Crosstalk Between Long Non-coding RNA MALAT1, miRNA-181a, and IL-17 in Cirrhotic Patients and Their Possible Correlation SIRT1/NF-Ƙβ Axis

Abstract

In liver diseases, the interplay of different noncoding RNA expressions, and inflammatory biomarkers show high context dependencies. Interrelations between these noncoding RNA and inflammatory biomarkers paved the way for the diagnosis of various diseases. Here, we analyzed the expression of MALAT1, miR-181a in liver cirrhosis and a panel of pro-inflammatory cytokines (IL-17, SIRT1 and NF-Ƙβ p65). The association between all measured parameters was monitored. Fifty healthy volunteers with normal liver function, hepatic ultrasonography, and negative results for HCV and HBV participated in our study as a healthy control group. In addition, hundred and fifty patients with liver cirrhosis were included. Compared with healthy controls, miR-181a expression was significantly decreased (p < 0.01), while MALAT1 expression was significantly elevated (p < 0.01) in patients with liver cirrhosis. IL-17 and NF-ƘB p65 were significantly increased (p < 0.001), while SIRT1 was significantly decreased (p < 0.001) in cirrhotic patients compared to controls. Serum expression of SIRT1 significantly positively correlated with miR-181a and negatively associated with MALAT-1, NF-Ƙβ p65, and IL-17expression levels. Our results pointed to alterations in the expression levels of miR-181a, and MALAT1 could serve as biomarkers in cirrhotic patients. Reduction of IL-17 and NF-Ƙβ p65 in combination with an elevation of SIRT-1 might refer to the dual effects of miR-181a and MALAT1 in controlling inflammation in liver cirrhosis.

Introduction

Liver cirrhosis has a poor prognosis; thus, a precise evaluation of fibrosis is essential, as liver fibrosis and hepatocellular carcinoma (HCC) progression are closely linked [1]. The correct staging of liver cirrhosis is critical for prioritizing treatment for HCC patients, especially for clinical decision-making in management and therapy, it is therefore critical to distinguish individuals with advanced cirrhosis from those with no or mild cirrhosis.

Biomarker assessments, including dysregulated noncoding RNA (ncRNA), are more objective and reproducible compared to imaging-based modalities and are considered adjunct tools [2]. Several abnormal non-coding RNAs (ncRNAs) have been identified in liver diseases, targeting genes that eventually contribute to tumorigenesis, proliferation, apoptosis, DNA repair, invasion, and metastasis. These findings have led to exploring miRNAs as potential biomarkers for early diagnosis and prediction of prognosis of numerous diseases and to be effective therapeutic targets, given their abundance in the plasma and easy detectability [3, 4].

MicroRNAs are a group of noncoding RNAs that are involved in many cellular processes [5, 6] that play a crucial role in hepatic fibrogenesis beyond activating hepatic stellate cells (HSCs), and depositing extracellular matrix (ECM); considering their roles microRNAs may even be used as indicators of disease progression [7]. The brain, liver, and lymphoid organs express miR-181 family members most frequently [8]. miR-181a and b are the most frequent in the etiopathogenesis of lymphoid, brain, and liver malignancies [8, 9]. miR-181a plays a crucial role in regulating the expression of both anti-inflammatory and pro-inflammatory cytokines [10]. Additionally, it demonstrates highly effective immune-modulatory functions [11]. Due to its capability to control the expression of genes involved in mitochondrial function, it holds great promise as a biomarker for diagnosing and predicting various metabolic disorders.

LncRNAs are a second family of non-coding RNAs that regulate gene expression by interfering with mRNA expression or competing at genomic loci for transcription factors [12]. They modulate multiple roles in the degradation of mRNA in a contextual manner. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) gene is 8.7 kb long and found on chromosome 11q13; which regulates the expression of many distinct genes by affecting various stages of their transcription and elongation [13]. It was first discovered in the early stages of non-small cell lung cancer, from which its name derived, and its dysregulation has associated with several human cancers, including breast, colorectal, and hepatocellular carcinoma [14].

