Abstract

General Background: Liver fibrosis, a precursor to cirrhosis, arises from chronic liver injury due to factors such as hepatitis infection, alcohol intake, obesity, and smoking. Specific Background: MicroRNAs (miRNAs), especially miR-21 and miR-98, are increasingly recognized for their regulatory roles in hepatic fibrogenesis. Knowledge Gap: Despite evidence of miRNA involvement in liver fibrosis, data on their gene expression patterns in specific populations, such as Iraqi patients, remain limited. Aims: This study aimed to evaluate the differential expression of miRNA-21 and miRNA-98 in liver fibrosis patients compared to healthy controls, and their correlation with clinical variables, particularly hepatitis B and C infections. Results: In a case-control design with 88 patients and 88 controls, RT-PCR analysis showed upregulation of miRNA-21 (1.15 vs. 0.32) and downregulation of miRNA-98 (1.67 vs. 6.33) in patients (P < 0.05). miRNA expression was higher in HBV patients and in females. Novelty: This is the first study in Iraq to reveal the opposing expression profiles of miRNA-21 and miRNA-98 in liver fibrosis, suggesting sex-specific and viral infection-related modulation. Implications: These findings highlight the potential of miRNA-21 and miRNA-98 as biomarkers for fibrosis severity and targets for therapeutic intervention, particularly in HBV-associated liver disease.
Highlight :

• miRNA-21 is upregulated in liver fibrosis patients, especially with HBV, suggesting a potential role in fibrogenesis.

• miRNA-98 is downregulated in liver fibrosis, indicating a protective or regulatory function in fibrosis progression.

• Gender and virus type influence miRNA expression—females and HBV patients show higher gene expression levels.

Keywords: miRNA 21, miRNA 98, RT- PCR, Gene expression, Liver fibrosis

Introduction

The term “liver disease” or “hepatic disease” encompasses a range of disorders and conditions that hinder or entirely prevent the liver from functioning properly [1]. Blood tests known as" liver function tests" are performed to track liver damage or illness and assist in determining the origin of symptoms. The tests quantify the blood's concentrations of specific proteins and enzymes [2]. Some of these tests assess the liver's ability to carry out its normal tasks of making protein and eliminating the blood waste product bilirubin. Enzymes released by liver cells in response to injury or illness are measured by other liver function tests [3]. Infections, genetic abnormalities, obesity, and alcohol misuse are the main causes of liver disease in humans. While some liver problems are temporary and may go away on their own, others progress into chronic conditions that can cause serious problems like liver fibrosis and eventually liver failure [4]. A complicated fibrogenic and inflammatory process, liver fibrosis is a precursor to liver cirrhosis and is brought on by long-term liver damage. Due to the dearth of efficient therapeutic options, cirrhosis is a significant global health concern [5]. A number of "miRNAs are linked to both systemic and organ-specific liver fibrosis. The identification of liver fibrosis can be aided by the individual expression of miRNAs in serum or plasma [4,6]. The RNA gene miR-98 (MicroRNA 98) belongs to the miRNA class. The let-7 miRNA family, of which MiR-98 is a member, was initially found to regulate the developmental timing of cell differentiation and proliferation in C. elegans". Endogenous noncoding RNAs of roughly 22 nucleotides, known as miR-98, bind complementary sequences in the 3-prime UTRs of target mRNAs, causing mRNA cleavage and/or translational suppression. A number of carcinomas have recently been discovered to exhibit altered miR-98 expression [5,7]. The miRNA 21 gene encode the mammalian microRNA 21 (sometimes referred to as has-miR-21 or miRNA21). "The human microRNA-21 (miR-21) gene is located on the plus strand of chromosome 17" at position 17q23.2 (coordinates 55273409–55273480), embedded within the coding region of the TMEM49 gene, also known as the vacuole membrane protein gene [8.9]. Many of its gene targets have been linked to both neoplastic and non-neoplastic diseases. Tropomyosin 1 (TPM1), Phosphatase and Tensin Homolog (PTEN), and Programmed Cell Death 4 (PDCD4) are three of miR-21’s primary targets [10,11]. Research has shown that hepatitis and liver cancer are associated with elevated expression of miRNAs, including miR-21. It has been demonstrated that miR-21 stimulates fibrogenesis in the heart, kidneys, lungs, liver, and muscles [11,12]. Unfortunately, studies about actual job of abnormal gene expression or polymorphism of miRNAs are very limited especially in Iraq therefore; we attempted to identify the potential role of miRNA 21 and 98 in development of hepatic fibrosis in Iraqi patients using RT-PCR dependent gene expression.

