Nonalcoholic fatty liver disease (NAFLD) is defined as the accumulation of fat in hepatocytes that is unrelated to severe alcohol consumption (1). Simple steatosis can progress to steatohepatitis and the appearance of liver fibrosis and hepatocellular carcinoma (HCC) (2). Therefore, patients with NAFLD have a higher risk of mortality from hepatic causes, as well as cardiovascular events (3, 4). It is estimated that the global prevalence is 25% in the adult population (5), and the global incidence of NAFLD ranges from 28 to 52 cases per 1,000 people/year (6), making it presumable that a large proportion of cardiovascular events, HCC, and deaths from liver disease will be related to NAFLD in the coming years. The most important factor for the development of NAFLD is obesity (7); however, there are other factors that have been shown to be associated with this pathology (8) and can serve as biomarkers.

The prevalence of NAFLD in people living with HIV (PLWH) is more frequent than in the general population, being approximately 34% according to a recent meta-analysis (9). In addition, PLWH with NAFLD have a higher prevalence of steatohepatitis development, as well as greater liver function impairment compared to HIV-uninfected NAFLD patients (10). In addition to factors inherent to HIV infection itself, numerous recent studies have reported weight gain in PLWH who switched from older antiretroviral therapy (ART) to integrase strand transfer inhibitor-based regimens (11, 12). For all these reasons, studies are needed to address the potential risk factors for the development and identification of biomarkers that could serve as diagnostic approaches for NALFD in this special population.

The gold-standard method for the diagnosis of NAFLD is the histological study of liver biopsy because it allows distinguishing between simple steatosis and steatohepatitis (13). However, due to its invasive nature, liver biopsy is not a useful tool for epidemiological screening or as a follow-up method (14). The controlled attenuation parameter (CAP), using Fibroscan^® (Echosens; Paris, France), was introduced as a new technique to detect fatty liver disease. This method is a non-invasive technique, has low inter- and intraobserver variability (15), and provides a numerical value that is highly correlated with the histological grade of NAFLD (16). However, the measurement by CAP has some limitations, since diagnostic performance decreases in the most severe cases of NAFLD and the correlation of CAP and liver biopsies in obese patients (Body mass index; BMI > 30 kg/m²) is lower (16). This highlights the need to identify new alternatives for the diagnosis of NAFLD. In recent years, new biomarkers with diagnostic potential for NAFLD have emerged, such as microRNAs (miRNAs), since they participate in the regulation of lipid metabolism, the development of obesity, the regulation of glucose homeostasis, and the pathogenesis of steatohepatitis (17). Different studies have found a relationship between the appearance of NAFLD and alterations in the expression levels of different miRNAs (18). Therefore, circulating miRNAs could be highly relevant biomarkers for the diagnosis and prognosis of NAFLD (17). Thus, miR-122, miR-34a and miR-192 have been identified as potential markers for NAFLD. In the PLWH population, only miR-200a has been suggested as a NAFLD biomarker, showing possible applicability to predict the progression of NAFLD and liver damage in this population (19). Nevertheless, wide evaluation of the whole spectrum of microRNAs as potential biomarkers in PLWH is lacking. Therefore, the aim of our study was to identify miRNA expression profiles associated with the presence of severe NAFLD in the PLWH population.

Our study does not show differences in the expression pattern of serum miRNAs in PLWH patients who could be associated with the presence of NAFLD. Analysis of the normalized data did not reveal any pattern of differential expression of miRNAs between patients who had severe NAFLD and those who did not. Therefore, our results suggest that serum miRNAs are not useful as biomarkers for the diagnosis of severe NAFLD in PLWH patients.

Although most of the studies that analyze the expression profiles of miRNAs in NAFLD have been carried out in the general population, the results of this study are contrary to what has been reported regarding some miRNAs linked to NAFLD (18, 25–27). However, these studies have great methodological heterogeneity among themselves. One of the most important methodological differences lies in the normalization of the data. In some of these studies, this normalization is inadequate or not even carried out; therefore, it can lead to biased conclusions. In this sense, data normalization is crucial in studies where miRNA expression profiles are analyzed (28, 29). However, to date, there is no consensus for the process of standardization of the results, and this fact could significantly hinder the identification of miRNAs that may be useful as biomarkers and reproduced between different studies. In addition, other factors may influence the heterogeneity of results, such as sample processing, RNA extraction, and the technology used to determine the different miRNA expression profiles.

