Assessment circulating micro-RNA-192 in Egyptian patients with non-viral liver cirrhosis and hepatocellular carcinoma

Abstract

Background

It is challenging to diagnose hepatic cirrhosis and hepatocellular carcinoma (HCC) at an early stage since non-invasive markers are not specific and do not identify the early stage.

Objective

Serum microRNA-192 could represent an early and critical pathogenetic factor in different liver diseases.

Methods

92 participants were allocated into four equal groups (n = 23): control, compensated liver cirrhosis, decompensated cirrhosis, and HCC groups.

Results

Decompensated cirrhosis and HCC showed significantly elevated Serum Alanine aminotransferase, aspartate aminotransferase, bilirubin, alpha-fetoprotein, and microRNA-192.

Conclusion

Serum microRNA-192 can predict decompensated liver cirrhosis and HCC with high sensitivity and specificity.

Introduction

Small RNAs called microRNAs (miRNAs) regulate the expression of many genes, which may play a role in organ development and cell death. In addition, several conditions, including inflammation, fibrosis, and malignancy, show changes in miRNA expression [1–6]. Extracellular circulating miRNAs may be encapsulated inside vesicles like exosomes or microvesicles, or may be attached to serum proteins and lipoproteins. Exosomes have been found to play a crucial role in hepatic cell-cell communication and in liver pathophysiology across a range of liver diseases. They can be secreted by various hepatic cells and transferred to recipient cells to regulate expression profiles in those cells [7–11].

Liver cirrhosis develops when chronic inflammation of the liver leads to diffuse hepatic fibrosis, followed by liver failure due to impaired normal liver structure and its replacement by regenerating hepatic nodules [12–15]. Both developing and developed countries have a high prevalence of liver cirrhosis attributable to high morbidity and mortality rates [16–18]. Patients with compensated cirrhosis don’t exhibit any symptoms. On the other hand, decompensated cirrhosis is represented with many complications that frequently require hospital admission, negatively impact patient life, and ultimately result in patient death [19].

Primary hepatocellular carcinoma (HCC), the most fatal complication of cirrhosis, can also result from architectural distortion and its concomitant dysplastic hepatocyte regeneration [12]. Liver cancer develops in about one-third of cirrhotic patients. First-stage treatment has been made possible by early diagnosis, which will significantly improve the patient’s overall survival [20]. Furthermore, because treatment options for advanced HCC have developed considerably recently, it is essential to discern prognostic factors that capture tumor growth rate and hepatic function loss to ensure more effective regimens [21].

Hence, there is a need to find other non-invasive options for early diagnosis and staging of liver cirrhosis. MicroRNA-192, predominantly expressed in hepatocytes, is associated with liver damage response, fibrotic development, and malignant transformation [22, 23]. It influences TGF-β-mediated fibrogenic signaling and hepatocarcinogenesis via regulating cell proliferation and death [24]. Studies have found higher levels of miR-192 in exosomes and plasma of individuals with hepatocellular carcinoma (HCC), implying that it has diagnostic and prognostic value [25–30].

Patients and methods

Study population

This cross-sectional comparative study was conducted on 92 patients from the Internal Medicine Department at Beni-Sueif University Hospital from June to November 2021. Before work initiation, permission from the Faculty of Medicine, Beni-Sueif University, research ethical committee (REC) was obtained with the number: FMBSUREC/06062021/Huzien.

Study design

Initial information about the study was given to the patients, allowing them to decide whether to participate or not. All participants undergo full history taking with a thorough clinical examination, including general (Weight, height, body mass index, temperature, arterial blood pressure, heart rate, edema, and nervous system examination) and a local examination of the abdomen for ascites.

Liver cirrhosis was determined by computed tomography/magnetic resonance imaging, laboratory findings, and clinical presentation. Diagnosis of HCC was determined by using definite imaging criteria according to recent guidelines [31].

Inclusion criteria

Individuals diagnosed with liver cirrhosis, irrespective of etiology, aged between 30 and 80 years.

