Circulating lncRNAs HOTTIP and HOTAIR as Potential Biomarkers in Crigler-Najjar Syndrome: A Preliminary Report from Shiraz Liver Transplant Research Center

Nasrin Motazedian ^1,†, Masoud Mokhtari ^1,2,†, Negar Azarpira ¹, Kimia Falamarzi ², Mohsen Mohammadi ^1,2, Seyed Mohsen Dehghani ¹, Sarina Pourjafar ^1,2, Maryam Ataollahi ¹, Elaheh Esfandiari ¹, Mahdokht Hossein-Aghdaie ¹, Seyed Ali Malekhosseini ^1,3, Tayebeh Kazemi ⁴, Mahintaj Dara ^5,*

1. Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

2. Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran

3. Abu-Ali Sina Organ Transplant Center, Shiraz University of Medical Sciences, Shiraz, Iran

4. Otolaryngology Research Center, Department of Otolaryngology, Shiraz University of Medical Sciences, Shiraz, Iran

5. Stem Cells Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

† These authors contributed equally to this work.

* Correspondence: Mahintaj Dara

Academic Editor: Rolf Teschke

Special Issue: Diagnostic Requirements Including Algorithms and Biomarkers in Liver Transplantation

Received: May 05, 2024 | Accepted: May 27, 2024 | Published: June 04, 2024

OBM Transplantation 2024, Volume 8, Issue 2, doi:10.21926/obm.transplant.2402217

Recommended citation: Motazedian N, Mokhtari M, Azarpira N, Falamarzi K, Mohammadi M, Dehghani SM, Pourjafar S, Ataollahi M, Esfandiari E, Hossein-Aghdaie M, Ali Malekhosseini S, Kazemi T, Dara M. Circulating lncRNAs HOTTIP and HOTAIR as Potential Biomarkers in Crigler-Najjar Syndrome: A Preliminary Report from Shiraz Liver Transplant Research Center. OBM Transplantation 2024; 8(2): 217; doi:10.21926/obm.transplant.2402217.

© 2024 by the authors. This is an open access article distributed under the conditions of the Creative Commons by Attribution License, which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is correctly cited.

1. Introduction

Crigler-Najjar syndrome (CNS) is a rare autosomal recessive disorder of bilirubin metabolism with a prevalence of 1 in a million live births worldwide, affecting both genders equally [1]. Impaired function of uridine 5′-diphosphate glucuronyltransferase (UGT1A1), an intracellular enzyme primarily expressed in hepatocytes responsible for bilirubin glucuronidation activity, results in neurotoxic unconjugated bilirubin (UCB) accumulation [1,2].

Crigler-Najjar syndrome has two distinct forms; type 1 is the most severe form due to the complete absence of UGT1A1 enzyme activity [1,3]. Patients with Crigler-Najjar syndrome type 1 have total serum bilirubin levels ranging from 20 mg/dl up to 50 mg/dl and severe jaundice. If left untreated, it can lead to severe neurological damage, including kernicterus, and eventually death [3,4]. Patients with Crigler-Najjar syndrome type 2 have residual UGT1A1 enzymatic activity, which is adequate for maintaining UCB concentration below the risk of developing severe neurologic impairment (these patients have total serum bilirubin levels of 3.5-20 mg/dl) [1,4]. The diagnosis of Crigler-Najjar syndrome is based on thorough clinical evaluation, laboratory findings, and molecular genetic study [5]. Intensive phototherapy, plasmapheresis, and pharmacological treatment are among the therapeutic methods available for Crigler-Najjar syndrome patients; however, these treatment options have limited efficacy and significant impacts on patients’ quality of life [4,5]. Currently, liver transplantation is the only effective treatment for Crigler-Najjar syndrome patients that restores UGT1A1 activity, reduces serum bilirubin levels, and significantly increases the 5-year survival rate of Crigler-Najjar syndrome patients. Yet, it has severe complications and shortcomings [4]. Therefore, understanding Crigler-Najjar syndrome molecular mechanisms is essential to identify new diagnostic and therapeutic targets.

Long non-coding RNAs (lncRNAs) are a subset of non-coding RNAs longer than 200 nucleotides [6]. They play significant roles in homeostatic and physiological functions such as cell cycle regulation, cell differentiation, and organogenesis by regulating transcription, translation, and chromatin modification (Figure 1) [7,8,9].

Figure 1 LncRNA function.