Due to the previously mentioned methodological heterogeneity of the studies, the meta-analysis carried out by Liu and colleagues is a study that includes all the studies that have analyzed miRNAs in the general population with the aim of identifying the potential usefulness of miRNAs as NAFLD biomarkers (18). Papers were included based on the diagnostic accuracy of NAFLD. Thus, the meta-analysis only included studies that reported data regarding the ability of certain miRNAs to diagnose NAFLD, as well as steatohepatitis. The manuscript highlighted miR-122 and miR-34a as the miRNAs with the greatest diagnostic capacity for NAFLD and steatohepatitis, with areas under the curve of 0.82 and 0.78, respectively. However, one of the main limitations of this meta-analysis is the fact that it includes studies whose analyses have not been adjusted for BMI, with being overweight possibly the greatest risk factor for the development of NAFLD (30, 31). An example of this limitation can be seen in the studies that highlight these two miRNAs as potential biomarkers for the diagnosis of NAFLD (26, 27, 32, 33). In these studies, differences in the expression profiles of miR-122 and miR-34a were observed between patients with NAFLD and healthy controls. The main limitation of these studies is that BMI was significantly higher in the NAFLD group. Therefore, the design of these studies does not allow us to conclude that the differential pattern of these miRNAs is associated with NAFLD, since they are comparing study subjects whose dietary habits could be significantly different. Along the same lines, Zarrinpar and colleagues found 6 pairs of discordant twins for NAFLD. The expression of these miRNAs failed to explain this mismatch (34). Therefore, the idea that environmental factors are the main factors involved in modifying the expression of these molecules is quite plausible. This fact has been reported in a review carried out by Ji and colleagues, in which they highlight that the expression level of these molecules is altered in obese and overweight patients (35). Specifically, in the case of miR-122, it has been described how in healthy individuals the liver produces this molecule; however, in conditions of obesity, adipose tissue increases the production and secretion of circulating miR-122 as a mechanism for maintaining hepatic function (35, 36). In the context of HIV infection, our study is contrary to the study carried out by Austermann and colleagues, which is one of the few studies that has analyzed the association of miRNAs with NAFLD in HIV infection (19). This study with a longitudinal design analyzed the role of miR-200a as a possible predictor of NAFLD, as well as a tool for its progression. However, as in the case of studies carried out in the general population, it has some limitations. One of them is the use of inadequate standardization of the results, which the author warns as a limitation of the study. The inclusion of patients with obesity (BMI greater than 30 kg/m²) denotes another limitation of the study, since the determination of CAP in these patients may be biased, as the technique is not indicated for these patients (16). Finally, the increased expression of miR-200a is closely linked to being overweight (p<0.01), so it cannot be concluded that this molecule is a useful biomarker to identify patients with NAFLD.

For all these reasons, since the design of this study has allowed the analysis of both study groups with a similar BMI, the results obtained confirm that the alteration of miRNAs reported in various studies in the general population could be more related to other factors such as obesity and diabetes than to the presence of NAFLD. Thus, diet and underlying weight factors would be more important risk factors than epigenetic mechanisms, such as miRNAs, in the development of NAFLD. However, there must be underlying risk factors for HIV infection that must be evaluated to elucidate the high prevalence of NAFLD in this special population compared to the general population. On the other hand, since the study was carried out in PLWH patients, we cannot completely rule out that this absence of differential patterns of miRNAs occurs in the same way in the general population. In fact, the results of this study suggest the existence of a hidden underlying classification for these patients who will be evaluated in future studies, given that the analysis showed a highly homologous profile among 7 patients (5 cases and 2 controls) whose classification was not associated with the degree of NALFD ( Figure 2 ).

Our study has three limitations. First, patients were diagnosed with NAFLD through CAP and not using the gold standard technique, which is the histological study of liver biopsy. Second, although patients with an alcohol consumption greater than 20 grams/day were not included, this was collected on a self-reported basis. Last, our study did not include a healthy volunteer group. This aspect is of great interest to assess the possible influence of HIV infection itself on the difference in expression of miRNAs and its relationship with severe NAFLD.

This study shows that the miRNA transcriptome profile of nonobese patients with severe NAFLD do not differ from the profile of PLWH without NAFLD. Therefore, miRNAs are not ideal markers for the diagnosis of severe NALFD. Further studies are needed to determine other -omic biomarkers that could be linked to the development of severe NAFLD in this special population.

The data presented in the study are deposited in public repository (ArrayExpress collection; Biostudies), with code E-MTAB-12974.

The studies involving humans were approved by Clinical Trials and Ethics Committee of Hospital Universitario Ntra. Sra. de Valme. The study was approved with code PI18/00606. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

Conceptualization: FM, R-JA and RA; Data curation: FM, R-JA, MM and RA; Formal Analysis: FM, R-JA, MM and RA; Funding acquisition: RA; Investigation: FM, C-GJ, L-LP, ACG, DC-M, R-CI, C-JM, P-VI and RA; Methodology: FM, R-JA, MM, C-GJ, P-VI and AR; Supervision: R-JA and RA; Validation: FM, R-JA and RA; Visualization: FM, R-JA and RA; Writing-original draft: FM, R-JA and RA; Writing-review & edition: All authors. All authors contributed to the article and approved the submitted version.