Material and methods

"Research design and samples collection: During this study which is a case-control study, researchers worked from June 10, 2023, through October 9, 2024. Samples were taken from the Gastrointestinal Unit (GIT) at Medical City in Baghdad, Al-Zhara Teaching Hospital and local outpatient clinics. 88 people with liver fibrosis and 88 healthy volunteers were part of the study as a control group. All participants were asked to give their consent before they filled in the questionnaire or gave their blood samples. Five milliliters of blood was collected from every participant so the required tests could be done.

Hepatitis Test: Hepatitis B surface antigen (HBsAg) and anti-hepatitis C virus antibodies (IgG, IgM, and IgA) in human serum can be qualitatively detected and differentiated using the one step HBsAg/HCVAb Rapid Diagnostic Test, a flow chromatographic immunoassay. One stage in this test is adding 0.5 cc of serum to the Raypo-Tech (USA) assay plate and watching for a color shift.

Liver function tests: Total albumin, glutamic oxalacetic transaminase/aspartate aminotransferase activity (AST-P Ⅲ), total serum bilirubin (TSB), alanine aminotransferase activity (ALT-P Ⅲ), and alkaline phosphatase activity (ALP-P Ⅲ) are all measured with FUJI DRI-CHEM SLIDE (China).

Molecular study:

"Following the company’s guidelines, total RNA was extracted from blood samples using the TRIzol® Reagent kit. The Nanodrop Spectrophotometer, which measures RNA concentration and estimates RNA purity by measuring absorbance at 260 and 280 nm, was used to examine the extracted genomic RNA. According to the protocol provided by Promega (USA), the extracted RNA was treated with DNase I enzyme using the DNase I enzyme kit to remove any residual genomic DNA from the eluted total RNA samples." The sequences of miR-98 (MIMAT0000096) and miR-21 (MIMAT0004495) were picked from the Sanger Center miRNA database registry and the miRNA Primer construct Tool was used to design qPCR primers for them. For this research, the qPCR housekeeping gene (GAPDH) (NM_001256799.3) was designed and created using the NCBI-Database and Primer3 Plus sites and not the traditional paper approach. According to Table (1), all the primers I used were supplied by the Macrogen Company which is located in Korea. To make the qPCR master mix, the kit known as GoTaq® qPCR Master Mix was used. After that, the master mix components were transferred to qPCR tubes, mixed for three minutes on the vortex centrifuge and inserted into the Miniopticon Real-Time PCR system. After loading the qPCR plate, the standard cycle of qPCR for GAPDH or miRNA genes was performed: There were 45 cycles and each cycle included 30 seconds at 60°C for annealing/extension/detection (scan), 20 seconds at 95°C for Denaturation Step 2 and 1 cycle at 95°C for 5 minutes for the initial denaturation TM step. "The relative quantification of gene expression levels (fold change) was performed using the ΔCT method with a reference gene, as described by Livak and Schmittgen" [12] were used to assess the data findings of q RT-PCR for the target and housekeeping genes. as the equation that follows:

Figure 1.

Statistical analysis

The data for this study were analyzed with version 22 of the Statistical Package for the Social Sciences. Excel 2010 was also used. A difference of less than 0.05 was deemed a statistically significant association[14].

Result

This research is a case-control study. 88 patients with liver fibrosis were included in the study; their ages ranged from 25 to 77 years, and their age mean ± standard deviation was 55.76 ± 9.16 years. As shown in Table (2), the majority of these patients were male (61%) compared to female (39%). Results from cases are contrasted with those of healthy people as a control group. 88 healthy people with an average age of 48.6 ± 11.55 years made up the control group.

