Circular RNA hsa_circ_0001666 and Its Target Protein ETV4 AS Potential Biomarkers for Crohn's Disease

Abstract

Introduction

Circular RNAs are stable non-coding RNAs that regulate key biological processes. Hsa_circ_0001666 was shown to be involved in several cancers and in chronic inflammatory diseases. It is overexpressed in Crohn's Disease (CD) and is associated with cell migration, invasion, epithelial-mesenchymal transition (EMT), and fibrosis. Hsa_circ_0001666 acts as a sponge for multiple microRNAs (miRNAs). Some of these miRNAs regulate the expression of ETS variant transcription factor 4 (ETV4), a protein regulating critical genes implicated in inflammation, invasion, and EMT.

Objectives

To assess the plasma relative expression levels of hsa_circ_0001666 and the ETV4 protein levels to evaluate their contribution to CD development and the possibility of utilizing them as potential non-invasive biomarkers for diagnosis of CD and assessment of the disease activity.

Material and Methods

The present study included 25 patients with active CD, 25 patients with inactive CD, and 25 healthy subjects as controls. The analysis of the relative expression of hsa_circ_0001666 in plasma was performed using the real-time quantitative polymerase chain reaction (qPCR) method. The ETV4 concentration in plasma was measured using an enzyme-linked immunosorbent assay (ELISA).

Results

Plasma expression levels of hsa_circ_0001666 and ETV4 protein were significantly higher in active and inactive CD patients than the healthy controls, and in active CD patients than in patients with inactive disease. Furthermore, there was a significant positive correlation between hsa_circ_0001666 to ETV4 expression in the three studied groups.

Conclusion

Higher levels of hsa_circ_0001666 and ETV4 in CD patients suggest their role in disease development and activity, and their potential use as diagnostic biomarkers and therapeutic targets for CD management.

Keywords

Hsa_circ_0001666 - ETV4 - Crohn's disease - epithelial-mesenchymal transition - fibrosis

Introduction

Crohn's disease (CD) is a type of inflammatory bowel disease, which is a chronic, immunologically mediated inflammatory disease affecting any part of the gastrointestinal tract, mostly the terminal ileum and right colon, with extraintestinal complications. The disease is characterized by a relapsing and remitting transmural inflammation extending through the whole thickness of the intestinal wall, from the mucosa to the serosa.[1]

The incidence and prevalence of CD differ according to geographic regions, with the highest epidemiological burden in the Western developed world. In the last two decades, the prevalence has increased in different developing Middle East and North African countries, including Egypt, making it a global health problem.[2] It is more prominent in urban than in rural areas. CD has a high incidence between 20–30 years of age.[2]

Its etiology remains unclear, but it is widely passable that multiple factors involving genetic predisposition, infectious, immunological, environmental, and dietary factors interact in a complex mechanism.[3]

Circular RNAs (circRNAs) are a new class of non-coding RNAs that are produced by the back splicing of precursor mRNAs with covalently closed loop structures. Because of their unique structure, circRNAs are extremely stable and can be easily detected in human peripheral blood, plasma, and other biological fluids. Therefore, they could serve as non-invasive stable biomarkers in different disease states.[4]

These molecules have been shown to regulate key biological processes by sponging microRNA (miRNA), interacting with RNA-binding proteins (RBPs), and transcriptional and post-transcriptional modification of gene expression. A total of 218 circRNAs with various degrees of expression alterations were found when CD patients' colon tissues were analyzed using circRNA microarray technology. This suggests that circRNAs might be implicated in the development of CD.[5]

Homosapien circular RNA 0001666 (hsa_circ_0001666), a newly discovered circRNA, originated from the linear gene family with sequence similarity (FAM) 120B, is associated with activities like migration, invasion, epithelial-mesenchymal transition (EMT), and fibrosis. Increasing evidence has shown that hsa_circ_0001666 is involved in several cancers, including colorectal cancer, and in chronic inflammatory diseases. Lately, hsa_circ_0001666 was shown to be upregulated in the colon tissues of CD patients by microarray screening of CD marker genes. However, the precise regulatory mechanism of hsa_circ_0001666 is still unknown.[6]

E26 transformation-specific (ETS) translocation variant 4 (ETV4), also called polyoma enhancer activator 3 (PEA3), is a member of the PEA3 subfamily of ETS transcription factors, present throughout the body and is involved in many functions, including the regulation of cell growth, differentiation, control of cell cycle, proliferation, angiogenesis, migration, and apoptosis. ETV4 was found to regulate critical genes implicated in inflammation and invasion, such as IL-8,[7] cyclooxygenase-2 (COX-2),[8] and matrix metalloproteases (MMPs).[9] Moreover, it was shown that ETV4 can induce expression of EMT markers, such as N-cadherin, TWIST1, and ZEB1.[10]

It has been shown that hsa_circ_0001666 acts as a sponge for multiple microRNAs (miRNAs). Some of these miRNAs were shown to regulate the expression of the ETV4 protein. These data suggest that hsa_circ_0001666, by functioning as a miRNA sponge, may participate in the development of CD by positively regulating.[6] [11]

Therefore, the present work aims to assess the plasma expression levels of hsa_circ_0001666 and ETV4 protein to evaluate their role in the development of CD and the possibility of using them as potential non-invasive biomarkers for the diagnosis of CD and assessment of the disease activity.

