34 ± 0.102, which was significantly lower than the control group (39.8 ± 0.11) (P= 0.012). Similarly, the mean ± SE value of microRNA-221 expression in the occult HB group was significantly lower compared to the control group (34.4 ± 0.1 vs 39.8 ± 0.11, P= 0.01). The sequencing reactions performed confirmed the precise identification of the analysed samples, which were found to be associated with the D genotype of HBV. Alignment of the nucleic acid sequences of samples H1 - H4 with the most closely related reference sequences of HBV (GenBank acc. no. KF170747.1) revealed the presence of two new nucleic acid variants (112T>C in H3 and 84C>A in H4) not present in these reference sequences. Translation of these variants showed that 112T>C leads to a missense effect in the protein encoded by the S gene (p.159Asp>Glu) that causes the conversion of p.159Asp to Glu. Similarly, 84C>A led to a missense effect, causing the conversion of p.169Ser to Pro in the large S protein (p.169Ser>Pro). The p.159Asp>Glu mutation involved the conversion of a negatively charged amino acid (Asp) at position 159 to a larger negatively charged amino acid (Glu). It can be concluded that the expression ratio of microRNA-101, microRNA-122 and microRNA-221 is highly significant in all clinical occurrences of HBV, especially those undergoing treatment. The positive correlation of these microRNAs with HBV markers suggested that they could be used as an auxiliary marker for determining the progression of HBV infection.