Exclusion criteria

All such patients who were below the group chosen with cardiovascular problems, chronic Hepatitis B or C viral infection, other forms of cancers excluding HCC, and patients who received chemotherapy were all excluded from our research.

The participants of the groups II-IV were randomly enrolled to the study based on the data from the hospital patients registry lists based on the final diagnosis. A minimum sample size for each group ws adopted as 23 for each. The randomization was performed via the systematic random technique (every third patient).

Study groups

After enrollment, participants were divided into four equal groups (n = 23): the healthy control group (I), the compensated cirrhotic group (II), the decompensated cirrhotic group (III), and the HCC Group (IV).

Biochemical study

A venous blood sample was obtained from the patients, and a clot was allowed to form and retract. All samples were centrifuged at 4000 RPM for 15 min to get a separate serum, which was then stored at -20 °C for subsequent analysis.

• (A)Assessing liver function

Serum alanine aminotransferase (ALT) enzyme activity was assayed using ALT activity assay kit (catalogue 700260, Cayman Chemical, Ann Arbor, USA) and aspartate aminotransferase (AST) enzyme using AST activity assay kit (catalogue MET-5127, Cell Biolabs, San Diego, USA). Serum albumin was assayed using the Human Albumin (Hu ALB) ELISA kit (catalogue ab108788, Abcam, Cambridge, UK). Serum bilirubin was assayed using the TOTAL AND DIRECT Colorimetric method [32].

• (B)Determination of serum alpha-fetoprotein (AFP)

Serum AFP was assayed using Human alpha-fetoprotein (AFP) ELISA Kit [33].

• (C)Determination of microRNA-192

MiRNeasy Serum/Plasma Advanced Kit (Catalog number 217204, QIAGEN Group, Germany) was used for purification of serum microRNA-192 [33].

Statistical data analysis

Data were gathered, edited, and coded into the Statistical Package for Social Science (IBM SPSS) software version 26. Shapiro-Wilk’s test was applied to verify the normality of the data. Data distribution was tested using the non-parametric Kruskal-Wallis test, followed by Dunn’s test (multiple comparisons). P values less than 0.05 were employed to conclude statistical significance. We used receiver operating characteristic (ROC) curve analysis, a measure of sensitivity and specificity, to examine the miRNA-192 serum level for predicting different liver diseases in this work. It was calculated as the area under the curve (AUC) with a 95% confidence interval (CI). The AUC ranged from 0.5 (indicating no predictive value) to 1 (indicating predictive value) [34].

Results

Clinical characteristics of the studied groups

The demographic data is discussed in Table 1.

Table 1.

Demographic data of the studied population

		Studied groups N = 23				Total 92
		Control	Cirrhosis compensated	Cirrhosis decompensated	Cirrhosis HCC
Age	Mean ±SD	60.91 ± 9.89	59.09 ± 9.88	58.91 ± 11.95	61.26 ± 11.37	60.04 ± 10.68
	Minimum	35.00	41.00	33.00	33.00	33.00
	Maximum	80.00	73.00	74.00	80.00	80.00
Gender n (%)	Female	10 (43.5)	11 (47.8)	9 (39.1)	10 (43.5)	40 (43.5)
	Male	13 (56.5)	12 (52.2)	14 (60.9)	13 (56.5)	52 (56.5)

Liver function tests of the studied groups (Fig. 1)

Fig. 1.

Liver function tests comparison among the study groups. Results are presented as median ± SEM. Statistically significant (P < 0.05) as compared to: (*) group 1, (^#) group 2, and (^@) group 3,

Serum AST, ALT, and bilirubin were statistically significantly (P < 0.0001) elevated in cirrhotic groups (decompensated, compensated, and HCC) compared to the control individuals. The decompensated cirrhotic patients had the highest values, and their serum bilirubin levels were statistically significantly (P < 0.018) higher than those of the compensated group. Conversely, the serum levels of AST in the HCC cirrhotic group were significantly higher (P < 0.0001) than in the compensated group.