Subjects and Methods

Subjects

The study was performed on 75 subjects, divided into three groups: 25 patients with active CD (Group IA), 25 patients with inactive CD (Group IB), and a healthy control group consisting of 25 sex- and age-matched healthy individuals (Group II). Patients were recruited from the Gastroenterology unit at the Internal Medicine Department in Alexandria Main University Hospital.

Exclusion criteria: indeterminate colitis, isolated upper gastrointestinal tract involvement, gastrointestinal malignancy, recent abdominal surgery in the last 3 months, autoimmune diseases, history of severe infectious diseases, concurrent gastrointestinal diseases, pregnancy and lactation, chronic renal and liver diseases, and refusal to be involved in the study.

Prior to enrollment in the study, each participant gave written informed consent, and the study was carried out in compliance with institutional procedures, adhering to the Declaration of Helsinki, and it was approved by the Ethics Review Board of Alexandria University, Faculty of Medicine, under number 0201932.

Methods

A comprehensive clinical history was taken, and a physical examination was performed on each patient. Routine laboratory tests included complete blood count (CBC), Erythrocyte sedimentation rate (ESR), quantitative C-reactive protein (CRP), serum albumin, liver enzymes (ALT and AST), renal function tests (urea and creatinine), and fecal calprotectin.

Ileocolonoscopy was done for all patients, and tissue specimens were taken for histopathological confirmation of diagnosis. Disease activity was assessed clinically by the Harvey-Bradshaw index (HBI),[12] and endoscopically by the Simple Endoscopic activity Score in Crohn's Disease (SES-CD).[13] Patients were categorized into inactive CD (HBI ≤ 4 and SES-CD ≤ 2) and active CD patients (HBI ≥ 5 and SES-CD ≥ 3)

Assessing the Relative Expression Level of hsa_circ_0001666 and ETV4 Protein Concentration in Plasma

Under extremely strict aseptic conditions, a venous blood sample was taken from the patients and controls. The plasma was then separated to measure the expression level of hsa_circ_0001666 and ETV4.

• Measuring the plasma hsa_circ_0001666 relative expression level using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR):

A plasma volume of 200 μL was used to extract total RNA, including circRNA, following the directions provided by the manufacturer of the Qiagen® miRNeasy Mini Kit (Cat. No. 217004) (Qiagen, Germany).

The RNA concentration and purity were estimated at 260, 280, and 230 nm using NanoDrop 2000/2000c Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A260/A280 and A260/A230 ratios of 1.8-2.1 suggested highly pure RNA.[14]

Synthesis of complementary DNA (cDNA) was performed using RevertAid First Strand cDNA Synthesis Kit (Cat. No. K1622) from Applied Biosystems (USA). Each reverse transcriptase (RT) reaction was carried out in a 20 μl reaction volume (10 μL of 2X RT master mix and 10 μL of RNA sample).

Real-time polymerase chain reaction (PCR) was carried out using Maxima SYBR Green qPCR Master Mix (2X) from Thermo Fisher Scientific (Cat.No. K0251), and specific primers for hsa_circ_0001666: forward (5′-CTGCCTAGCTGTCAAGGAGTGG-3′) and reverse (5′-TCCGGGAAAGGATCTGGAATG-3′). After 10 minutes of initial denaturation at 95°C, a three-step cycling process was carried out: denaturation (95°C, 15 seconds), annealing (55°C, 30 seconds), and extension (72°C, 30 seconds). Each PCR reaction was carried out in a 16 μl reaction volume (10 μL master mix, 1 μL forward Primer, 1 μL reverse primer, 0.1 μL ROX solution, 3.9 μL nuclease-free water). A melting-curve analysis was then conducted to confirm the identity and specificity of the PCR products. Quantitative real-time PCR was performed in duplicate for each cDNA sample. To avoid misinterpretation, a blank control without DNA was included in each PCR run as a negative control. The relative expression of hsa_circ_0001666 was calculated using the comparative cycle threshold (CT) formula (2^−ΔΔCT) using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control.[15]

• Measuring the plasma ETV4 concentration using enzyme-linked immunosorbent assay (ELISA):

This ELISA kit was provided by Chongqing Biospes Co., Ltd, China (cat. No. BZEK2235), (Website: www.biospes.com, Email: [e-mail]/[e-mail]).

This kit is based on standard sandwich ELISA technology. A human ETV4 antibody has been pre-coated onto the Micro ELISA strip plate included in the kit. The sensitivity of the kit is 1 pg/ml, and the detection range is 10-160 pg/ml.

Samples and standards were run in duplicates. The optical density (OD) was determined using spectrophotometry at a wavelength of 450 nm. The concentration of ETV4 was related to the OD value. The standard curve was constructed, and the relative O.D.450 value for each sample was used to determine the corresponding concentration of ETV4 from the standard curve.

