Circulating microRNAs as predictors of response to sofosbuvir + daclatasvir + ribavirin in in HCV genotype-4 Egyptian patients | BMC Gastroenterology

HCV is a global health problem and the major etiological cause of chronic hepatitis and liver disease worldwide. More than 50% of HCV-infected cases develop CHC which can lead to many sequels like fibrosis, cirrhosis, end‐stage liver disease, and HCC [12]. Nowadays, the study of non-invasive diagnostic and prognostic biomarkers of the diseases has become an interesting and rapidly-growing field in clinical research.

Patients who are more or less likely to attain SVR are identified by doctors using predictors of response to therapy as decision-making aids. In the current study, we assessed a number of variables related to HCV infection that affect the prognosis of the illness and how well it responds to therapy. These variables include the amount of expression of a panel of four liver-associated miRNAs in the blood in CHC Egyptian patients getting DAA therapy (SOF/DAV + RIB), as well as in healthy controls.

Regarding HCV replication and infection, microRNAs are thought to be an intriguing area of research. They could also serve as novel targets for the creation of antiviral drugs. Depending on a patient’s treatment reaction, miRNA levels vary. In order to reduce unsuccessful therapies, miRNAs can be employed as predictors of patient medication responses before combination therapy [13].

In the current study, A significant overexpression of circulating miR-122 and miR-155 levels was reported in CHC patients compared with healthy controls. These findings are in agreement with other findings that reported increased miR-122 expression [14,15,16] and increased miR-155 expression [9, 17] in CHC patients. On the other hand, these findings are inconsistent with other data that reported decreased miR-122 expression with advanced fibrosis [18]. This overexpression of miRNAs in serum of HCV‐infected patients may be caused by inflammation and liver cells damage due to viral infection. Inflammation due to viral infection causes damage to hepatocytes resulting in release significant amounts of miRNAs into the circulation, which may explain its high level in serum [19]. miR‐122 facilitates viral RNA replication and plays an important role in the progression of liver disease, therefore it may be considered as a biomarker for HCV infection [20].

On the other hand, the current study has recorded decreased the expression of both miR-29 and miR-196 in CHC patients compared with healthy controls. These findings are consistent with data recorded in previous studies [16, 21] for miR-29 and miR-196 respectively. In contrast to our data El Guendy et al., reported that members of miR-29 and miR-196 families are overexpressed in CHC patients [18]. Downregulation of miR-29 could be explained as HCV might downregulate miR-29 to its advantage. Remarkably, miR-29 overexpression decreased HCV RNA replication. Thus, miR-29 can be deleterious to HCV, providing a possible motivation for the virus to downregulate miR-29 [11]. Roderburg et al. also explained miR-29 down-regulation in liver fibrosis of mice models, where downregulation of miR-29 is mediated by transforming growth factor beta (TGF-β), inflammatory signals lipopolysaccharide (LPS) and nuclear factor kappa B (NF-κB). TGF-β, LPS and NF-κB stimulation leads to decreased miR-29 levels which is associated with increased collagen production leading to fibrosis [22].

Circulating miR-196 is significantly reduced in CHC patients, via decreased release of miR-196 from HCV-infected hepatocytes [21]. Following activation by infection with the hepatitis C virus (HCV), the interferon (IFN) pathway activates miR196 expression. This miRNA either directly inhibits viral replication via a target site on the HCV RNA or indirectly via Bach1 down-regulation and up-regulation of heme oxygenase-1 (HMOX1) [23]. As a result, HCV has evolved to counteract the effect of this antiviral miRNA, however such mechanism remains to be fully investigated. Regulation of miR-196b expression through the (IFN) pathway and inhibition of this pathway by HCV virus particles may explain the lower expression of miR-196 in the cell line after virus transfection and also in the PBMCs of HCV-infected persons [10].

In 2016, the World Health Organization (WHO) updated its guidelines for the screening, care, and treatment of HCV-infected patients to recommend DAA-based regimens instead of IFN-based regimens [24]. Since the publication of the 2016 guidelines, DAA regimens successfully resolve HCV infection in over 85% of treated persons across all six major genotypes. Despite this very high response rate, complete elimination of HCV infection is still not achieved and about 1–15% of HCV-infected patients are still resistant to treatment [25].

In this study, out of 150 patients 141 (94%) have accomplished SVR while 9/150 patients (6%) were non-responders to DAAs treatment. Since treatment is expensive and often has several adverse effects, viral and host factors which impact on the severity of disease and response to treatment becomes more significant [26]. Host miRNAs expressions have been reported to vary with different treatment responses. As a result, miRNAs can be used as biomarkers for predicting response prior therapy, therefore avoiding adverse effects of ineffective treatments, and reducing unnecessary cost. Furthermore, they tend to be new targets for the development of antiviral treatment [27].

