Al-Rawaf et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-024-00514-8(2024) 25:18

BMC Molecular and Cell
BiologyOpen Access

RESEARCH

Circulating microRNAs as potential biomarkersof physical activity in geriatric patientswith HCV
Hadeel A. Al-Rawaf1, Sami A. Gabr2, Amir Iqbal2* and Ahmad H. Alghadir2
Abstract
Background Circulating microRNAs have been implicated in a diverse array of biological and pathologicalphenomena. Their potential utility as noninvasive biomarkers for screening and diagnosing various diseases has been
proposed.
Objective This study aimed to explore the potential role of the miRNAs miR-122 and miR-486 as molecularbiomarkers in the pathogenesis of hepatitis C virus (HCV) infection. Thus, miR-122 and miR-486 were detected in theserum of HCV patients and healthy controls. Moreover, the potential correlations of miR-122 and miR-486 with viral
complications, such as physical activity, pain, muscle fatigue, and HCV infection, were identified.
Methods A total of 150 subjects aged 30 to 66 years were included in this study. The patients were classified as
patients with chronic hepatitis C virus (CHC) (n = 110) or healthy controls (n = 40). Real-time polymerase chain reaction(PCR) analyses were performed to determine miR-122 and miR-486 expression. Physical activity (PA), pain score, HCV
genotyping, viral overload, aspartate transaminase (AST), alanine transaminase (ALT), lactic acid dehydrogenase(LDH), creatine kinase (CK), and antioxidant status were also estimated by using prevalidated questionnaires, PCR, andspectrophotometric analyses.
Results Compared with those in normal controls, significant increases in the serum levels of miR-122 and miR-486
were reported in patients with CHC. In physically active CHC patients, there was a significant correlation between
the expression of miRNAs and increased alanine transaminase (ALT), aspartate transaminase (AST), fibrosis scores,
and inflammation activity, but no association was reported for hepatitis C virus (HCV) RNA or viral load. Additionally,significant decreases in LDH, CK, GSSG, and pain scores and increases in TAC, GSH, and the GSH/GSSG ratio werereported. Moreover, the expression of miR-122 and miR-486 was positively correlated with changes in body massindex (BMI) and liver fibrosis stage, as well as negatively correlated with sex, PA, TAC, GSH, GSSG, and the GSH/GSSG
ratio.
Conclusion MiR-122 and miR-486 expression levels were strongly correlated with physical activity, pain perception,and muscle fatigue biomarkers in HCV-infected patients. These miRNA levels were associated with elevated AST, ALT,
fibrosis scores, LDH, CK, and antioxidant status, thus suggesting their potential as biomarkers for disease severity and*Correspondence:Amir Iqbal
physioamir@gmail.com; ajamaluddin@ksu.edu.sa
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Al-Rawaf et al. BMC Molecular and Cell Biology(2024) 25:18

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oxidative stress. However, no correlation was observed with viral load or HCV-RNA expression, thus implying thatthese miRNAs may impact disease progression and symptoms through host factors, rather than directly affecting
viral replication. In summary, the results demonstrated that molecular studies of miR-22 and miR-468 and theirassociations with PA, pain, adiposity, sex differences, and muscle fatigue, as well as routine biomarkers, could be usefulas prognostic nanoninvasive biomarkers, thus providing novel therapeutic targets for CHC infection.
Keywords MicroRNAs, Pain, HCV, Physical activity, Antioxidant status, Muscle capacity

Introduction
Chronic liver conditions, including liver cirrhosis,
hepatic failure, and hepatocellular carcinoma, have been
linked to chronic hepatitis C virus (HCV) infections in an
estimated 170 million individuals worldwide [1, 2]. The
progression of infection subsequently leads to severe livercomplications, which can necessitate transplantation in
many patients [3]. Thus, eradication of HCV infection
significantly reduces liver-related morbidity and mortality [4–6].
HCV infection has its most pronounced impact onpatients’ social and physical functioning, overall wellbeing, and vitality [7]. Current medical advice recommends the management and correction of health-related
behaviors, including nutritional or physical activity,
during viral infection to prevent further liver diseaseprogression. Importantly, this refers to the influence oflifestyle on disease development [8–10].
Patients who are afflicted with HCV infection havereported experiencing diminished self-assurance anda decrease in their physical capabilities [11, 12]. Thisdecrease in quality of life, which is linked to chronic hepatitis, may be influenced by both comorbid conditions
and the patient’s awareness of their diagnosis rather thanbeing solely dependent on the severity of the viral infection, liver function, or the extent of fibrosis [7, 13].
The advantages of physical exercise are multifacetedand extend to mitigating risks in individuals with various
chronic conditions [14–17]. Indeed, physical exercise canplay a crucial role in the management of chronic diseases[18]. Earlier studies have suggested the incorporation ofphysical activity as an integral component in supporting
individuals who are experiencing chronic HCV infection,with an aim of bolstering their self-assurance and overall capabilities [19, 20]. In physically active HCV patients,
their bodies manufacture ATP more often, thus indirectly
boosting the body’s glutathione levels.
This scenario correspondingly improves the resilienceof the liver against free radicals and hepatitis C damage[21]. Using the aforementioned data, we can determinethat there is no direct correlation between viral infection,disease progression, or physiological changes; moreover,
only a correlation with antioxidant activity has beenreported in patients with HCV [7, 13, 18, 21].
Thus, in our hypothesis, we suggest that there is a relationship between physical activity and HCV infection,