Statistical Analysis

Version 20.0 of the Statistical Package for Social Sciences for Windows (SPSS, IBM Corp., Armonk, NY, United States) software was used for the statistical analysis.

When comparing two groups for quantitative data, the Mann-Whitney test was used for non-normal distributions, and the student's t-test was used for normal distributions. To compare between more than two groups, F for one-way ANOVA was used for normally distributed quantitative variables, and Post Hoc test (tukey) for pairwise comparisons. While the Kruskal Wallis test was used for non-normally distributed quantitative variables, and Post Hoc (Dunn's multiple comparisons test) for pairwise comparisons.

Results

1. Demographic and Laboratory Data: ([Table 1])

Table 1 Comparison between the three studied groups according to demographic data and laboratory investigations

	Group Ia (n = 25)		Group Ib (n = 25)		Group II (n = 25)		Test of Sig.	p
	No.	%	No.	%	No.	%
Sex
Male	9	45.0	5	25.0	10	50.0	χ2= 2.917	0.233
Female	11	55.0	15	75.0	10	50.0
Age (years)
Min – Max.	20.0–50.0		18.0–46.0		27.0–47.0		F= 2.757	0.072
Mean ± SD.	32.0 ± 8.0		32.0 ± 8.17		36.90 ± 6.59
Median (IQR)	30.50 (26.0–38.0)		29.50 (25.50–39.0)		37.0 (31.0–44.0)
Hemoglobin (g/dl)
Min – Max.	9.20–15.90		10.0–14.60		10.90–14.40		F = 2.388	0.101
Mean ± SD.	12.80 ± 1.64		12.03 ± 1.07		12.89 ± 1.33
Median (IQR)	12.80(11.75–13.80)		12.05(11.65–12.40)		13.15(11.70–14.10)
WBCs
Min – Max.	3.90–21.50		3.80–13.0		4.0–7.60		F = 3.660[*]	0.032[*]
Mean ± SD.	7.99 ± 3.90		7.67 ± 2.30		5.87 ± 0.96
Median (IQR)	7.30 (5.80–8.15)		7.65 (6.50–8.80)		5.75 (5.20–6.50)
Sig. bet. grps.	p1 = 0.924, p2 = 0.039[*], p3 = 0.093
Platelets
Min – Max.	155.0–544.0		194.0–506.0		190.0–485.0		F = 0.027	0.974
Mean ± SD.	319.5 ± 109.7		320.6 ± 87.06		313.6 ± 110.4
Median (IQR)	300.5(240.5–386.0)		318.5(253.0–378.5)		290.5(213.0–432.0)
ESR (mm/hr.)
Min – Max.	15.0–32.0		6.0–16.0		3.0–10.0		F= 114.63[*]	<0.001[*]
Mean ± SD.	21.45 ± 4.55		9.25 ± 2.86		6.50 ± 2.06
Median (IQR)	21.0 (17.0–24.0)		8.0 (7.0–10.50)		6.50 (5.0–8.0)
Sig. bet. grps.	p1 < 0.001[*], p2 < 0.001[*], p3 = 0.030[*]
CRP (mg/l)
Min – Max.	4.0–57.0		0.16–3.0		0.10–2.0		H= 45.273[*]	<0.001[*]
Mean ± SD.	13.25 ± 11.34		1.79 ± 0.91		0.69 ± 0.56
Median (IQR)	10.50(8.50–13.0)		1.95 (1.1–2.55)		0.58 (0.24–1.0)
Sig. bet. grps.	p1 < 0.001[*], p2 < 0.001[*], p3 = 0.015[*]
albumin(g/dl)
Min – Max.	3.60–5.20		3.0–5.30		3.60–5.0		H= 4.988	0.083
Mean ± SD.	4.38 ± 0.56		4.01 ± 0.66		4.17 ± 0.55
Median (IQR)	4.15 (3.90–4.90)		3.80 (3.55–4.40)		3.95 (3.80–4.90)
ALT (U/L)
Min – Max.	11.0–32.0		7.0–24.0		9.0–16.0		F= 12.864[*]	<0.001[*]
Mean ± SD.	18.15 ± 4.45		13.65 ± 3.98		12.50 ± 2.44
Median (IQR)	17.50(16.0–19.50)		12.50 (11.0–16.0)		12.0 (11.0–15.0)
Sig. bet. grps.	p1 = 0.001[*], p2 < 0.001[*], p3 = 0.594
AST (U/L)
Min – Max.	11.0–27.0		9.0–22.0		10.0–19.0		F= 6.095[*]	0.004[*]
Mean ± SD.	17.25 ± 4.44		14.60 ± 3.03		13.60 ± 2.48
Median (IQR)	16.50(14.0–19.50)		14.0 (13.0–16.0)		13.0 (12.0–15.0)
Sig. bet. grps.	p1 = 0.045[*], p2 = 0.004[*], p3 = 0.627
Urea (mg/dl)
Min – Max.	11.0–30.0		13.0–28.0		10.0–24.0		F= 3.885[*]	0.026[*]
Mean ± SD.	20.35 ± 5.30		19.90 ± 5.13		16.20 ± 5.06
Median (IQR)	20.0 (17.0–23.0)		19.5(15.50–24.50)		15.50(12.0–20.0)
Sig. bet. grps.	p1 = 0.959, p2 = 0.036[*], p3 = 0.069
Creatinine (mg/dl)
Min – Max.	0.57–1.10		0.45–1.05		0.37–1.02		F= 4.029[*]	0.023[*]
Mean ± SD.	0.79 ± 0.14		0.75 ± 0.17		0.63 ± 0.25
Median (IQR)	0.79 (0.69–0.88)		0.78 (0.61–0.86)		0.55 (0.39–0.91)
Sig. bet. grps.	p1 = 0.767, p2 = 0.022[*], p3 = 0.113
Fecal calprotectin (ug/g)
Min – Max.	40.0–549.0		20.0–388.0		16.0–45.0		H= 33.380[*]	<0.001[*]
Mean ± SD.	248.9 ± 178.7		80.45 ± 111.0		28.40 ± 9.37
Median (IQR)	214.5 (70.0–403.0)		36.0 (28.0–52.0)		25.50 (21.0–33.50)
Sig. bet. grps.	p1 < 0.001[*], p2 < 0.001[*], p3 = 0.039[*]
ETV4 (pg/ml)
Min – Max.	159.5–249.0		123.0–185.0		92.50–137.5		F= 82.381[*]	<0.001[*]
Mean ± SD.	198.3 ± 28.63		142.6 ± 17.03		112.3 ± 16.59
Median (IQR)	199.5 (171.0–217.0)		140.8 (127.8–150.3)		103.3 (98.75–129.5)
Sig. bet. grps.	p1 < 0.001[*], p2 < 0.001[*], p3 < 0.001[*]
Hsa_circ_0001666 relative expression (Fold change)
Min – Max.	102.5–10022.8		0.97–275.4		0.002–7.97		H= 43.771[*]	<0.001[*]
Mean ± SD.	1584.4 ± 2600.3		65.21 ± 102.5		2.93 ± 3.32
Median (IQR)	786.4 (402.6–1160.4)		8.79 (5.47–81.88)		0.21 (0.02–6.03)
Sig. bet. grps.	p1 < 0.001[*], p2 < 0.001[*], p3 = 0.005[*]