Comparison of serum Alpha-Fetoprotein (AFP) between the studied groups (Fig. 2a)

Fig. 2.

Comparison of (a) Alpha Fetoprotein (AFP) Tumor Marker and (b) microRNA 192 serum levels among the study groups. Results are presented as median ± SEM. Statistically significant (P < 0.05) as compared to: (*) group 1, (^#) group 2, and (^@) group 3,

Serum levels of AFP demonstrated statistically insignificant (P > 0.001) values between the control and the compensated cirrhotic groups. However, there was a statistically significant (P < 0.0001) variation between the decompensated and HCC cirrhotic groups compared to the control individuals, and between the HCC cirrhotic and decompensated cirrhotic patients.

Serum miRNA192 levels in the study (Fig. 2b)

Serum miRNA192 of the cirrhotic group with HCC was statistically significantly (P < 0.0001) higher compared to the other three groups. Moreover, the decompensated cirrhotic group showed statistically significant (P < 0.001) raised serum miRNA192 levels in comparison with the compensated cirrhotic group.

ROC curve analysis of serum miRNA-192 level for prediction of different liver conditions (Fig. 3)

Fig. 3.

Receiver operating characteristic (ROC) curve analysis – microRNA192 serum level to predict; a decompensated liver disease (0.769) and b cirrhotic liver disease with HCC (1.000)

Through ROC curve assessment, the sensitivity and specificity of the serum level of miRNA-192 in predicting different liver conditions in our study were determined. The area under the curve (AUC) of serum level of miRNA-192 in the prediction of decompensated liver disease was (AUC = 0.769, SE = 0.078, 95% CI: 0.617–0.921).

Area under the curve (AUC) of the level of serum miRNA-192 to forecast cirrhotic liver disease with HCC was (AUC = 1.000, SE = 0.000, 95% CI: 1.000–1.000).

The Multivariant logistig regression analysis for HCC predectors shows that ALT (0.004), AST (0.001), AFP (0.014) and microRNA-192 (0.0001) were found to be predectors for HCC (Table 2).

Table 2.

Multivariant Logistig regression analysis for predectors of HCC

Model	Standardized coefficients beta	t	95.0% confidence interval for B		Sig.
			Lower bound	Upper bound
(Constant)		1.231	- 0.558	2.285	0.226
Age	0.018	0.457	- 0.009	0.014	0.650
Gender	0.061	1.463	- 0.071	0.439	0.152
ALT	- 0.236	-3.060	- 0.016	- 0.003	0.004*
AST	0.374	3.591	0.003	0.013	0.001*
Albumin	- 0.086	-1.372	- 0.437	0.084	0.178
Bilirubin	- 0.005	-0.094	- 0.086	0.079	0.926
AFP	0.169	2.587	0.000	0.001	0.014*
microRNA-192	0.636	8.431	0.652	1.064	0.0001*

Discussion

Finding a trustworthy, accurate indication of the severity of liver cirrhosis and HCC stage has been challenging, despite the tremendous advancements in conventional biomarkers. In recent years, microRNAs have gained attention as possible options.

Serum miR-192 was found as a viable non-invasive biomarker for stratifying compensated, decompensated cirrhosis, and HCC stages. The robust discriminative ROC performance (AUC = 1.00 for HCC prediction) demonstrates its diagnostic value, exceeding AFP, which showed no significant variations among cirrhotic subgroups.

Past research has shown an overwhelming quantity of evidence indicating the involvement of miRNAs in initiating and developing chronic hepatic disorders, cirrhosis, and ultimately HCC [11]. Various biomarkers have been investigated to detect liver cirrhosis severity and HCC stage noninvasively. However, biomarkers that represent progressive hepatocytic inflammation lack sufficient accuracy and specificity. In our work, we hypothesize that circulating miRNA-192 may be used as a biomarker to detect liver cirrhosis and HCC.