which may be clarified by investigating other biomarkers, such as microRNAs (miRNAs). MicroRNAs, which
are short noncoding RNAs consisting of approximately
22 nucleotides, are involved in posttranscriptional gene
expression regulation. They have been demonstrated toparticipate in a broad spectrum of biological and pathological processes [22–24].
Recently, there has been growing interest in assessing
the potential of miRNAs as being noninvasive biomarkers for the screening and diagnosis of various diseases.
The ability of circulating miRNAs to serve this purpose
may be attributed to their presence in exosomes, which
provide stability [25–28]. In the human genome, over
2,500 miRNAs have been identified, and they are likely
involved in a wide range of cellular processes, including (but not limited to) development, differentiation,
proliferation, apoptosis, disease pathology, and antiviral
defense [29, 30].
In the context of HCV infection, several miRNAs are
significantly upregulated in different HCV genotypes,
with notable examples being serum miR-134, miR-198,
miR-320c, and miR-483-5p, which show promise as being
diagnostic biomarkers for HCV infection [31]. Among
all human miRNAs, microRNA 122 (miR-122) is one of
the most highly expressed and abundant miRNAs, constituting more than 70% of the total miRNA content in
the human liver. Consequently, it plays a pivotal rolein the regulation of hepatic functions [32]. Moreover,research has indicated that miR-122 is indispensable for
HCV replication and may serve as a target for anti-HCV
therapy [33]. Additionally, there is a body of evidence
linking physical activity to skeletal muscle development[34–36], and miRNAs are believed to act as physiological mediators of the adaptations induced by exercise [37].
Thus, miRNAs represent interesting factors in the studyof host-HCV interactions, ranging from HCV infection
and related consequences such as low physical activity,
fatigue, and depression to potential novel targets for antiviral therapy [38, 39].
Additionally, several studies have shown that themolecular signatures of cellular miRNA expression may
significantly control the classification, diagnosis, andprognosis of cancer [40, 41]. However, the diagnostic andprognostic accuracy of the miRNA genetic fingerprint
for more than 16,000 mRNAs were significantly different in different cancers, including the incidence rates of

Al-Rawaf et al. BMC Molecular and Cell Biology(2024) 25:18

hepatocellular carcinoma among patients at high risk ofhepatitis C virus infection [41, 42]. The biological functions and targets of several miRNAs, such as miR-618
and miR-650, have been reported as being noninvasive diagnostic markers [42] that can be identified inurine samples and are valuable approaches for developing screening methods that can reduce mortality rates
among HCV-infected patients [43–47].
In addition, several studies have shown that cellular
miR-486 is a multifunctional miRNA involved in many
cancers, including the tumorigenesis of hepatocellular
carcinoma, with additional functional roles as a modulator for regulating drug resistance, such as sorafenib
resistance, by targeting FGFR4 and EGFR, thus offering
a potential target for HCC treatment [44–47]. Although
the potential roles of both miR-22 and miR-486 as prognostic markers in the diagnosis, treatment, and prevention of drug resistance have been addressed in manystudies [40–47], little is known about their association
with physical activity, pain, and muscle fatigue as complications in patients with HCV infection. Thus, this study
aimed to elucidate the potential role of miR-122 andmiR-486 in relation to physical activity, pain, and muscle
fatigue biomarkers in patients with HCV infection or its
Table 1 Clinical and demographics characteristics of control andgeriatric CHC patientsParameters

CHC patients(n = 110)Age (years) #Sex (male/female) #BMI (kg/m2) #AST (IU/mL) # #ALT (IU/mL) # #Albumin (gm/dL) #Bilirubin (mg/dL) #AFP (ng/mL)
Platelets (109/L) #Glucose (mg/dL)
HOMA-IR
Duration of HCV (years)
HCV RNA (IU/mL)
HCV genotypes
Viral load
Length of liver-biopsy core
Portal tracts
ASC/fibrosis/cirrhosis
Necroinflammation; n (%)
A0–A1
A2–A3

46.5 ± 5.8
75/35
24.3 ± 4.6*
69.4 ± 9.5**
115.4 ± 21.5**
2.8 ± 0.89**
3.9 ± 0.96**
38.1 ± 3.6
175 ± 25.4**
91.7 ± 2.5*
4.25 ± 1.6*
11.5 ± 3.1
9.5 × 105 ±2.25 × 10 5G4441.8 ± 3.5
22.5 ± 0.75 cm
16 ± 3.8
48/35/27
45 (40.9%)
65 (59.1%)

Healthy
controls(n = 40)
46.9 ± 5.1
25/15
22.8 ± 6.3
18.3 ± 3.5
23.4 ± 5.8
4.3 ± 0.75
0.81 ± 0.65
2.8 ± 3.1
254 ± 34.7
82.5 ± 3.9
1.7 ± 0.53-

Notes: All values represent mean ± standard deviation or n (%). These
parameters are related to viral estimation and hepatic pathology; #Student’s
t-test; ##Mann–Whitney U-test; *P < 0.05; **P < 0.01. Abbreviations: CHC, chronic
hepatitis C; BMI, body mass index; HCV, hepatitis C virus; HOMA, homeostatic
model assessment; IR, insulin resistance; ASC, asymptomatic CHC; AST,
Aspartate aminotransferase; ALT, Alanine aminotransferase

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complications, as well as to provide valuable insights into
the differences between patients with HCV infection andhealthy individuals.