Abbreviations: ALT, Alanine aminotransferase; AST, Aspartate transaminase; CRP, C-reactive protein; ESR, Erythrocyte sedimentation rate; ETV4, ETS variant transcription factor 4; F, F for One way ANOVA test; H, H for Kruskal Wallis test; IQR, Inter quartile range; SD, Standard deviation; WBCs, White blood cells; χ², Chi-squared test.

Notes: Pairwise comparison between each 2 groups was done using Post Hoc Test (Tukey), Pairwise comparison between each 2 groups was done using Post Hoc Test (Dunn's for multiple comparisons.

p: p value for comparing between the three studied groups.

p₁: p value for comparing between Group Ia and Group Ib.

p₂: p value for comparing Group Ia and Group II.

p₃: p value for comparing Group Ib and Group II.

*Statistically significant at p ≤ 0.05.

Group Ia: Patients with active Crohn's disease.

Group Ib: Patients with inactive Crohn's disease.

Group II: Control group.

There were no significant differences in the distribution of age and gender among the study groups (p >0.05).

Regarding routine laboratory testing, WBC count was significantly higher in active CD patients than in healthy controls.

ESR and CRP plasma levels were significantly higher in active and inactive CD patients than in healthy controls, and in active CD patients than in patients with inactive disease.

ALT and AST plasma levels were significantly higher in active CD patients than in patients with inactive disease and healthy controls.

Plasma levels of Urea and creatinine were significantly higher in active CD patients than in healthy controls.

The level of Fecal calprotectin was significantly higher in active and inactive CD patients than in healthy controls, and in active CD patients than in patients with inactive disease.

2. Harvey-Bradshaw index (HBI) and Simple Endoscopic Score for Crohn's Disease (SES-CD) in the patient group:

Active CD patients demonstrated significantly higher HBI and SES-CD than patients with inactive disease (P <0.001).

3. Plasma relative expression of hsa_circ_0001666 and ETV4 level: ([Table 1])

Plasma relative expression levels of hsa_circ_0001666 and ETV4 concentrations were significantly higher in both active and inactive CD patients than in the control group. Additionally, active CD patients had a significantly higher level than inactive individuals.

4. Correlation between plasma ETV4 level and other parameters in each group: ([Figs. 1] and [2])

Fig. 1 Correlation between plasma levels of ETV4 and Circ_0001666 relative expression in group I (Patients group).

Fig. 2 Correlation between plasma ETV4 level and fecal calprotectin level in group I (Patients group).

There is a significant positive correlation was observed between plasma levels of ETV4 and hsa_circ_0001666 relative expression in active CD patients, inactive CD patients, and in healthy controls as determined by Pearson correlation coefficient (p <0.001).

Also in the patient group (group I), plasma ETV4 level was positively correlated to the fecal calprotectin level (p =0.002), HBI and SES-CD (p <0.001), ESR and CRP levels (p < 0.001), serum albumin level (p = 0.048), and serum ALT level (p = 0.031).