HOXA transcript at the distal tip (HOTTIP) is a lncRNA involved in the HOX gene network, leading to epigenetic changes and promoting tumorigenesis [9]. Additionally, HOTTIP is involved in developmental processes [10]. Several studies have identified HOTTIP as an oncogenic lncRNA and a non-invasive biomarker for various human malignancies, including colorectal, pancreatic, and gastric cancer, as well as renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC) [7,10].

The lncRNA HOX transcript antisense RNA (HOTAIR) contributes to normal mammalian development, including skin, lumbosacral, urogenital system development, and osteogenesis [11]. HOTAIR plays a crucial role in initiating and progressing numerous human cancers by promoting cellular proliferation, migration, and invasion while inhibiting cancer cell apoptosis and regulating immune signaling and inflammation [11,12]. Previous studies have explored the role of lncRNA HOTAIR in various cancers, such as breast, pancreatic, gastric, and non-small cell lung cancers, as well as cholangiocarcinoma and HCC, suggesting HOTAIR as a potential diagnostic and therapeutic target for these malignancies [12,13,14].

As lncRNAs can be detected in body fluids, recent studies have suggested that they could serve as a promising non-invasive biomarker for early diagnosing diseases like cancer [15].

The purpose of this study is to assess the expression of lncRNA HOTTIP and HOTAIR in the serum of Crigler-Najjar syndrome patients and compare the findings with those of healthy individuals to create a non-invasive diagnostic tool for this rare yet challenging disease.

2. Materials and Methods

2.1 Participants and Ethics Statement

This cross-sectional study involved 6 Crigler-Najjar syndrome patients and 26 healthy individuals. The patients were from the Shiraz Pediatric Liver Cirrhosis Cohort Study (SPLCCS) (IR.SUMS.REC.1398.142) [16], and their clinical and laboratory data were obtained from the pediatric liver cirrhosis registry (IR.SUMS.REC.1399.530). Blood samples were collected and stored at -80°C in the Shiraz Transplant Research Center biobank. The control group was comprised of patients admitted to surgery wards for minor procedures, such as tonsillectomy, with no history of liver disorders. Written informed consent was obtained from each individual's parents before they were enrolled in the study. The Ethics Committee of Shiraz University of Medical Sciences approved the study protocols (IR.SUMS.MED.REC.1401.457).

2.2 Circulating lncRNAs Extraction and Quantitative Real-Time Polymerase Chain Reaction

lncRNA was extracted from the serum samples of Crigler-Najjar syndrome patients and the control group using RNX-Plus (Cinnagen, Iran) following the manufacturer’s instructions. Complementary DNA was synthesized by the RB-lncRNA Synthesis kit (RNA biotechnology company, Iran) with specific stem-loop primers using 300 ng total RNA in a 20-μL total reaction. The reaction included 100 pMol stem-loop primer for lncRNA, 10 mM dNTP, 100 pMol RT Primer, 5× Reverse Transcriptase Buffer, and M-MLV reverse transcriptase enzyme. It was conducted in a thermal cycler machine under the following conditions: 60°C for 10 minutes, 55°C for 45 minutes, 72°C for 20 minutes, and 12°C for 10 minutes. Real-time PCR was carried out using the Step-One ABI applied Biosystem (Life Technologies). U6 snRNA served as a control for the analysis of mi expression. Reactions were performed following these parameters: 95°C for 15 minutes, 95°C for 15 seconds, and 62°C for 60 seconds for 40 cycles. Each sample underwent three measurements for subsequent analysis, and the mean cyclic threshold (Ct) values were utilized. The Livak (2−ΔΔCt) method was used to determine the samples' lncRNA HOTAIR and HOTTIP expression.

2.3 Statistical Analysis

Data normality was assessed using the Shapiro-Wilk test, skewness, and kurtosis indices. Average data were expressed as mean ± standard deviation. Non-normal variables were presented as the median and interquartile range (IQR) (25 and 75 percentile) with numbers (%) for categorical variables. The Mann-Whitney and Chi-square tests were employed to compare the two groups. Furthermore, the relationship between lncRNAs’ expression and laboratory and baseline data, as well as gender in the cases, was analyzed using Spearman’s and Mann-Whitney tests. The receiver operating characteristic (ROC) curve for each lncRNA was plotted, and sensitivity, specificity, and area under the curve (AUC) were determined. The data was analyzed using SPSS software (Version 21, SPSS Inc., Chicago, United States). A p-value < 0.05 was considered statistically significant.