Silent information regulator 1 (SIRT1) is a protein that regulates cellular processes like aging, inflammation, and stress resistance [15]. In addition, activation of SIRT1 has been found to reduce liver fibrosis by inhibiting the activation of hepatic stellate cells, which are responsible for liver fibrosis. SIRT1 activation can suppress the production of extracellular matrix proteins and slow down the progression of liver fibrosis. Overall, SIRT1 plays a protective role in liver health, and its activation has shown potential as a therapeutic target for various liver diseases in preclinical studies. However, further research is needed to understand the molecular mechanisms underlying the effects of SIRT1 on liver diseases.

IL-17 is the most important cytokine representing Th17 and can induce pro-inflammatory cytokines and chemokines and cause tissue cell infiltration and tissue destruction [16]. The biological actions of IL-17 may stimulate the development of neutrophils and macrophages at the site of inflammation, escalating liver injury, which plays a role in pathogenesis and the body’s response to many diseases. Moreover, IL-17 plays a crucial role in hepatocytes by facilitating systemic inflammation and attracting inflammatory cells to the liver. It is also associated with the development of fibrosis and insulin resistance [17]. Elevated levels of IL-17 have been linked to the progression of NAFLD to steatohepatitis, cirrhosis, and even hepatocellular carcinoma [17].

To our knowledge, miR-181a and MALAT-1 expression levels were dysregulated in cirrhotic patients with varying grades. However, their correlation with IL-17 and their crosstalk with the SIRT1/NF-κβ axis were not explored. Thus, we assessed whether circulating miR-181a / MALAT1 and IL-17 could be used as biomarkers for monitoring the grade of liver cirrhosis. Moreover, we examined the correlation between the cross-linked SIRT1/NF-Ƙβ axis and the grade of liver cirrhosis.

Materials and Methods

Fifty healthy volunteers with normal liver function, hepatic ultrasonography, and negative results for HCV and HBV participated in our study as a healthy control group. Healthy volunteers, matched by age and sex and with no history of liver cirrhosis or inflammatory disease, were selected for the study. In addition, hundred and fifty patients with hepatic diseases were included and classified according to the Child–Pugh classification [18, 19] into the Child A group (50 patients), Child B group (40 patients), and Child C group (60 patients).

Every patient had a clinical evaluation as well as laboratory tests. Based on imaging investigations, including abdominal ultrasonography with Doppler and upper endoscopy for the functional assessment of decompensated cases, patients are diagnosed as having liver cirrhosis. The study excluded patients with active schistosomiasis, hepatitis virus, cardiovascular illness, thyroid dysfunction, diabetes mellitus (DM), and alcohol consumption. The study proposal was complied with by the Ethics Committee of the Faculty of Medicine, Beni-Suef University (FMBSUREC/07062022), in accordance with the Declaration of Helsinki and best clinical practice recommendations.

Following an overnight fast, each participant had blood samples (10 mL) taken. Two aliquots of blood samples were taken. The serum for all routine and serological assays was separated from the first aliquot using centrifugation, and kept at − 80 °C.

Transaminase enzymes (ALT and AST) and total bilirubin levels were determined in serum samples using dedicated kits from Roche Diagnostics GmbH (Penzberg, Germany), according to Huang et al. [20] and Landis et al. [21], respectively. Interleukin 17 (IL-17) was assayed using an ELISA kit from Cloud-Clone Corp.

For RNA isolation, serum samples were mixed with Direct-zol RNA Miniprep Plus (Cat# R2072, ZYMO RESEARCH Corp., USA), then following the manufacturer’s recommendations, reverse transcription using the SuperScript IV One-Step RT-PCR kit (Cat# 12,594,100, Thermo Fisher Scientific, Waltham, MA, USA), followed by real-time PCR (Step One Applied Biosystems, USA). Each quantitative PCR reaction was tested twice for accuracy. The PCR data sheet included cycle threshold (Ct) values of the assessed genes has-miRNA-181a versus the corresponding housekeeping gene, RUN U6B. MALAT1 versus the corresponding housekeeping gene (GAPDH). miRNA-181a and MALAT1 expression levels were calculated using the 2 − ΔΔCt formula, According to Livak and Schmittgen [22].