Materials and methods
Study design

This study employed a case‒control observational design
to determine the relationships between miRNA-122
and miR-486 expression and physical activity, pain, andmuscle fatigue biomarker levels in patients with HCV
infection or its complications, as well as to compare
these findings with those observed in the healthy control
group.
Ethical considerations

The study protocol underwent review and received
approval from the ethics subcommittee at King Saud
University, Riyadh, Saudi Arabia, with the assigned filenumber ID: RRC-2016-045. The research adhered to ethical principles outlined by the local institutional review
board and was conducted in accordance with the Declaration of Helsinki (2010). Prior to the commencementof the study, all of the participants were provided withcomprehensive information about the research, and eachparticipant provided their informed consent through awritten and signed document.
Subjects

A total of 150 older adults, ranging in age from 30 to 66
years, met the eligibility criteria for participation in thisstudy. The study included a control group consisting of
40 healthy HCV-negative individuals who exhibited normal levels of physical activity (with a PA score ≥ 2,500
MET minutes/week). Additionally, a total of 110 patientswith chronic HCV infection, as well as with a confirmed
disease duration of more than 10 years, viremia, HCV
antibodies as indicated by the RIBA-II test, elevatedserum transaminase levels surpassing the upper normal
limit, HCV-RNA positivity, and genotype determinations, were included in the current study. The exclusion
criteria included individuals with viral coinfections (HIVand/or HBV), advanced stages of other liver diseases(such as cirrhosis), hepatocellular carcinoma, prior livertransplantation, obesity (BMI ≥ 25 and ≥ 30 kg/m2), ironoverload, or insufficient liver biopsy results. Heparinizedsyringes were utilized to collect blood samples from allof the subjects either on the day of the biopsy or within
5 days following the biopsy procedure. Subsequently,
serum samples were extracted through centrifugation,
which lasted for 1 min at 1,400 rpm. These samples wereassigned coded study identification numbers and werethen promptly frozen at -20 °C for subsequent analysis.
A comprehensive summary of the participants’ demographic and clinical information can be found in Table 1.

Al-Rawaf et al. BMC Molecular and Cell Biology(2024) 25:18

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The levels of liver enzymes, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin(ALB), and total bilirubin (TB), were measured in thesera of both healthy controls and HCV-infected patients.
In addition, complete blood platelet count, glucose, and
HOMA-IR were also estimated according to the manufacturer’s instructions. Serum AFP levels were measured via sandwich enzyme-linked immunosorbent assay(ELISA; R&D Systems, Minneapolis, MN, USA).

reviewed and graded blindly by a senior pathologist. The
mean length of liver biopsies and the number of portal
tracts were assessed (including only the complete, intact
portal tracts) [54–56].
The degree of fibrosis was scored according to theMetavir system, and no fibrosis was defined as F0, mild
fibrosis as F1, moderate fibrosis as F2, severe fibrosis
as F3, and cirrhosis as F4. Significant fibrosis was also
defined as F2–F4. Hepatic inflammatory activity was alsoscored [54, 55].

Assessment of HCV infection

Isolation of miRNAs and RT–PCR

As a measure of diagnosis, HCV-RNA was also estimatedqualitatively by using a nested PCR Qiagen RNA-extraction kit (Thermo Fisher Scientific, Waltham, MA, USA)and quantitatively by using Smart Cycler II real-time PCR(Cepheid, Sunnyvale, CA, USA) with HCV RNA quantification kits (Sacace Biotechnologies, Como, Italy) for
estimation of HCV RNA-positive subjects, as previously
described [48, 49]. Moreover, subjects with an HCV viralload lower than 250 IU/ml were considered negative. In
addition, reverse hybridization was achieved to estimate
the HCV genotype by using a line-probe assay (InnoLiPA HCV II kit; Innogenetics, Ghent, Belgium) [50].
In this regard, patients with alanine aminotransferase (ALT) levels higher than the upper limit of normal,
elevated titers of anti-HCV antibodies estimated with athird-generation enzyme immunoassay (AxSym HCV
3.0; Abbott Laboratories, Abbott Park, IL, USA), andconfirmed levels of HCV-RNA by PCR analysis werediagnosed with chronic hepatitis C (CHC).

Total RNA from the serum samples of each subject was
extracted by using TRIzol LS reagent (Invitrogen, Carlsbad, CA) and subsequently subjected to reverse transcriptase PCR (RT‒PCR) analysis. A ready-made solution
containing primers and probes for human miR-122 andmiR-486 (Applied Biosystems, Foster City, CA) andreal-time RT–PCR was generated by using an ABI 7300
system (Applied Biosystems) [46]. After the addition ofphenol solution to inhibit RNase activity, chemically synthesized cel-miR-39 (Sigma‒Aldrich Japan, Tokyo, Japan)
was added as an exogenous and endogenous control,and miR-16, which is a representative miRNA enrichedin blood, was added as an endogenous control. For each
serum sample, the ratio of the miRNA signal to that ofendogenous (miR-16) and exogenous (cel-miR-39) controls was calculated as previously reported [57].