5. Correlation between plasma hsa_circ_0001666 relative expression level and other parameters in the different studied groups: ([Fig. 3])

Fig. 3 Correlation between plasma Circ_0001666 relative expression level and fecal calprotectin level in group I (patients' group).

In the patient group (I), there was a significant positive correlation relating plasma hsa_circ_0001666 level to fecal calprotectin level (p =0.002), plasma ETV4 level (p < 0.001), HBI and SES-CD (p <0.001), ESR and CRP levels (p < 0.001), serum albumin level (p = 0.003) and serum ALT level (p = 0.002).

6. Diagnostic performance of plasma hsa_circ_0001666 and ETV4 and fecal calprotectin to discriminate inactive CD patients from healthy controls: ([Fig. 4])

Fig. 4 ROC curve for fecal calprotectin, plasma ETV4 and Circ_0001666 relative expression to discriminate group Ib (inactive CD patients) from group II (healthy controls).

The optimal cut-off values for plasma hsa_circ_0001666 and ETV4 and fecal calprotectin were determined using Youden's index (J = sensitivity + specificity – 1), which identifies the threshold that maximizes the test's discriminatory power.[16]

At the cut-off value of 5.35-fold change, the sensitivity of the plasma relative expression level of hsa_circ_0001666 in detecting CD patients has been estimated to be 75% while its specificity has been shown to be 70% (AUC = 0.865, 95%CI = 0.755–0.975, P < 0.001).

At the cut-off value of 128 pg/ml, the sensitivity of the plasma ETV4 level in detecting CD patients was 75% while its specificity was 70% (AUC = 0.871, 95%CI = 0.765–0.977, P < 0.001).

At the cut-off value of >30 ug/g, the sensitivity of the fecal calprotectin level in detecting CD patients has been estimated to be 65% while its specificity was 65% (AUC = 0.735, 95%CI = 0.580–0.890, P < 0.011).

Combined together, plasma hsa_circ_0001666 and ETV4 and fecal calprotectin showed 80% sensitivity and 85% specificity in detecting CD patients (AUC = 0.935, 95%CI = 0.864–1.000, P < 0.001).

7. Diagnostic performance of plasma hsa_circ_0001666 and ETV4 and fecal calprotectin to discriminate active from inactive CD patients: ([Fig. 5])

Fig. 5 ROC curve for fecal calprotectin, plasma ETV4 and Circ_0001666 to discriminate group Ia (active CD patients) from group Ib (inactive CD patients).

At the cut-off value of 108-fold change, the sensitivity of the plasma relative expression level of hsa_circ_0001666 in detecting active CD patients has been estimated to be 85% while its specificity has been shown to be 80% (AUC = 0.955, 95%CI = 0.899–1.000, P < 0.001).

At the cut-off value of 166 pg/ml, the sensitivity of the plasma ETV4 level in detecting active CD patients was 85% while its specificity was 90% (AUC = 0.961, 95%CI = 0.908–1.000, P < 0.001).

At the cut-off value of >51 ug/g, the sensitivity of the fecal calprotectin level in detecting active CD patients has been estimated to be 80% while its specificity was 75% (AUC = 0.880, 95%CI = 0.771–0.989, P < 0.001).

Combined, plasma hsa_circ_0001666 and ETV4 and fecal calprotectin showed 90% sensitivity and 90% specificity in detecting active CD patients (AUC = 0.970, 95%CI = 0.927–1.000, P < 0.001).

Discussion

Crohn's disease (CD) is an inflammatory bowel disease (IBD) characterized by remission and relapses.[17] Fibrosis is a common complication of CD. Epithelial-mesenchymal transition (EMT) might play a major role in the pathogenesis of fibrosis in CD, where activated fibroblasts are being recruited in the inflamed intestinal tract.[6]

Crohn's disease is continuously increasing worldwide, making it a global health concern.[18] Early diagnosis and treatment is important because CD can lead to complications; the most serious of which is colorectal cancer (CRC).[19] The diagnosis of CD still relies on non-specific markers and invasive endoscopy.[20]

CircRNAs represent one of the non-coding RNAs that are implicated in various diseases such as chronic inflammation and malignant tumors. These molecules are highly stable both intracellular and in biological fluids as blood. These findings suggest the possibility of using circRNAs as promising non-invasive biomarkers and effective targets for disease treatment.[21]

The circRNAs in CD are not well studied, and little is known about their roles. However, research results suggest that these molecules can play an important role in CD. It has been discovered that certain circRNAs are expressed differently in healthy individuals and those with IBD. Some of these circRNAs are thought to regulate IBD development. Though the precise mechanism of their involvement in CD is still unclear.[6]

Has_circ_0001666 is thought to participate in the development of CD by mechanisms such as miRNA sponging. It has been shown that hsa_circ_0001666 acts as a sponge for miR-330-5p, miR-193a-5p, and miR-326. These three miRNAs were shown to have binding sites with ETV4 mRNA, a protein involved in inflammation, invasion, EMT, and fibrosis.[11]

In the current study, we investigated the plasma relative expression levels of hsa_circ_0001666 and ETV4 protein concentration in 50 patients (25 diagnosed with active CD and 25 with inactive CD) and 25 healthy controls to determine the possibility of using them as non-invasive biomarkers for diagnosis of CD and assessment of the disease activity.