NF-Ƙβ p65 and SIRT1 Expression analysis

From Bio-Rad Inc., the Protein Extraction Kit ReadyPrepTM. (catalog #163–2086) added to each sample of lysed cells from each group. Protein analysis was performed for each sample using the Bradford assay (Bio Basic Inc., Canada), followed by polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, BioRad Trans-Blot Turbo was used to transfer protein bands from the gel to the membrane. The primary antibodies for SIRT1 and NF-Ƙβ were incubated on the membrane for an entire night (Rockland, Philadelphia, USA). Then, a secondary antibody with HRP-conjugated was applied to the membrane. Chemiluminescent substrate (Catalog#170–5060)was then incubated and applied to the blots. The Harlow and Lane [22] method of protein normalization was used to detect the target protein on the ChemiDoc MP imager in comparison to the control sample beta-actin (housekeeping protein).

Statistical Analysis

Comparisons between multiple groups were done using one-way ANOVA followed by Tukey’s post-hoc for pairwise comparisons. p < 0.05 implies a significant value.

Results

This cross-sectional controlled study conducted one hundred and fifty cirrhotic patients with different Child–Pugh classes (A, B, and C) who were divided into three groups. Group I included 50 patients (33 males and 27 females; mean age, 42.13 ± 3.98). Group II included 40 patients, including 25 males and 15 females (mean age, 40.73 ± 4.97), while Group III also included 60 patients, including 35 males and 25 females (mean age, 42.83 ± 5.20). Fifty patients with healthy physical and laboratory examinations were enrolled as controls, including 24 males and 26 females (mean age, 39.33 ± 4.18). The ages and gender composition between the groups did not differ statistically (p > 0.05). Compared to the healthy control participant, patients in group I, II, and III had significantly higher elevation in ALT, AST, total bilirubin, and INR as shown in Table 1.

Table 1.

The demographic data age, gender, and liver function profile among study participants

Variables	Healthy control (50)	GI(50)	GII(40)	GIII(60)
Age
(mean ± SD)	39.33 ± 4.18a	42.13 ± 3.98ab	40.73 ± 4.97ab	42.83 ± 5.20b
Gender
Male (n,%)	24 (48%)	33 (66%)	25 (62.5%)	35 (58.33%)
Female (n, %)	26 (52%)	27 (44%)	15 (37.5%)	25 (41.67%)
ALT	14.73 ± 0.64a	31.96 ± 0.70b	51.33 ± 0.70c	68.78 ± 0.83d
AST	15.73 ± 0.92a	31.13 ± 0.80b	53.08 ± 0.74c	69.90 ± 0.60d
Total bilirubin	0.49 ± 0.09a	0.85 ± 0.02b	1.75 ± 0.07c	2.81 ± 0.04d
Albumin	4.60 ± 0.05d	3.95 ± 0.03c	3.21 ± 0.05b	2.53 ± 0.07a
INR	1.00 ± .001a	1.24 ± 0.01b	1.88 ± 0.02c	2.01 ± 0.01d

ALT, AST, total bilirubin, and albumin levels are represented as Mean ± SEM. GI (child A patients): GII (child B patients); and GIII (child C patients). According to the Tukey’s multiple range test, the different letters indicate statistical significance (p < 0.05). Values which share the same superscript symbol are not significantly (p > 0.05) different

ALT, alanine transaminase; AST, aspartate transaminase; INR, international normalized ratio

The expression of IL-17, and MALAT-1 was investigated, and the results are shown in Table 2. The expression of NF-κβ p65, and SIRT-1 was investigated, and the results are shown in Table 2 and Fig. 1. Comparison of serum IL-17, MALAT-1, and NF-Ƙβ p65 expression levels between different studied groups displayed an increasing tendency towards statistically significant fold elevation in the serum of cirrhotic patients in groups I, II, and III in comparison to healthy control participants. Moreover, cirrhotic patients in group III showed significant upregulation of IL-17, MALAT-1, and NF-κβ compared with cirrhotic patients in groups I and II. Additionally, serum expression levels of miR-181a, and SIRT-1 in cirrhotic patients in groups I, II, and III displayed a significant decline compared to healthy control participants. Table 2 demonstrates that miR-181a and SIRT-1 were considerably downregulated in group III cirrhotic patients in comparison to groups I and II.