3. Results

3.1 Clinical and Demographic Characteristics of the Participants

A total of 6 Crigler-Najjar syndrome patients and 26 healthy control subjects participated in this cross-sectional study. Their baseline demographic data are presented in Table 1. 50% of Crigler-Najjar syndrome patients were female with a median age of 2.5 years. In contrast, the median age of the control group was 6 years, with 38.5% being female. All Crigler-Najjar syndrome cases' parents were relatives, and 50% of cases had a family history of liver diseases. One out of six patients underwent liver transplantation, and no deaths were reported during the study. Crigler-Najjar syndrome patients exhibited significantly higher levels of total and direct bilirubin and liver enzymes (AST, ALT, ALKP) compared to the control group. The participants' laboratory findings are detailed in Table 2.

Table 1 Demographic features of case and control groups.

Table 2 Laboratory findings of case and control groups.

3.2 Expression of HOTTIP and HOTAIR lncRNAs in CNS Patients

This research examined the serum levels of HOTTIP and HOTAIR lncRNAs in both the case and control groups. The findings indicated that lncRNA HOTTIP was reduced in Crigler-Najjar syndrome patients (mean fold change of 0.2334) compared to healthy individuals (mean fold change of 0.8895). However, the difference was not statistically significant (p = 0.09). Similarly, the expression of lncRNA HOTAIR was lower in Crigler-Najjar syndrome cases (mean fold change of 0.3492) than in healthy controls (mean fold change of 0.9016), but this disparity was not statistically significant (p = 0.24). These results are illustrated in Figure 2.

Figure 2 LncRNA HOTTIP and HOTAIR expression levels in CNS patients and healthy subjects.

3.3 ROC Curve

Receiver operating curves [17] were utilized to assess the diagnostic accuracy of HOTAIR and HOTTIP lncRNAs in Crigler-Najjar syndrome patients (Figure 3). For HOTAIR, to distinguish between Crigler-Najjar syndrome and the healthy group, an expression level of 0.266 was identified as the cut-off value, with a sensitivity of 83.3% and specificity of 100% (area under the curve (AUC) of 0.867 and p = 0.008). Regarding HOTTIP, to differentiate between Crigler-Najjar syndrome cases and the control group, a plasma level of 0.202 was determined as the cut-off value, with a sensitivity of 83.3% and specificity of 100% (AUC of 0.867 and p = 0.008).

Figure 3 ROC curves for HOTAIR and HOTTIP lncRNAs.

3.4 Relationship between lncRNA HOTAIR and HOTTIP Expression and Baseline and Laboratory Data of CNS Patients

Furthermore, we examined the association between lncRNA HOTAIR and HOTTIP expression levels and the baseline and laboratory findings of Crigler-Najjar syndrome cases. Our findings indicated no link between HOTAIR and HOTTIP expression and patients’ demographic data, such as gender, parental relationship, and history of liver transplantation. Additionally, our results showed no correlation between serum levels of HOTAIR and HOTTIP and laboratory findings, including liver enzymes, total, and direct bilirubin among Crigler-Najjar syndrome cases. Nevertheless, we observed a significant inverse correlation between HOTAIR levels and serum albumin (r = -0.928, p = 0.008) and a direct correlation with platelet counts (r = 0.943, p = 0.005). The detailed data are presented in Table 3 and Table 4.

Table 3 Relationship between HOTAIR and HOTTIP expression and CNS patients’ demographic data.

Table 4 Correlation between HOTAIR and HOTTIP levels and laboratory findings of Crigler-Najjar syndrome cases.

4. Discussion

Crigler-Najjar syndrome (CNS) is an infrequent condition caused by a complete or partial loss of UGT1A1 activity leading to unconjugated hyperbilirubinemia [18]. Despite current standard treatments like phototherapy and liver transplantation, Crigler-Najjar syndrome poses a significant burden and dramatically impacts patients' quality of life. Therefore, innovative diagnostic and therapeutic approaches are essential [2,18]. Long non-coding RNAs (lncRNAs) are increasingly recognized for their regulatory roles in various biological processes and diseases such as cancer [11,19]. Hence, we conducted this research to assess the levels of lncRNAs HOTAIR and HOTTIP in individuals with Crigler-Najjar syndrome compared to healthy controls. Our findings revealed a decrease in the expression of lncRNAs HOTAIR and HOTTIP in the plasma of Crigler-Najjar syndrome patients compared to the control group. However, this decrease did not reach statistical significance. Previous studies have indicated that HOTTIP is one of the most significantly upregulated lncRNAs in hepatocellular carcinoma (HCC) and is associated with poor overall survival [7,17]. Suppression of HOTTIP has been linked to the inhibition of HCC cell proliferation, tumor formation, reduced cell migration, and decreased pulmonary metastasis [7]. Kim et al. have suggested that HOTTIP, as a serum small extracellular vesicle-derived lncRNA, shows promising potential as a biomarker for early HCC diagnosis [20]. Additionally, individuals with higher levels of HOTTIP expression in tumors face an increased risk of post-transplantation recurrence [9].