Laboratory assessment

Histologic examination

The degree of liver inflammation in CHC patients wasassessed through the use of the Batts and Ludwig scoring system [51]. After informed consent documents had
been submitted, hepatic biopsies were obtained from allof the patients by a surgeon after computed tomography
or magnetic resonance imaging. The histological diagnosis of cirrhosis and HCC was based on an elevated serum
AFP level (≥ 400 ng/mL) and computed tomography ormagnetic resonance imaging scans, as recommended byinternational criteria. Thus, a preoperative clinical diagnosis of primary liver cancer and hepatocellular carcinoma (HCC) was made as described previously [52, 53].
For histological examination, liver biopsies wereobtained by using an automatic 16-gauge Trucut needle(biopsy gun), which provides adequate specimens forevaluation and fewer cases with tissue fragmentation. The
analyzed liver biopsy specimens were at least 15–25 mmlong with complete portal tracts (> 10). Formalin-fixed,
paraffin-embedded sections were stained with hematoxylin and eosin and Masson’s trichrome. Slides were labeledwith patient identification numbers and subsequently

Serum biochemical analyses

A standard commercially available enzymatic assay(Granutest 15, EMD Millipore, Billerica, MA, USA) wasused to calculate the serum creatine kinase (CK) concentration. Lactate dehydrogenase (LDH) activity wasestimated by using an ultraviolet method provided byRandox Laboratories Ltd. (Antrim, UK). Serum alaninetransaminase (ALT), aspartate transaminase (AST), albumin, bilirubin, blood platelet count, and fasting blood
glucose were estimated in serum and blood samples byusing standard methods. The serum total antioxidant
capacity (TAC) was calculated by using a colorimetric assay kit (K274-100; BioVision, Milpitas, CA, USA).
The data were measured and calculated as previously
reported [58, 59]. The serum samples were utilized forspectrophotometric assays of glutathione (GSH) and glutathione disulfide (GSSG) levels, which was conductedby employing an enzyme-linked immunosorbent assay(ELISA) reader (Tecan GENios, A-5082, Tecan, Salzburg,
Austria) and a glutathione assay kit (Cayman Chemical
Company, Ann Arbor, MI, USA). The concentrations of
GSH and GSSG were determined at 405 nm based on thecolor development of the TNB (5-thio-2-nitrobenzoicacid) product resulting from the reaction between GSH
and DTNB (5,5’-dithio-bis-2-nitrobenzoic acid) [60].

Al-Rawaf et al. BMC Molecular and Cell Biology(2024) 25:18

Assessment of physical activity (PA)A prevalidated global PA questionnaire (GPAQ) was used
to calculate the PA scores of all participants regarding theintensity of exercise performed in minutes or hours per
day for each participant, as previously reported [61, 62].
In relation to the intensity and frequency of PA achieved
per week, energy expenditure was measured in the formof the metabolic equivalent (MET) of all of the participants. Thus, according to energy expenditure, PA scores
were classified into three scores: physically inactive (MET
minutes/week of ≤ 500), moderate PA (MET minutes/
week of 500–2,500), and physically active (≥ 2,500 MET
minutes/week) [61, 62]. Using the PA scores as a criterion, HCV patients were divided into two categories:
those who were physically active (≥ 2,500 MET minutes/
week, n = 85) and those who were PA inactive (MET minutes/week of ≤ 500, n = 25).
Assessment of VAS score

A reliable standard 100 mm visual analog scale (VAS),
with 0 mm indicating “no pain” and 100 mm indicating“most severe pain”, was used to measure the pain intensity of all of the participants, as previously reported [63,64].
Statistical analyses

The Shapiro–Wilk test was used to assess the normalityof the distribution of the data, and logarithmic transformation was applied for subsequent statistical analyses.
To evaluate differences between subject groups, bothStudent’s t test and ANOVA analyses were utilized, followed by Bonferroni’s multiple comparison analysis.
To compare microRNA levels among different groups,
adjustments were made using univariate analysis througha general linear model. Multiple stepwise regression andPearson’s correlation analysis were employed to determine the relationships between microRNA levels, TAC,
LDH, CK, GSH/GSSG, HCV, and metabolic parameters.

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Results
A study involving 150 participants was conducted, withthe majority being male (66.66%). The clinical and demographic characteristics of both the control group and the
CHC patients are presented in Table 1. CHC patients
exhibited significantly greater values (P = 0.001) in termsof BMI, AST, ALT, platelet count, and glucose status than
did control subjects.
In this study, LDH, CK, TAC, GSH, GSSG, and the
GSH/GSSG ratio were measured as predictors of muscle
function capacity and physical activity in controls andpatients with CHC (Table 2). In physically active CHC
patients, LDH, CK, and GSSG were significantly lower,
whereas TAC, GSH, and the GSH/GSSG ratio were significantly greater than those in CHC patients with lower
PA scores (P = 0.001) or in the control group (P = 0.01).
Additionally, pain was significantly greater in patientswith CHC who had lower PA scores than in both controls(p = 0.01) and PA patients with CHC (P = 0.001).
To explore the expression levels of miRNAs and theirpotential associations with physical activity patterns in
CHC patients, we examined whether miR-122 and miR486 were detectable and modulated in the serum of CHC
patients. The serum concentrations of miR-122 andmiR-486 were notably greater in all of the CHC patients(n = 110) than in the HCs (n = 40) (miR-122: 11.5-fold vs.
2.3-fold, respectively; P = 0.0001; miR-486: 42.6-fold vs.
21.9-fold, respectively; P = 0.0001) (Fig. 1A-B).
Among CHC patients who engaged in regular physical activity, the serum expression levels of both miR-122
and miR-486 were significantly lower than those in CHC
patients with lower physical activity levels (miR-122: 5.2fold vs. 9.5-fold, respectively; p = 0.0001; miR-486: 18.5fold vs. 31.9-fold, respectively; p = 0.0001) (Fig. 1A-B).
The correlations of miR-122 and miR-486 withdemographic, physical activity, muscle capacity, antioxidant, and viral parameters were further studied byusing stepwise regression and Pearson’s correlation
analyses (Table 3). Both miR-122 and miR-486 wereTable 2 Change in muscle and antioxidant biomarkers, and pain scores (VAS) of control and geriatric CHC patients based up on PA(Mean ± SD).
Parameters