Table 2.

Changes in the levels of IL-17, and relative expression of MALAT1 and miR-181a among study participants

Variables	Healthy control (50)	GI(50)	GII(40)	GIII(60)
IL-17 (pg/mL)	36.29 ± 1.39a	143.65 ± 2.20b	293.14 ± 2.46c	394.76 ± 2.06d
MALAT1	0.98 ± 0.01a	3.06 ± 0.17b	4.98 ± 0.34c	8.33 ± 0.52d
MiR-181a	1.03 ± 0.03d	0.43 ± 0.01c	0.26 ± 0.01b	0.08 ± 0.01a
NF-Ƙβ	0.97 ± 0.01a	2.17 ± 0.04b	3.12 ± 0.05c	4.26 ± 0.05d
SIRT-1	0.97 ± 0.01d	0.73 ± 0.01c	0.49 ± 0.01b	0.20 ± 0.01a

All data are represented as Mean ± SEM. GI (child A patients): GII (child B patients); and GIII (child C patients). According to the Tukey’s multiple range test, the different letters indicate statistical significance (p < 0.05). Values which share the same superscript symbol are not significantly (p > 0.05) different

IL-17, Interleukin 17; MALAT1, metastasis-associated lung adenocarcinoma transcript-1; miR-181a, microRNA-181a; NF-Ƙβ, nuclear factor-kabba beta; SIRT-1, silent information regulator-1; SEM, standard error mean

Fig. 1.

SIRT1 and NF-Ƙβ expression levels among study participants

Correlation Analysis Between IL-17; MALAT1; miR-181a; NF-Ƙβ p65, and SIRT1 Among Study Participants

Serum expression of IL-17 was positively correlated with serum NF-Ƙβ, whereas MALAT-1 expression in cirrhotic patients was significantly negatively correlated with serum expression of SIRT1 and miR-181a, as shown in Table 3. NF-κβ expression levels were negatively associated with serum expression of miR-181a (p < 0.01) and SIRT1 (p < 0.01) and positively correlated with MALAT-1 (p < 0.01) and IL-17 (p < 0.01). Moreover, serum expression of SIRT1 significantly positively correlated with miR-181a and negatively associated with MALAT-1, NF-Ƙβ p65, and IL-17expression levels, as shown in Table 3. Moreover, our results revealed a negative correlation between serum expression levels of MALAT1 and miR-181a (p < 0.01) (Table 3).

Table 3.

The correlation between IL-17; MALAT1; miR-181a; NF-Ƙβ, and SIRT1 among study participants

Parameter	IL-17		SIRT-1		NF-Ƙβ		MALAT-1
	R	p	R	p	R	P	R	p
miR-181a	− 0.893**	< 0.01	0.867**	< 0.01	− 0.895**	< 0.01	− 0.834**	< 0.01
MALAT-1	0.948**	< 0.01	− 0.929**	< 0.01	0.931**	< 0.01	–	–
NF-Ƙβ	0.964**	< 0.01	− 0.941**	< 0.01	–	–	0.931**	< 0.01
SIRT-1	− 0.953**	< 0.01	–	–	− 0.941**	< 0.01	− 0.749**	< 0.01
IL-17	–	–	− 0.953**	< 0.01	0.964**	< 0.01	0.769**	< 0.01

**Significant linear correlation at p < 0.01 (2-tailed)

Diagnostic Performance of Serum miR-181a, and MALAT-1

ROC analysis revealed that differentially expressed serum MALAT-1 and miR-181a could significantly discriminate cirrhotic patients from healthy participants with AUC values of 0.990 and 1.00, respectively, and a sensitivity of 97.5% and 100%, and specificity of 100%, at a cutoff value of 1.225 and 0.715, respectively (Fig. 2a, b). Notably, the diagnostic accuracy of serum mir-181a was most significant than that of serum MALAT-1 in discriminating patients with cirrhosis. Serum MALAT-1, and miR-181a levels could also differentiate patients with grade III from those with grade I and II, with an area under the curve of 0.840 and 0.996, respectively. At the cut-off values of 8.375, 0.185, with a sensitivity of 65%, 100%, and specificity of 100% and 93.75%, respectively, as shown in Fig. 2c and d.