The study revealed that plasma relative expression level of hsa_circ_0001666 and ETV4 protein concentration, together with fecal calprotectin level, were significantly higher in both active and inactive CD patients in comparison to the healthy controls, and in active CD patients than in patients with inactive disease. A significant positive correlation was observed between hsa_circ_0001666 and ETV4 levels in the three studied groups. The upregulated hsa_circ_0001666 might contribute to increased ETV4 expression, potentially by miRNA sponging.[11]

Moreover, correlation between hsa_circ_0001666 and ETV4 levels and the markers of disease activity in the patient group (group I) showed that plasma relative expression of hsa_circ_0001666 and ETV4 levels were positively correlated to the fecal calprotectin level, HBI, and SES-CD, ESR, and CRP levels. The higher expression in active disease may indicate that hsa_circ_0001666 plays a role in driving or modulating inflammatory responses in active CD, contributing to disease flare-ups.

ROC curve was done to compare the diagnostic role of plasma hsa_circ_0001666 and ETV4 and fecal calprotectin to discriminate inactive CD patients from the control group. Each of hsa_circ_0001666 and ETV4 showed higher sensitivity and specificity (75% and 70% respectively, for both) than fecal calprotectin (both sensitivity and specificity were 65%). The highest sensitivity and specificity were obtained by using the three markers together (sensitivity 80% and specificity 85%).

Furthermore, ROC curve analysis for discriminating active from inactive CD patients revealed that each of hsa_circ_0001666 and ETV4 had higher sensitivity (85% for both) and specificity (80% and 90% respectively) than fecal calprotectin (sensitivity 80% and specificity 75%). While using the three markers together, they showed the highest sensitivity and specificity (both were 90%).

Our findings agree with those of Li J et al,[22] who found that hsa_circ_0001666 was overexpressed in CD children's colonic mucosal tissues and in the CD cell model induced by TGF-β1. Moreover, the expression of hsa_circ_0001666 was correlated with the expression of EMT marker proteins in the colonic mucosal tissues of CD patients. It was positively correlated with N-cadherin and negatively correlated with E-cadherin (an adhesion molecule that was significantly downregulated in the colonic mucosa of patients with CD, weakening the cell-cell connection).[22]

The results of this study demonstrated that hsa_circ_0001666 enhanced intestinal epithelial cell migration, invasion, EMT, and fibrosis by interacting with the splicing factor SRSF1 (Serine/Arginine-Rich Splicing Factor 1), decreasing the stability of BMP7 mRNA, a protein known for its anti-fibrotic properties. Silencing hsa_circ_0001666 has been shown to inhibit TGF-β1-induced EMT and intestinal epithelial cell fibrosis.[22]

Another research by Yu- an Hu et al[6] showed that, compared to ulcerative colitis (UC) and healthy controls, hsa_circ_0001666 expression was significantly higher in colon tissues of CD, while the level did not differ between UC and controls. This speculated that hsa_circ_0001666 is CD-associated circRNA. The AUC of hsa_circ_0001666 was 0.858, indicating its favorable diagnostic value. Moreover, they observed that the predicted microRNAs of hsa_circ_0001666 are related to several cellular processes, such as EMT and carcinogenesis.[6]

To the best of our knowledge, no other research has studied the exact mechanism of hsa_circ_0001666 in the development of CD yet.

However, several studies have evaluated hsa_circ_0001666 expression in various cancers. Zhang R et al[23] found that hsa_circ_0001666 was upregulated in pancreatic cancer tissues and cell lines, and high expression correlated with poorer prognosis. The results of this study suggested that hsa_circ_0001666 promotes EMT and invasion by sponging miR-1251, leading to overexpression of SOX4, a transcription factor that directly controls the expression of many genes promoting EMT, tumor growth, and metastasis.[23]

Another study revealed that hsa_circ_0001666 was overexpressed in non-small cell lung cancer (NSCLC) cell lines and tissues, and it promoted migration and invasion of NSCLC cells both in vitro and in vivo. Hsa_circ_0001666 could directly sponge miR-1184 and miR-548I to upregulate the expression of eukaryotic initiation factor AGO1 and consequently activate the PI3K/AKT/mTOR signaling pathway.[24]

In contrast, Na Su et al[25] found that hsa_circ_0001666 was downregulated in breast cancer tissues and cell lines, leading to overexpression of miR-620 and downregulation of serine/threonine kinase WNK2, a key regulator of cell growth and apoptosis, which has a suppressive tumor effect. Overexpression of hsa_circ_0001666 inhibited tumor proliferation and invasion by sponging miR-620 and upregulating WNK2.[25]