LDH(IU/L)CK (UI/L)TAC (nmol/µL)
GSH (µmol/L)
GSSG (µmol/L)
GSH/GSSG ratio(×10 − 2)
VAS score

PA control (n = 40)

18.9 ± 3.1
168.6 ± 8.4
45.8 ± 12.3
1320 ± 412
86.5 ± 22.8
26.3 ± 11.4
12.3 ± 2.5

CHC patients (n = 110)
PA-Active, n = 85(≥ 2,500 MET min/week)
29.5 ± 6.4a
156.7 ± 5.9 a
36.4 ± 6.7 a
815 ± 251 a
135.7 ± 51.2 a
9.5 ± 4.5 a
16.7 ± 4.7 a

PA-Inactive, n = 25(≤ 500 MET min/week)
48.6 ± 9.1 a, b
210.2 ± 5.3 a, b
18.1 ± 3.8 a, b
521 ± 125 a, b
215 ± 65.9 a, b
4.6 ± 2.6 a, b
48.2 ± 12.4 a, b

Notes: Values are expressed as mean ± SD; aP<0.01 (CHC vs. control), bP<0.001 (PA-inactive vs. PA active). Significance at P < 0.05. Abbreviations: CK, creatine kinase;
LDH, lactate dehydrogenase; MET, metabolic equivalent; PA, physical activity; SD, standard deviation; TAC, total antioxidant capacity; VAS, visual analog scale; GSH,
reduced glutathione; GSSG, oxidized glutathione

Al-Rawaf et al. BMC Molecular and Cell Biology(2024) 25:18

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Fig. 1 Elevation in serum miR-122 and miR-486 expression in both physically active and non-active chronic hepatitis C. Serum levels of miR-122 (A) andmiR-486 (B) were compared between control subjects and CHC patients. The CT values of serum miRNA expression levels were transformed into absolutevalues using standard curves. The bars represent the levels corresponding to CT values ranging from 10 to 12 (2.9 × 104 − 0.75 × 104 copies/ml) for miR122 and 40 to 45 (1.75 × 104 − 0.68 × 104 copies/ml) for miR-486, respectively

positively correlated with glucose levels (P = 0.01) andBMI (p = 0.001) and negatively correlated with sex(p = 0.002).
Furthermore, we assessed the activities of ALT and
AST in CHC patients and observed a significant increase
compared to those in control subjects. Additionally,
we found that the relative expression of both miRNAs(miR-122 and miR-486) exhibited a moderately positive
correlation with the serum ALT and AST levels (miR122: r = 0.98, P = 0.001 and r = 0.78, P = 0.01, respectively;
miR-486: r = 0.89, P = 0.001 and r = 0.92, P = 0.01, respectively) (Table 3). However, no correlation was detected
between the expression levels of these miRNAs andeither HCV-RNA (r = 0.215; P = 0.21) or the viral load

Al-Rawaf et al. BMC Molecular and Cell Biology(2024) 25:18

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Table 3 Stepwise regression analyses and Pearson’s correlation of miR-122 and miR-486 with indices of HCV infectivity and muscle
functional capacity in CHC patients
Covariables

Pearson’s correlation

Age
Sex
BMI
HCV RNAHCV viral load
ALT (IU/L)
AST(IU/L)
Fibrosis score
Inflammation activity
Glucose (mg/dl)
LDH
CK
TAC
GSH
GSSG
GSH/GSSG ratio
PA-scores
VAS scoremiR-122r
0.032-0.156
0.325
0.215
0.298
0.98
0.78
0.112
0.58
0.35
0.258
0.35
-0.25
-0.45
-0.78
-0.34
-0.48
0.96

P-value
0.365
0.002
0.001
0.214
0.568
0.001
0.01
0.001
0.001
0.01
0.001
0.001
0.001
0.001
0.01
0.01
0.05
0.001

StepwisemiR-486r
0.124-0.163
0.325
0.152
0.325
0.89
0.92
0.311
0.61
0.39
0.368
0.39
-0.035-0.054-0.084-0.042-0.056
0.085

P-value
0.312
0.002
0.001
0.145
0.665
0.001
0.01
0.001
0.001
0.01
0.001
0.001
0.001
0.001
0.01
0.01
0.05
0.001

miR-122β
0.045-0.054
0.037
0.058
0.075
0.051
0.042
0.045
0.031
0.28
0.025
0.054
-0.043-0.036-0.075-0.051-0.0320.074

miR-486
P-value
0.524
0.317
0.112
0.614
0.234
0.01
0.05
0.001
0.02
0.01
0.001
0.01
0.001
0.001
0.01
0.01
0.01
0.001

β
0.031
-0.042
0.025
0.081
0.081
0.061
0.075
0.084
0.054
0.36
0.057
0.012
-0.058-0.065-0.095-0.074-0.071
0.068

P-value
0.325
0.128
0.613
0.215
0.128
0.01
0.05
0.001
0.02
0.02
0.001
0.01
0.001
0.001
0.01
0.01
0.01
0.001

r, Pearson’s correlation coefficient; β, standardised regression coefficient; ‘–’, indicates that the variable was not included in the multiple stepwise regression
modelling. Abbreviations: CK, creatine kinase; LDH, lactate dehydrogenase; MET, metabolic equivalent; PA, physical activity; SD, standard deviation; TAC, total
antioxidant capacity; VAS, visual analog scale; GSH, reduced glutathione; GSSG, oxidized glutathione