Fig. 2.

ROC analysis regarding a, b MALAT1, and miR-181a discrimination between cirrhotic patients and healthy participants. c, d MALAT1, and miR-181a discrimination between grade III from those with grade I and II

Discussion

Liver cirrhosis (LC) is a major global health concern among the top 20 causes of disability and fatalities. 1.6% of the global burden is attributable to LC, which is among the top 20 causes of disability and fatalities [23]. Egypt had the world’s highest rate of age-standardized death from cirrhosis in 2017, with 103.3 deaths per 100,000. However, the rate decreased by 22.4% due to the rapid drop in the HCV death rate [24]. Therefore, effective bioassays for the diagnosis, prognosis, and surveillance of the illness are required to track the development of liver cirrhosis and may alleviate its progression.

Imaging and liver biopsy are the two primary types of current approaches for LC diagnosis. For clinical presentations that are not immediately apparent, the diagnostic and prognostic values of CT, MRI, ultrasonography, and liver biopsy are constrained [25]. This opens up new avenues for investigating and examining new candidate biomarkers that will satisfy the steady requirements for a perfect surrogate fibrosis marker in LC.

In liver diseases, the interplay of different noncoding RNA expressions, and inflammatory biomarkers show high context dependencies. Interrelations between these noncoding RNA and inflammatory biomarkers has paved the way for the diagnosis of cirrhosis owing to their expression specificity and constancy in body fluids. In our current study, we revealed whether circulation miR-181a / MALAT1 and IL-17 can be used as biomarkers to monitor the grade of liver cirrhosis and scrutinized their correlation with the cross-link SIRT1/NF-Ƙβ axis.

Regarding the laboratory data of our participants, statistically significant differences were noted in ALT, AST, INR, albumin, and total bilirubin levels between healthy and cirrhotic patients. These results are consistent with the findings of Ahmed et al. 2018 [26].

IL-17 plays a critical role in autoimmune diseases by promoting the secretion, release, and chemotaxis of various inflammatory factors to the site of inflammation and by synergizing with them to amplify their biological effects [27]. In line with the literature [28, 29], the current investigation found a substantial rise in serum IL-17 levels in cirrhotic patients compared to healthy participants, indicating that elevated cytokine IL-17 levels are related to crosstalk of hepatocyte inflammatory damage. It is easy to induce the differentiation and release of Th17 cells, which reach the liver through the blood circulation and mediate the development of an autoimmune response. Simultaneously, they secrete numerous inflammatory factors that promote liver inflammatory reactions.

Sirtuin-1 regulates the activity of numerous transcription factors crucial for immunological functions in mammals and acts as an epigenetic regulator [30]. In our investigation, Sirtuin-1 levels were considerably lower in the cirrhotic group than in the control group, which was consistent with the results of Wu et al. (2014), who proved that as liver disease progresses, sirtuin expression levels are downregulated [31]. Our findings suggest that serum levels of SIRT1 may indicate the severity of liver disease as there is a significant inverse association between the expression level of SIRT1 and the degree of cirrhosis as per the Child–Pugh classification.

Inflammatory mediators, which activate or are targeted by NF-κB, unequivocally contribute to the progression of chronic liver disease. The role of NF-κB in regulating the liver’s inflammatory signaling pathways is vital and indisputable. NF-κB is activated in almost all types of chronic liver disease, including alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD), viral hepatitis, and biliary liver disease, leaving no room for doubt. Furthermore, NF-κB regulates essential functions in hepatocytes, Kupffer cells, and hepatic stellate cells (HSCs). Finally, it is unquestionable that genetic inactivation of different components of the NFκB signaling pathway leads to liver damage, fibrosis, and carcinogenesis. Thus, it is an undeniable fact that NF-κB plays an essential role in liver homeostasis processes [32, 33]. In our study, we found significantly elevated levels of serum NF-κβ p65 in patients with liver cirrhosis compared with controls. Furthermore, our data revealed that the level of NF-ĸβ p65 increased gradually among cirrhotic patients as the Child–Pugh classification increased. This is in agreement with [34, 35], who reported that NF-κβ acts as a master regulator of inflammation and cell death in liver cirrhosis.