The present study reported that the expression levels of miR-122 and miR-155 were significantly higher in non-responders to DAAs than in responders. In contrast, the expression levels of miR-29 and miR-196 were significantly higher in responders to DAAs than in non-responders. ROC analysis showed that all the studied miRNAs could serve as valuable biomarkers for prediction of response to DAAs with AUC 0.973 for miR-122 (95% CI 0.933–0.993, p < 0.0001), 0.878 for miR-155 (95% CI 0.815–0.926, p = 0.0003), 0.808 for miR-29 (95% CI 0.736–0.868, p < 0.0001) and 0.874 for miR-196 (95% CI 0.810–0.922, p < 0.0001) respectively. The optimal sensitivity and specificity to differentiate SVR from NR were (85.8 and 100% at a cut-off value ≤ 689.8) for miR-122, (94.3 and 88.9% at a cut-off value ≤ 71.3) for miR-155, (78 and 77.8% at a cut-off expression value > 0.067) for miR-29, (76.6 and 88.9% at a cut-off value > 0.116) for miR-196, respectively. These results suggest that all studied miRNAs could be potential predictive biomarkers differentiating SVR and NR in CHC patients receiving DAAs.

Univariate logistic regression analysis revealed that miR-196 level is positive predictor for SVR, while miR-122,155 levels are negative predictors of response. Multivariate logistic regression analysis revealed that miR-196 is the most important marker in predicting response to treatment (p value = 0.011).

Meissner et al., as well investigated differential expression of our four-studied miRNAs before and after SOF/RBV therapy, they reported an increase in expression of miR-122 during treatment-induced HCV clearance (1.4-fold increase, p = 0.034), with no change of the other three miRNAs over the course of therapy [28].

Hyrina et al., reported also an increase of miR-122 expression in the serum of NR compared to patients achieving SVR after Interferon-based therapy with first-generation direct-acting antivirals, theses data is consistent with our findings [29]. Although several studies have investigated the role of our four-studied miRNAs as predictors of response to IFN-based regimens [30]. There aren’t many studies evaluating their predictive value with DAA-based regimens, according to Meissner and colleagues [28].

To the best of our knowledge, despite the relatively small sample size of the current study, it provided the first clinical evidence of the use of circulating miRNAs (miR; 122, 155, 196, and 29) as diagnostic and predictive biomarkers of CHC in HCV- genotype 4 patients receiving the new DAA regimen (SOF/DAV + RIB). However, more research with a bigger sample size is necessary to thoroughly assess the potential worth of these miRNAs as a useful biomarker.

Circulating miRNAs have become attractive biomarker candidates and are increasingly used in the prevention, diagnosis, prognosis, therapeutic monitoring and even treatment of various human diseases [31].

Bivariate correlation analysis was assessed in the current study between miRNAs expressions and different biochemical Parameters using spearman’s correlation test, A significant moderate positive correlation was observed between mirRNAs (miR-122, 155) and liver transaminases levels; [(miR-122): ALT (rho = 0.497); AST (rho = 0.606)], [(miR-155): ALT (rho = 0.532); AST (rho = 0.632)].A significant moderate and weak negative correlation was reported between mirRNAs (miR-29, 196) and liver transaminases levels respectively;[(miR-29): ALT (rho = − 0.529); AST (rho = − 0.520)], [(miR-196): ALT (rho = − 0.410); AST (rho = − 0.407)]. In contrast, bivariate correlation analysis revealed a significant negative weak correlation between mirRNAs 122, 155 and serum albumin level (rho = − 0.234, − 0.323) respectively and a significant positive weak correlation between mirRNAs 29, 196 and serum albumin level (rho = 0.221, 0.150) respectively. The previous observations suggest presence of correlation between the four studied miRNAs and liver function.

Our findings are consistent with some previous studies which reported a positive correlation between miR-122 and liver transaminases levels [19, 32]. Although our data are on contrary with LIU and coworkers’ results which observed no correlation between miR-196 and liver transaminases [21].

We also investigated whether there is any correlation between levels of our four-studied miRNAs with baseline HCV RNA levels; Bivariate correlation analysis revealed positive significant strong correlation between mirRNAs 122, 155 and HCV RNA levels (r = 0.856, p < 0.001), (r = 0.798, p < 0.001) respectively. A negative significant strong and moderate correlation between mirRNAs 29, 196 and HCV RNA levels was observed (r = − 0.808, p < 0.001), (r = − 0.725, p < 0.001) respectively.

Our findings agree with a previous study that reported positive correlation between miR-122 and HCV RNA levels [32]. Our results are also in consistent with a previous report showing that overexpression of miR-196b decreases HCV replication by approximately 60% [32]. In contrast to our findings, Motawi et al. [27], reported a negative correlation between miR-122 and HCV RNA levels (r = − 0.278. p = 0.023). Sendi et al. [33]. also reported that miR-122 expression level is negatively correlated with the baseline HCV RNA levels (r =  − 0.43, p = 0.03), while they found no correlation between miR- 29 and HCV RNA levels. Our data also disagree with LIU et al. [21], who observed no correlation between miR-196 and baseline HCV RNA levels.

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