Fig. 2 Expression levels of miR-122 and miR-486 in serum of CHC patientswith early (F0-F1) and advanced (F3-F4) liver fibrosis. Serum levels of miR122 and miR-486 were elevated in patients with advanced liver fibrosis(miR-122, 10.8-fold; miR-486, 8.6-fold) compared to those with early liver
fibrosis scores (miR-122, 6.1-fold; miR-486, 5.7-fold)(r = 0.298, P = 0.56) in CHC patients. Notably, both miR122 and miR-486 exhibited positive correlations withliver fibrosis score (P = 0.001) and inflammatory activity(P = 0.001) in both physically active and nonactive CHC
patients.
Regarding physical activity and muscle capacity, theexpression of miRNAs in CHC patients was positively
correlated with LDH and CK and negatively correlatedwith PA score, TAC, GSH, GSSG, and the GSH/GSSG
ratio (Table 3). In addition, pain intensity was significantly correlated with the levels of both miR-122(P = 0.001) and miR-486 (P = 0.001).
Based on histological grading, the patients were categorized into two groups: those with an early stage offibrosis (F0-F1) and those with advanced fibrosis (F3-F4).
We found that the serum levels of miR-122 and miR486 were significantly greater (P = 0.001) in patients withadvanced liver fibrosis (miR-122, 10.8-fold; miR-486,
8.6-fold) than in those with early liver fibrosis (miR-122,
6.1-fold; miR-486, 5.7-fold) (Fig. 2). Notably, the increasein miR-122 expression was predominantly observed inpatients with F2-F4 disease, whereas the increase in miR486 expression was consistent with that in patients with
F0-F3 disease and further elevated in patients with F4
disease. In addition, lower expression of the two miRNAs
was reported in the livers of physically active patients
than in those of patients with lower PA or sedentary lifestyles at comparable fibrosis stages (Table 4).

Discussion
MicroRNAs (miRNAs) are negative gene regulators withmore than 2,500 copies in the human genome [21–28]
and have been shown to control many crucial biological processes, such as cellular proliferation, cellular differentiation and apoptosis. The location of miRNAs in
exosomes provides sufficient stability, thus underscoringAl-Rawaf et al. BMC Molecular and Cell Biology(2024) 25:18

Page 8 of 13

Table 4 Correlation between serum levels of miR-122 and miR-486 and histological liver severity in CHC patients
CHC patients
PA-patients
miR-122
miR486
PA-inactive patients
miR-122
miR486