In colorectal cancer (CRC), a study by Bai F et al[26] showed that hsa_circ_0001666 was downregulated in colorectal cancer (CRC) tissues and cell lines. They proved that hsa_circ_0001666 suppresses CRC cell proliferation, invasion, and increases apoptosis by targeting miR-1229, consequently inhibiting the Wnt/β-catenin signaling pathway, a well-known cancer-promoting pathway.[26] Similarly, Zhou J et al[27] found that hsa_circ_0001666 was downregulated in CRC, and higher expression levels were associated with a better clinical prognosis. The results of this study revealed that hsa_circ_0001666 inhibits proliferation, invasion, metastasis, and EMT and induces apoptosis of CRC cells by sponging miR-576-5p, reducing its suppression on PCDH10, a tumor suppressor protein acting through Wnt/β-catenin and PI3K/AKT signaling pathways.[27]

These findings highlight that hsa_circ_0001666 might have either oncogenic or tumor suppressive actions by targeting variable miRNA molecules affecting different signaling pathways. Therefore, hsa_circ_0001666 might be a promising therapeutic target for cancer treatment

ETV4 has been studied in various cancers, such as hepatocellular carcinoma and intrahepatic cholangiocarcinoma, where it is known to promote tumor progression through mechanisms like EMT and the upregulation of matrix metalloproteinases (MMPs).[9] Other research revealed that ETV4 serves a tumor-promoting role in other cancers, including colorectal cancer.[28]

According to our knowledge, no research has linked the transcription factor ETV4 directly to Crohn's disease. However, related factors like ETV5 have been implicated in promoting CD4+ T cell–mediated inflammation and fibrosis through upregulating IL-9 in IBD models.[29]

The findings of the present study are in line with those of Ying QI et al[11] who revealed that hsa_circ_0001666 and ETV4 protein were upregulated in human papillary thyroid carcinoma (PTC) with downregulation of miR-330-5p, miR-193a-5p, and miR-326, and these findings were associated with lymph node metastasis. Hsa_circ_0001666 knockdown suppressed proliferation and promoted apoptosis of PTC cells both in vitro and in vivo. Specific inhibitors targeting miR-33-5p, miR-193a-5p, or miR-326 reversed the effect of hsa_circ_0001666 silencing and resulted in ETV4 overexpression.[11] These results suggested that hsa_circ_0001666 promotes the progression of PTC by functioning as a sponge for miR-330-5p, miR-193a-5p, and miR-326 to enhance proliferation and suppress apoptosis of PTC cells by positively regulating ETV4.[11]

These findings, together with the results of our study, suggested that hsa_circ_0001666 may have a role in the development of CD and regulation of the disease activity via the miR-330-5p/miR-193a-5p/miR-326/ETV4 pathway.

Conclusions

The results of this study suggest that hsa_circ_0001666 can participate in the development of CD by functioning as a miRNA sponge to positively regulate ETV4. In addition to fecal calprotectin, hsa_circ_0001666 and ETV4 could be used as potential non-invasive biomarkers to improve overall diagnostic accuracy and to monitor CD progression or flare-ups. Furthermore, targeting this pathway with specific inhibitors could offer a promising therapeutic approach for managing CD.

Limitations and Recommendations

Because of the participants' restricted geographic distribution, these results require confirmation with a larger sample size from several different locations. The circRNA/miRNA/mRNA and protein network were only predicted by bioinformatics analysis. Therefore, further investigations and experimental research are required in the future to measure the expression level of circRNAs, together with associated RNAs and proteins. As a result, their functional mechanisms will be better understood, and patients with Crohn's disease may be able to access new therapy options.

Conflict of Interest

The authors have no conflicts of interest to declare.

Authors' Contribution

WNR: writing and editing; FDA: writing and editing; AFK: review and approval of the final version; NAB: review and approval of the final version; AMI: review and approval of the final version. All authors contributed equally to data collection, implementation of the experiment, analysis, and interpretation of results.