* significant association ( P <0.05), SD: Standard deviation, SE: standard error

According to Table (3), the mean BMI of patients was higher (33.66 ± 8.14 kg/m2) than that of controls (24.31 ± 3.11 kg/m2) (t=7.255, 95% CI=7.620 to 13.362, P=0.049). Additionally, it was shown that hepatitis, particularly type HBV (11%), affects 17% of patients. 89% of patients had chronic conditions, with obesity and hypertension accounting for the bulk of cases (34% and 26%, respectively), followed by diabetes (7%). There have also been reports of certain patients experiencing multiple chronic diseases at the same time. According to Table (4), 53% of patients were found to be alcoholics, and 60% of patients were found to smoke.

* significant association ( P <0.05), SD: Standard deviation, SE: Standard error, CI Confidence interval

* significant association ( P <0.05)

The gene expression analysis results (Figure 1 & 2) revealed that patients had a higher mean fold change (2-∆∆CT) for miRNA 21 when compared to controls (0.32) and a lower mean fold change for miRNA 98 when compared to healthy individuals (1.67). These findings resulted in the emergence of significant differences (P<0.05) as shown in Table (5). We discovered that the mean fold change (2-∆∆CT) of the genes miRNA 21 and miRNA 98 increased in females (1.081 and 1.897) when we sorted the gene expression rate of the examined genes by patient sex. Additionally, the current findings revealed that patients infected with HBV had higher gene expression of miRNA 21 and miRNA 98 (1.902 and 2.984, respectively) than those infected with HCV (0.884 and 0.311, respectively) (P value < 0.05), as shown in Table (6). Additionally, there was an ambiguous (weak) Pearson association (r = 0.065) between miRNA 21 and miRNA 98 gene expression (Figure 3).

* significant association in compared with controls (P < 0.05)

Figure 2. The miRNA 21 gene expression analysis by Real Time PCR melting plot demonstrating qPCR specificity at 85°C

Figure 3.The miRNA 98 gene expression analysis by Real Time PCR melting plot demonstrating qPCR specificity at 83°C

* significant association in compared with controls (P < 0.05) , χ 2 : chi square

Figure 4.Pearson correlation (r = 0. 0.65 ) among gene ex pression of miRNA 21 and miRNA 98 of patients with liver fibrosis