Liver fibrosis scores
F0

F1

F2

F3

F4

2.8 × 104±0.65 × 104
1.3 × 104±0.35 × 104

3.1 × 104±1.2 × 104
2.6 × 104±0.62 × 104

5.4 × 104±1.6 × 104
2.9 × 104±0.91 × 104

6.1 × 104±3.1 × 104
3.1 × 104±0.41 × 104

7.9 × 104±2.1 × 104
3.5 × 104±1.1 × 104

3.1 × 104±0.22 × 104
1.9 × 104±0.81 × 104

4.7 × 104±0.89 × 104
3.9 × 104±1.2 × 104

6.1 × 104±2.3 × 104
4.3 × 104±1.3 × 104

7.6 × 104± 1.5 × 104
5.3 × 104±1.3 × 104

8.6 × 104±2.3 × 104
6.9 × 104±0.81 × 104

their role in diverse cellular processes, including disease
pathogenesis and antiviral defense [29, 30]. In addition,
an understanding of the pathogenesis of human virusassociated complications is important for developing
effective means of preventing and treating liver diseases[65, 66].
Currently, the identification of noninvasive biologicalbiomarkers, such as miRNAs, that can be used to screen
high-risk patients for diagnosis is urgently needed [67,68]. Changes in miRNA expression profiles have been
documented in liver diseases, particularly in the context
of HCV infection, spanning both the early and advanced
stages of liver fibrosis [67, 68]. However, there is limited
or no available information regarding the detection of
miRNAs and their potential association with physicalactivity and muscle capacity in individuals with CHC.
Thus, the aim of our biomarker discovery approach
was to investigate the potential of using miRNA expression in HCV-infected patients and its correlation with
HCV complications in terms of physical activity, pain,
and muscle capacity for potential use as noninvasive biomarkers for the early diagnosis of high-risk HCV.
In this study, we initially quantified the serum concentrations of both miR-122 and miR-486 in control subjects
and individuals with CHC. Notably, patients with CHC
exhibited markedly elevated serum levels of miR-122
and miR-486 in comparison to those in healthy controls.
Our findings demonstrated positive correlations between
miR-122, miR-486, alanine transaminase (ALT), aspartate transaminase (AST), fibrosis stage, and inflammation
activity. However, we did not observe any significant correlations with HCV-RNA levels or viral load. Intriguingly,
patients with advanced liver fibrosis (F3-F4) displayed
higher expression levels of these measured miRNAs than
did those with early liver fibrosis (F0-F1).
Our results align with previous studies reporting ofincreased circulating levels of miR-122 and miR-486 inpatients with hepatitis B virus (HBV) [69–75] and hepatitis C virus (HCV) [74, 75] infections. These elevatedlevels were found to be significantly associated with liver
fibrosis, inflammation scores, and alanine transaminase(ALT) and aspartate transaminase (AST) activity [69–75].
Importantly, no substantial correlation was establishedbetween the assessed miRNAs and HCV-RNA levels inhuman liver biopsies [76–80].
The expression of distinct miRNAs, including serummiR-122, is upregulated in patients with various HCV
genotypes, thus suggesting the potential utility of thesemiRNAs as diagnostic biomarkers for HCV infection[31]. Notably, our study demonstrated correlations
between miR-122, miR-486, fibrosis, inflammation, andglucose levels, which could be linked to the progressionof hepatic steatosis [75, 81–84].
The release of miRNAs into the serum can occur
through passive mechanisms, such as liver cell apoptosis and necrosis, or active processes involving the secretion of exosomes and viral particles. Consequently, thequantification of specific miRNA levels in serum may
serve as a significant indicator of ongoing activity withinliver cells [83, 84]. Although miRNAs are typically foundwithin exosomes or macrovesicles, they can also exist in
the form of free ribonucleoprotein complexes or be transported via the HBV surface antigen (HBsAg) [84–88].
Recently, it was postulated that the presence of miRNAs enriched in muscles in the circulation may be
associated with the modulation of metabolic responses
induced by physical exercise [89]. Previous studies havedemonstrated that the levels of certain miRNAs inskeletal muscle can be altered by physical activity andexercise programs in humans [90] and experimental
models [91]. Previous reports have also suggested that toenhance the self-confidence and overall muscle capacityof patients with CHC, improving the physical activity ofthese patients is a promising option [19, 20]. The bodies
of physically active patients with HCV manufacture more
ATP, thus indirectly boosting the body’s glutathione levels, which correspondingly improves the resilience of theliver against free radicals and hepatitis C damage [21].
In the current study, we attempted to determine
whether there was an association between the expression of miRNAs and both physical activity and muscle
capacity in controls and patients with CHC. In patientswith CHC with a lower physical activity score, the serum
levels of miR-122 and miR-486 were significantly lower
than those in control subjects and more physically active
patients with CHC. Consistent with our results, miR486 was demonstrated to be significantly reduced inAl-Rawaf et al. BMC Molecular and Cell Biology(2024) 25:18

physically active subjects following focused and regularexercise [92]. However, in those with a sedentary lifestyleor who exhibit lower physical performance, increased
levels of miR-486 were reported in the circulation [93,94].
The decrease in the release of miR-486 from muscle
tissue into the bloodstream of physically active individuals may be linked more to the utilization of lipids rather
than glucose as the primary energy source during physical activity [92, 93]. This phenomenon could involve theuptake of miR-486 from the blood into muscle tissue.
Furthermore, this concept has been supported by previous research that explored the connection between HCV
infection and lipid metabolism in the host. Notably, HCV
proteins and circulating infectious particles have been
associated with and shown to interact with lipid droplets
and structures resembling very low-density lipoproteins(VLDLs), which are commonly referred to as lipoviralparticles (LVPs). These interactions play a role in facilitating virion assembly and production [93, 94].
In addition, miR-122 was shown to be closely correlated with obesity, as were the levels of lipids, glucose andinsulin in patients with a lower physical activity score;
this effect correspondingly demonstrated a significant
correlation with viral infection. In active patients, lipid
utilization and decreased insulin resistance require theuptake of miR-122 from the blood into liver tissues [95,96]. A wide range of miRNAs were up- or downregulated
based upon individual participant, exercise type and theintensity of physical training, thus raising the possibilityof manipulating and understanding tissue development,function, and disease progression [97, 98]. Furthermore,
it was previously reported that miR-122 is one of themiRNAs that is solely expressed in the muscle, liver andbrain [99–101].
In the present study, the levels of LDH, CK, TAC, GSH,
and GSSG, as well as the GSH/GSSG ratio, were estimated as potential markers of physical activity and muscle capacity in patients with CHC and control subjects.
In physically active patients with CHC, significantly
lower levels of LDH, CK, and GSSG and elevated levelsof TAC and GSH were observed, as was a higher GSH/
GSSG ratio and a reduction in pain intensity compared topatients with CHC who had lower PA scores (P = 0.001)
and those in the control group (p = 0.01). In addition,
miR-122 and miR-486 expression in patients with CHC
was positively correlated with LDH and CK levels andnegatively correlated with PA scores; TAC, GSH, and
GSSG levels; and the GSH/GSSG ratio. In physically
active patients with HCV, their bodies manufacture
more ATP, thus indirectly boosting the body’s glutathione levels. This correspondingly improves the resilienceof the liver against free radicals and hepatitis C damage[21]. Normally, in liver disease, the antioxidant status isPage 9 of 13