The aberrant expression of IL-17, SIRT-1, and NF-κβ p65 represents a stepping stone in the progression of liver cirrhosis. Previous studies have revealed a complex mechanism related to inflammation and fibrosis through the upregulation of the expression of inflammatory mediators and non-coding RNA [36]. NF-κB p65 modulates hepatic fibrogenesis predominantly by regulating hepatocyte injury, which is the primary trigger for fibrogenic responses in the liver. Inflammatory and chemotactic factors are secreted frequently by hepatocytes as NF-kβ p65 is activated, which enhances hepatic fibrosis and inflammation [35, 37].

The use of ncRNAs as serum biomarkers for inflammation and fibrosis has gained attention in recent years. Because of the incoherence of the findings and the inadequacy of the studies on the differential expression schemes of miR-181a on liver disorders, we aimed to explore the biochemical contribution of miR-181a expression in cirrhotic subjects and clarify their role in the early diagnosis of liver cirrhosis. Our findings demonstrated that miR-181a expression levels noticeably declined in the blood of patients with cirrhosis relative to healthy participants. Consistent with our results, Lima et al. 2021 [38] reported an extraordinary drop in miR-181a levels in the blood of patients with clinically substantial fibrosis. Gupta et al. found higher miR-181a expression in cirrhotic liver tissues, which may be related to enhanced fibrogenesis via TGF-β [39]. As we detected miR-181a in the blood but not in the liver tissue, these data do not dispute our findings.

Extensive research has highlighted the link between long non-coding RNAs (lncRNAs) and liver fibrosis development, suggesting that lncRNAs have potential value in the diagnosis, prognosis, and treatment of liver fibrosis [40]. lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was reported to be a negative regulator of sirtuin 1 [41]. Accordingly, the expression of serum MALAT1 was significantly higher in patients with liver cirrhosis than in healthy controls. Furthermore, serum MALAT1 expression gradually increased with the fibrosis stage, thus indicating that serum MALAT1 can help in the staging of liver fibrosis.

Further analysis showed that serum MALAT1 expression was negatively correlated with the inflammatory factors SIRT-1/ interleukin-17 (IL-17) and miR-181a. Serum MALAT1 expression positively correlates with NF-Ƙβ p65. According to our findings, MALAT1 may influence liver fibrosis and inflammation by reducing the release of anti-inflammatory mediators (such as SIRT-1 and IL-17), which in turn causes exacerbated inflammation. Additionally, via controlling downstream pathways (including the p38 MAPK/p65 NF-B signaling cascade), lncRNA MALAT1 alteration may harm liver tissue and cause tissue damage.

Collectively, our study provides evidence that serum levels of IL-17, miR-181a, and MALAT1 and their possible correlation SIRT1/NF-Ƙβ axis may have great clinical value as accurately promising candidate biomarkers in liver diseases screening, these biomarkers play a role in the pathogenesis of liver cirrhosis disease. Additionally, miR-181a and MALAT1 are promising therapeutic targets for liver fibrosis.

Certain limitations of our study are as follows: (i) the population of enrolled patients and controls was relatively small, which requires a larger sample size for further study to verify our results; (ii) our future work will cover the inclusion of a disease control group, which is crucial to ensure accurate analysis and conclusive results, and (iii) more investigations are required to determine the exact molecular mechanisms by which miR-181a and MALAT1 participate in liver fibrosis pathophysiology.

Funding

The authors declare that no funding for the research received.

Declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethical Approval

Our study was conducted in compliance with the Declaration of Helsinki, and the Research Ethical Committee, Faculty of Medicine, University of Beni-Suef, Egypt provided its approval (FMBSUREC/07062022).

Informed Consent

All study participants provided their informed consent permission for participation in this study.