Discussion

The results of the current study showed that the average age of those with liver cirrhosis was approximately 55.76 years, and the majority of them were males. This may be due to unhealthy habits common among men in our society, such as smoking and alcoholism, which often continue into adulthood. This makes the average age of liver fibrosis patients 55.76 years. We also found that the most important causes or chronic diseases associated with liver fibrosis are excess BMI, body weight and high blood pressure [13,15]. These diseases may be the primary cause or contributing factor in the development of liver fibrosis. A study of the Midspan cohorts in Scotland [15] revealed that body mass index (BMI) influences the impact of alcohol consumption on liver disease, with obese individuals showing greater vulnerability to alcohol-related liver damage compared to those with lower BMI. Similarly, findings from the Million Women Study [16] supported this interaction, highlighting that the combined effect of high BMI (above 30) and alcohol intake exceeding 150 g/week significantly increases the risk of developing liver cirrhosis. These results also suggest a potential interaction with patterns of alcohol consumption [17]. Numerous biological phenomena, such as cancer, fibrosis, and inflammation, are associated with increased expression of miR-21. There is growing evidence that miR-21 has a significant role in a number of liver disorders [18]. MiR-21 dysregulation is prevalent in a number of chronic liver disorders. According to the current study, miR-21 gene expression rose in liver fibrosis patients and fell in healthy ones. However, based on the research, the expression of miR-21 varies and is heterogeneous in each kind of liver illness [19]. The utilization of various animal models and the absence of established practices and ways to normalize its level could be the root cause. Using "miR-21 as a therapeutic target" or as a possible biomarker for particular liver disease types carries a number of risks [20]. Previous studies have demonstrated that host miRNAs can either favorably or negatively regulate viral infection by targeting cellular components or viral genomes [21]. According to our findings, miRNA 21 and 98 gene expression rose in HBV infection and fell in HCV infection. Viral infections can alter the amount of miRNA expression in cells and provide an environment that is conducive to their survival and harmful effects. Research revealed that patients with Hepatitis B virus (HBV) have elevated serum levels of miR-21 [21,22]. Several studies have shown that miR-21 plays a critical role in the transformation from non-tumor to tumor state induced by the hepatitis B virus X protein (HBx) [22,23]. This action is mediated at the molecular level by the phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway [24], despite the lack of direct evidence tying miR-21 to HBV infection or replication. According to a previous study, HCV raises the expression of miR-21 in primary human hepatocytes and hepatocyte cell lines, which is consistent with our findings [25]. According to clinical evidence, the degree of fibrosis and viral load in liver biopsies of individuals infected with HCV were linked to miR-21 expression in liver tissues [26]. Chen et al. demonstrated that miR-21 suppresses the expression of myeloid differentiation primary response 88 (MyD88) and interleukin-1 receptor-associated kinase 1 (IRAK1) during hepatitis C virus (HCV) infection, thereby exerting a negative regulatory effect on interferon-alpha (IFN-α) signaling. [27]. The current study found that patients, particularly men, had lower levels of miRNA 98 gene expression. In the same vein, Wang et al. reported that miR-98 expression declined in various liver fibrotic models as well as activated HSCs. By blocking the HIF-1α/TGF-β/Smad2/3 signaling cascade, miR-98 overexpression targeted HLF and prevented the activation of HSCs and the production of profibrotic markers [28]. MiR-98-5p was found to be a potential predictor of the course of liver fibrosis in the Ma et al. investigation. Activated LX2 cells have lower levels of miR-98, and overexpression of miR-98-5p prevented the activation of HSCs. By specifically targeting TGFbR1 and inhibiting the TGFb1/Smad3 signaling cascade, MiR-98-5p mechanically stops liver fibrosis. Additionally, a total of 70 patients with persistent HBV infection and 29 healthy people served as controls, and their serum miR-98 levels were assessed. Compared to healthy controls and HBV carriers, individuals with liver fibrosis had significantly reduced serum "miR-98 levels "[29]. Research has demonstrated that miR-98-5p may limit hepatoma cell growth while causing cell death, at least partially, by suppressing its target gene IGF2BP1[30].Another study found that miR-98 directly targets the IκB kinase IKKε in glioma cells, suggesting that miR-98 has an anti-invasion function by preventing glioma cell movement and invasion [31]. On the other hand, little is known regarding the role of miR-98-5p in liver fibrosis. By targeting activin receptor-like kinase-4 and matrix metalloproteinase-11, Siragam and his colleagues discovered that miR-98 decreased the growth, invasion, angiogenesis, survival, and proliferation of breast cancer cells [32,33]. Yang et al. discovered that miR-98 is down-regulated in non-small cell lung cancer, while" P21-activated protein kinase 1 (PAK1)" is substantially expressed [34]. According to our research, females had higher mean fold changes (2-∆∆CT) for the miRNA 21 and miRNA 98 genes. In general, we could not find enough research to explain this, but this result demonstrates that the genetic expression of these genes is either directly or indirectly linked to sex hormones. In his study on miRNAs, Szabo and Bala found that the gender of the patient was related to the miRNA gene expression [35].

Conclusion

The recent study's findings demonstrated that the primary causes of liver fibrosis are chronic illnesses linked to smoking and obesity. Genetically, miRNAs 21 and 98 have been connected to liver fibrosis particularly in HBV-infected patients but they play opposing roles since fibrosis is correlated with either an increase in miRNA 21 gene expression or a decrease in miRNA 98 gene expression.

Ethical approval: Prior to beginning work, the hospital legally granted approval, and before collecting samples, patients gave their verbal agreement.

"Declarations of interest: No conflict of interest exists."

"Formatting of funding sources: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors."

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