For demographic data in the current work, participants’ age was between (33) and (80), and their mean age was (60.04 ± 10.68) years old. There were 52 males (56.5%) and 40 females (43.5%) participating, and they were roughly evenly divided among the four studied groups. That is in line with a previous study that included 237 Egyptian patients with cirrhosis, gender [35]. However, males dominated, with a median age of 67 years, in an earlier study of all 134 liver patients [36].

In our study, serum AST and ALT levels were the highest in group (IV) (Cirrhosis HCC) compared to the other three study groups, and the lowest in group (I) (Healthy Control). ALT was much more significant in groups (II and III) (Cirrhosis compensated and decompensated, respectively) when compared to group (I) (Healthy Control). Our findings were in accordance with Tan et al., who found that ALT and AST were considerably higher among HCC patients [37]. Apart from that, in their earlier cross-sectional study, they found that ALT, AST, and AFP were significantly higher among hepatic cirrhosis patients [38]. But ALT and serum cytokines are unstable under severe forms lacking tissue specificity, hence the need for more sensitive, stable, and specific biomarkers of liver injury [39].

In the present study, serum albumin was significantly highest in group (I) when compared with the other three studied groups, and was lowest in group (III). Similarly, serum bilirubin was significantly lowest in the healthy control group and highest in group (III) compared to the other study. This agrees with the findings of Motawi et al. [35] who indicated that albumin was found to be significantly elevated in the controls in comparison to other study groups. In contrast, however, bilirubin was significantly elevated in hepatic than the other healthy patients.

Serum alpha-fetoprotein (AFP) is utilized in the diagnosis of HCC. In chronic hepatic patients, a persistently high serum level was shown to be one of the risk factors for HCC. Patients with cirrhosis experience a change in serum AFP with deterioration of their liver condition or the occurrence of HCC [40]. In the current study, serum AFP was significantly highest in group (IV) in comparison to the remaining three groups. Apart from that, there was an insignificant difference among groups (I), (II), and (III) compared to AFP. This agrees with a previous study, which determined fluctuating AFP levels in decompensated cirrhosis and HCC [40]. In addition, AFP elevation can be observed in hepatocyte regeneration, hepatocarcino-genesis, and embryonic [41]. However, AFP diagnostic sensitivity is limited by non-AFP-secreting tumors [42].

MiRNA 192 is a hepatocyte-specific miRNA, specific for liver injury detection. The elevated serum miRNA 192 level in the liver is considered to be a marker for liver cirrhosis [43]. In this study, serum miRNA-192 was significantly the highest in group (IV) compared to the other three studied groups. It was lower in groups (I) and (II) compared to group (III) (Cirrhosis Decompensated). There was no noteworthy variation between groups (I) and (II). In agreement with our results, serum miRNA-192 level was considerably higher in cirrhotic groups than in the controls [44]. In addition, Xue and colleagues [45] illustrated higher serum exosomal miR-192 levels in HCC. Additionally, our results agree with Frundt et al. [36], where miR-192 was aberrantly regulated in HCC patients.

On the other hand, the decompensated liver cirrhosis groups showed a lower level of miRNA-192 than the compensated liver cirrhosis patients [46]. In agreement with our results, Cermelli et al. [47] found elevated expressions of miRNA-192 in both cirrhotic and fibrotic patients. In his article, miRNA-192 was linked to AST/ALT levels, fibrotic disease stage, and inflammatory activity, indicating that circulating miRNA levels can reflect some characteristics in the pathophysiology of chronic liver diseases. In contrast to our results, high miRNA-192 levels were predictive of higher inflammatory activity but not of the extent of liver fibrosis [48].

Variations in circulating miRNA-192 observed in some research when comparing decompensated cirrhosis to compensated stages could be attributed to exosomal release dynamics and hepatic reserve [49, 50]. According to Jin et al., severe liver failure is associated with reduced hepatocytes production and altered vesicular miRNA secretion, which may result in lower levels of circulating miRNA-192 even as the disease worsens [25]. These discrepancies underscore the importance of evaluating miR-192 expression in relation to hepatic functional capacity when employing it in clinical settings for prognosis or disease staging.