LncRNA HOTAIR was significantly overexpressed in tumor tissue and peripheral blood of HCC patients, and patients with lower levels of lncRNA HOTAIR had more prolonged overall survival and progression-free survival [14]. HOTAIR enhances human liver cancer stem cell growth and knockdown of lncRNA HOTAIR induces apoptosis and prevents HCC cell proliferation, epithelial-mesenchymal transition, and migration [21,22]. Additionally, overexpression of HOTAIR was significantly associated with tumor size, TNM stage, and postoperative recurrence in patients with cholangiocarcinoma (CCA), and its knockdown led to decreased invasion and increased apoptosis of CCA cells [13]. Tang and colleagues demonstrated that HOTAIR modulates autophagy through the miR-20b-5p/ATG7 axis in hepatic ischemia-reperfusion injury (IRI) [23].

A study conducted by Roshdy et al. on patients with chronic liver disease induced by hepatitis C virus (HCV) with or without cirrhosis and HCC revealed that lncRNA HOTAIR and HOTTIP were significantly upregulated in diseased groups and were able to distinguish between cirrhotic and HCC cases compared to healthy controls. Furthermore, they reported a significant inverse correlation of HOTAIR and HOTTIP with albumin (r = -0.304, p = 0.007 and r = -0.392, p = 0.001 respectively) [15]. Our results also demonstrated that HOTAIR had an inverse correlation with patients' age but no association with patients' gender. A recent meta-analysis indicated that HOTAIR expression had no association with the age and gender of patients with HCC but showed a significant relation with tumor stage and overall survival [24]. These discrepancies in results might be due to the limited sample size and different liver pathologies evaluated in these experiments. Additionally, several studies have revealed the contribution of HOTAIR and HOTTIP to liver fibrosis [19,25]. HOTAIR might enhance liver fibrosis by regulating DNMT1, MEG3, and the p53 pathway in hepatic stellate cells (HSC) and induce the expression of fibrosis-related genes such as matrix metalloproteinase 2 (MMP2) and MMP9 [19,26]. Previous investigations have shown that HOTTIP promoted mouse hepatic fibrosis by activating HSC as a competing endogenous RNA (ceRNA) for miR-148a and miR-150 [27,28,29].

The limitation of this study is the small sample size of patients, although this limitation is inherent when studying such a rare disease. This study is preliminary. Further studies in larger populations are needed to confirm or rule out the significant role of HOTTIP and HOTAIR as potential biomarkers in Crigler-Najjar Syndrome.

5. Conclusion

In conclusion, the results of this study showed a decrease in lncRNAs HOTAIR and HOTTIP in the serum of patients with Crigler-Najjar syndrome. However, it did not reach statistical significance. The absence of an increase in the level of ncRNA in patients with CNS may confirm that no oncogenic process is expected during the evolution of the CNS. This is the first study to investigate the expression of lncRNAs HOTAIR and HOTTIP in Crigler-Najjar syndrome patients compared to healthy controls. As mentioned earlier, HOTAIR and HOTTIP are implicated in various liver diseases, such as HCC, hepatic ischemia-reperfusion injury (IRI), and liver fibrosis. They have been proposed as potential diagnostic and therapeutic targets. Therefore, further research with a larger population is necessary to elucidate their precise roles in the pathology of Crigler-Najjar syndrome and assess the potential of these two lncRNAs as non-invasive biomarkers for diagnosing and treating Crigler-Najjar syndrome.

Acknowledgments

The authors wish to thank Ms. Firooze. Dara, Language editor, for her valuable comments in editing this manuscript.

Author Contributions

All of the authors contributed substantially to the concept and design of the study. Material preparation, data collection, and analysis were performed by N.M., M.M., N.A., K.F., M. Mo, S.P., M.A., E.S., M.H., S.A.M., T.K., and M.D. The primary draft of the manuscript was written by N.M. All authors read and approved the final manuscript.

Funding

This work is supported by Shiraz University of Medical science.

Competing Interests

The authors declare that they have no conflict of interest.

Data Availability Statement

All data are available in manuscript.