• References

• 1 Lightner AL, McKenna NP, Alsughayer A. et al. Anti-TNF biologic therapy does not increase postoperative morbidity in pediatric Crohn's patients. J Pediatr Surg 2019; 54 (10) 2162-2165
• 2 Cockburn E, Kamal S, Chan A. et al. Crohn's disease: an update. Clin Med (Lond) 2023; 23 (06) 549-557
• 3 Chen P, Zhou G, Lin J. et al. Serum Biomarkers for Inflammatory Bowel Disease. Front Med (Lausanne) 2020; 7: 123
• 4 Jeck WR, Sharpless NE. Detecting and characterizing circular RNAs. Nat Biotechnol 2014; 32 (05) 453-461
• 5 Qiao YQ, Cai CW, Shen J, Zheng Q, Ran ZH. Circular RNA expression alterations in colon tissues of Crohn's disease patients. Mol Med Rep 2019; 19 (05) 4500-4506
• 6 Hu YA, Zhu Y, Liu G. et al. Expression profiles of circular RNAs in colon biopsies from Crohn's disease patients by microarray analysis. J Clin Lab Anal 2021; 35 (06) e23788
• 7 Iguchi A, Kitajima I, Yamakuchi M. et al. PEA3 and AP-1 are required for constitutive IL-8 gene expression in hepatoma cells. Biochem Biophys Res Commun 2000; 279 (01) 166-171
• 8 Subbaramaiah K, Norton L, Gerald W, Dannenberg AJ. Cyclooxygenase-2 is overexpressed in HER-2/neu-positive breast cancer: evidence for involvement of AP-1 and PEA3. J Biol Chem 2002; 277 (21) 18649-18657
• 9 Kapila S, Xie Y, Wang W. Induction of MMP-1 (collagenase-1) by relaxin in fibrocartilaginous cells requires both the AP-1 and PEA-3 promoter sites. Orthod Craniofac Res 2009; 12 (03) 178-186
• 10 Pellecchia A, Pescucci C, De Lorenzo E. et al. Overexpression of ETV4 is oncogenic in prostate cells through promotion of both cell proliferation and epithelial to mesenchymal transition. Oncogenesis 2012; 1 (07) e20
• 11 Qi Y, He J, Zhang Y. et al. Circular RNA hsa_circ_0001666 sponges miR–330–5p, miR–193a–5p and miR–326, and promotes papillary thyroid carcinoma progression via upregulation of ETV4. Oncol Rep 2021; 45 (04) 50
• 12 Harvey RF, Bradshaw JM. A simple index of Crohn's-disease activity. Lancet 1980; 1 (8167) 514
• 13 Brodersen JB, Kjeldsen J, Knudsen T, Jensen MD. Endoscopic severity and classification of lesions with pan-enteric capsule endoscopy and ileocolonoscopy in ileocolonic Crohn's disease. Endosc Int Open 2023; 11 (01) E32-E38
• 14 Sriram H, Khanka T, Kedia S. et al. Improved protocol for plasma microRNA extraction and comparison of commercial kits. Biochem Med (Zagreb) 2021; 31 (03) 030705
• 15 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25 (04) 402-408
• 16 Šimundić AM. Measures of diagnostic accuracy: Basic definitions. EJIFCC 2009; 19 (04) 203-211
• 17 Dolinger M, Torres J, Vermeire S. Crohn's disease. Lancet 2024; 403 (10432): 1177-1191
• 18 Long D, Wang C, Huang Y, Mao C, Xu Y, Zhu Y. Changing epidemiology of inflammatory bowel disease in children and adolescents. Int J Colorectal Dis 2024; 39 (01) 73
• 19 Lichtenstein GR, Shahabi A, Seabury SA. et al. Increased Lifetime Risk of Intestinal Complications and Extraintestinal Manifestations in Crohn's Disease and Ulcerative Colitis. Gastroenterol Hepatol (N Y) 2022; 18 (01) 32-43
• 20 Li L, Cheng R, Wu Y, Lin H, Gan H, Zhang H. Diagnosis and management of inflammatory bowel disease. J Evid Based Med 2024; 17 (02) 409-433
• 21 Conn VM, Chinnaiyan AM, Conn SJ. Circular RNA in cancer. Nat Rev Cancer 2024; 24 (09) 597-613
• 22 Li J, Xu JZ, Dou B. et al. Circ_0001666 upregulation promotes intestinal epithelial cell fibrosis in pediatric Crohn's disease via the SRSF1/BMP7 axis. Kaohsiung J Med Sci 2023; 39 (10) 966-977
• 23 Zhang R, Zhu W, Ma C, Ai K. Silencing of circRNA circ_0001666 Represses EMT in Pancreatic Cancer Through Upregulating miR-1251 and Downregulating SOX4. Front Mol Biosci 2021; 8: 684866
• 24 Wang X, Li R, Feng L. et al. Hsa_circ_0001666 promotes non-small cell lung cancer migration and invasion through miR-1184/miR-548I/AGO1 axis. Mol Ther Oncolytics 2022; 24: 597-611
• 25 Su N, Liu L, He S, Zeng L. Circ_0001666 affects miR-620/WNK2 axis to inhibit breast cancer progression. Genes Genomics 2021; 43 (08) 947-959
• 26 Bai F, Zuo C, Ouyang Y, Xiao K, He Z, Yang Z. Circular RNA 0001666 inhibits colorectal cancer cell proliferation, invasion and stemness by inactivating the Wnt/β-catenin signaling pathway and targeting microRNA-1229. Oncol Lett 2022; 23 (05) 153
• 27 Zhou J, Wang L, Sun Q. et al. Hsa_circ_0001666 suppresses the progression of colorectal cancer through the miR-576-5p/PCDH10 axis. Clin Transl Med 2021; 11 (11) e565
• 28 Fonseca AS, Ramão A, Bürger MC. et al. ETV4 plays a role on the primary events during the adenoma-adenocarcinoma progression in colorectal cancer. BMC Cancer 2021; 21 (01) 207
• 29 Shi Y, Ma C, Wu S. et al. ETS translocation variant 5 (ETV5) promotes CD4⁺ T cell-mediated intestinal inflammation and fibrosis in inflammatory bowel diseases. Mucosal Immunol 2024; 17 (04) 584-598

Publication History

Received: 13 May 2025

Accepted: 04 August 2025

Article published online:
25 September 2025