significantly reduced as a result of viral infection or hepatotoxic agents [102, 103].
Reports indicate that HCV infection alone or in combination with HBV or HIV is significantly correlated witha reduction in cellular antioxidant mechanisms and elevated oxidative stress, which causes liver cell damage anda decrease in the number of mitochondrial DNA copies[104]. Hence, the cumulative impact of oxidative stressand the depletion of antioxidants may play a crucial rolein the progression of liver diseases [105, 106]. Generally,in patients with HCV infection, an increase in GSSG levels and a reduction in TAC and GSH levels and the GSH/
GSSG ratio have been reported in response to increased
free radical oxidative species [107].
Additionally, physical activity through exercise has
demonstrated effectiveness in decreasing inflammation and markers of oxidative stress in individuals with
impaired liver function and liver fibrosis, as reported inprevious research [86]. Other studies have reported ofelevated LDH and CK levels in HCV patients [85–87],
and the release of miR-122 and miR-486 was shown to belinked to a noticeable increase in the peak LDH and CK
levels in the muscle tissues of infected patients [108, 109];
this miRNA expression was delayed following exercise or
physical activity [89–91].
Concerning pain, previous research has indicated aconnection between HCV infection and musculoskeletal
pain, as well as general pain [92, 93]. In our investigation,
we noted a substantial decrease in pain severity among
CHC patients who were physically active. Previous studies have shown that physical inactivity is an additional
negative parameter affecting chronic pain conditions[94–98], and patients or healthy persons who are regularly physically active may use moderate exercise as asignificant tool for the prevention of chronic disease [14,15].
Moreover, our results suggested that the expressionof both miR-122 and miR-468 was negatively correlatedwith sex among the examined CHC patients, whichsuggests that miRNA expression could be influenced
by sex differences. Previous studies have shown thatsome miRNAs expressed in certain tissues are sex specific and are significantly more strongly associated with
the X chromosome in females than with the Y chromosome [110–113]. Gender differences may be due to inactivation of one of the two X chromosomes occurringin men to maintain balanced expression of the X chromosomes, whereas chromosome inactivation leads tobiallelic expression of miRNAs [110–113]. In addition,
significant sex differences in miRNA expression haveemerged in many patients with liver diseases, including
liver cancer and viral hepatitis, and are closely correlatedwith sex hormones [114, 115]. In addition, our results
provided evidence that the expression of miR-122 andAl-Rawaf et al. BMC Molecular and Cell Biology(2024) 25:18

miR-468 was significantly associated with an increase in
BMI, which is an adiposity marker, in CHC patients withpoor physical activity compared to that in patients whoare physically active. Previous studies have shown that
adipose-derived exosomal miRNAs can regulate gene
expression in distant tissues such as the liver and could
be considered a novel class of adipokines [116–118]. The
importance of circulating miRNAs as novel adipokines in
HCV infection remains to be clarified.
Obviously, further studies are necessary to clarify themechanisms of action and targets of the identified miRNAs miR-122 and miR-486, which are justified noninvasive biomarkers of the pathogenesis of HCV, as wellas to correlate their expression levels with poor physical activity, obesity, sex, pain, and muscle fatigue, which
are potential prognostic factors for CHC, and which can
respond to therapeutic parameters.

Conclusions
In conclusion, this study demonstrated significant associations between miRNA-122 and miRNA-486 expression levels and various aspects of health in patients with
HCV infection and other related viral complications interms of physical activity, pain perception, adiposity, andmuscle fatigue biomarkers. In CHC patients, the expression of cellular miR-122 and miR-486 was shown to playa crucial role in influencing physical activity, pain perception, adiposity, and muscle fatigue biomarkers within thisCHC population. Furthermore, elevated levels of miR122 and miR-486 were associated with increased levels of
aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and creatine
kinase (CK). Additionally, these miRNA expression levels
were correlated with fibrosis scores and antioxidant status, thus suggesting their potential utility as biomarkers
for disease severity and oxidative stress in patients with
HCV infection or its complications. Interestingly, no significant correlation was found between miR-122/miR486 expression and viral load or HCV RNA expression
in the same patients, thus suggesting that these microRNAs may be more closely associated with overall healthstatus and complications of HCV infection than directly
impacting viral replication or RNA expression.
Overall, our study provides valuable insights into thepotential of miR-122 and miR-486 as noninvasive biomarkers for evaluating the pathogenesis of CHC infection in terms of their correlation with physical activity,
pain perception, adiposity, sex, and muscle fatigue inindividuals with HCV infection. Further research should
explore the mechanistic pathways underlying these associations and their clinical implications for managing
HCV-infected patients.

Page 10 of 13

Acknowledgements
The authors are grateful to the Researchers Supporting Project number(RSP2024R382), King Saud University, Riyadh, Saudi Arabia for funding thisresearch.
Author contributions
H.A.R. A.H.A. and S.A.G. provided the research idea, conception, and design.
H.A.R. and S.A.G. conducted practical work and collected the data. A.H.A. and
A.I. analyzed and interpreted the data. H.A.R. A.H.A. S.A.G. and A.I. carried out
the initial manuscript drafting and preparation. H.A.R. A.H.A. S.A.G. and A.I.
critically edited and formatted the manuscript. All authors read, reviewed, andapproved the final manuscript version to be submitted or published.
Funding
This project was funded by the Researchers Supporting Project number(RSP2024R382), King Saud University, Riyadh, Saudi Arabia.
Data availability
The datasets used and/or analyzed during the current study are available fromthe corresponding author on reasonable request.

Declarations
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Ethical approval and consent to participate
The ethics sub-committee at King Saud University, Riyadh, Saudi Arabia
reviewed and approved the study protocol assigned under file number ID:
RRC-2016-045. This study was conducted following the ethical guidelines
of local institutional review board and the Declaration of Helsinki (2010). A
signed informed-consent form was obtained from each participant as a proof
of consent to participate before the start of this study.
Author details
1
Department of Clinical Laboratory Sciences, College of Applied Medical
Sciences, King Saud University, Riyadh 11433, Saudi Arabia
2
Department of Rehabilitation Sciences, College of Applied Medical
Sciences, King Saud University, P.O. Box 10219, Riyadh 11433, Saudi Arabia
Received: 22 January 2024 / Accepted: 9 July 2024

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