This work assessed the sensitivity and specificity of miRNA-192 serum levels in predicting different hepatic diseases by ROC curve analysis. This agrees with Tan et al. [37], where the serum miRNA panel (hsa-miR-192-5p, hsa-miR-122-5p, hsa-miR-1290, and hsa-miR-27b-3p) showed high accuracy in diagnosing hepatic disease. Regarding the miRNA panel AUC values, they clarified that this miRNA panel could predict liver disease with a sensitivity = 69.9% a specificity = 83.7%.

In addition, one report showed an upregulation of miRNA-192 in the blood sera of the chronic liver disease group compared to the normal controls. More importantly, the ROC curve revealed a diagnostic value of miRNA-192 in diagnosing liver cirrhosis [51]. Moreover, Frundt and others [36] illustrated that plasma exosomal miR-192 levels were of diagnostic and prognostic value in HCC patients.

For many years, the AFP biomarker has been the most utilized for HCC monitoring [52]. However, it has low sensitivity and specificity, particularly in early or AFP-negative tumors, limiting its self-sustaining usage [53]. Circulating miR-192 could improve early HCC identification, since conventional biomarkers like AFP are insensitive in non-AFP-secreting tumors [54, 55]. Unlike AFP, which has low sensitivity in early HCC or non-AFP-secreting tumors, circulating miR-192 is more stable and can be identified in exosomal or protein-bound forms [56]. This makes it a prospective option for early identification and staging of a wide range of hepatic diseases, including compensated cirrhosis and HCC.

Circulating MiR-192 has been proven to provide significant advances over AFP for liver cancer screening and clinical categorization [57]. MiR-192 primarily functions as a tumor suppressor in liver cancer, blocking XIAP, targeting TRIP25 and SLC39A6, and inducing autophagy and death via CYR61 [58, 59]. Integrating miR-192 measurement into existing diagnostic tests may improve early HCC detection and dynamic surveillance for individuals with hepatic cirrhosis. Thus, miR-192 has the potential to augment AFP and improve diagnostic accuracy and prognosis in clinical settings.

Conclusion

We reached the conclusion that cirrhosis of the liver is difficult to diagnose early owing to unreliable biomarkers, thus highlighting the need for new early diagnostic biomarkers. High levels of serum miRNA-192 could predict decompensated cirrhotic liver disease with high sensitivity and specificity. Circulating levels of miRNA also had potential for early detection of hepatocellular carcinoma.

Limitations of the study

The limited sample size limits the description of the correlations. Our observations need to be validated in higher populations. Sources and routes underlying the generation of serum miRNAs remain unresolved. Finally, additional research studies are required to investigate the molecular mechanisms of miRNA-192 and ascertain their possible therapy targets for HCC and cirrhosis of the liver.

Author contributions

Research conception: ASS, AAI, AHH and SSG; Methodology ASS, HIS, AAI, AHH and SSG; Experiments: MAA‏, AME, ASS, HIS, AAI, AHH, JZA, AAD and SSG; Investigation, MAA‏, AME, ASS, HIS, AAI, AHH, JZA, AAD and SSG;Data curation, JZA and SSG; Data interpretation and manuscript writing: MAA‏, AME, ASS, HIS, AAI, AHH, JZA, AAD and SSG; Work revision and ultimate approval: MAA, HIS, and SSG.

Funding

This study was funded by Prince Sattam Bin Abdulaziz University, project number (PSAU/2023/R/1444).

Data availability

all the datasets analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All experimental protocols were approved by the Faculty of Medicine, Beni-Sueif University research ethical committee (REC) with the number: FMBSUREC/06062021/Huzien.

Methodology

All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects.

Consent for publication

All authors grant permission to reproduce and distribute this work.

Competing interests

The authors declare no competing interests.